HPPD VARIANTS AND METHODS OF USE

VARIANTS HPPD ET LEURS PROCEDES D'UTILISATION

English Abstract

The present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, wherein in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence comprises at least one amino acid selected from certain amino acids at specific positions important for conferring an increased HPPD inhibitor tolerance. The present invention also relates to proteins encoded by the nucleic acid of the invention, to chimeric genes, plant cells comprising the nucleic acid of the invention operably linked to a plant- expressible promoter and optionally a transcription termination and polyadenylation region, plants essentially consisting of the plant cells of the invention and methods of obtaining transgenic plants.

French Abstract

La présente invention concerne une molécule dacide nucléique isolée qui comprend une séquence nucléotidique qui encode une protéine de HPPD mutée où cette protéine de HPPD mutée a une activité de HPPD où au moins un acide aminé de cette protéine de HPPD mutée a été remplacé pour que la séquence d'acides aminés résultante comprenne au moins un acide aminé sélectionné parmi certains acides aminés à certaines positions importantes pour apporter une tolérance augmentée aux inhibiteurs de HPPD. La présente invention concerne également les protéines codées par lacide nucléique de linvention, les gènes chimères, les cellules végétales qui comprennent lacide nucléique de linvention liée fonctionnellement à un promoteur d'expression chez les végétaux et, facultativement, une terminaison de transcription et une zone de polyadénylation. Les plantes consistent essentiellement de cellules végétales de linvention et les procédés pour obtenir des plantes transgéniques.

Claims

118
CLAIMS:
1. An isolated nucleic acid comprising a nucleotide sequence encoding a
mutated
HPPD protein,
wherein said mutated HPPD protein has HPPD activity, and
wherein said mutated HPPD protein comprises a replacement of the amino
acid at the position corresponding to position 421 of SEQ ID No. 2 with Asp or

Asn.
2. The isolated nucleic acid of claim 1, wherein said HPPD protein is
derived from
Streptomyces avermitilis (Genebank SAV11864), Daucus carota (Genebank
DCU 87257), Arabidopsis thaliana (Genebank AF047834), Mycosphaerella
graminicola (Genebank AF038152), oryza sativa / rice [BAD26248], Zea mays
/ corn [ACN36372], Avena sativa [ABZ23427], Pseudomonas fluorescens
[ABF50055], Synechococcus sp. [YP_473959], Blepharisma japonicum
[BAF91881], Rhodococcus RHA1 sp. r00240 [YP_702005], Rhodococcus
RHA1 sp. ro0341 [YP_703002], Picrophilus torridus [YP_024147], Kordia
algicida [ZP_02161490], Sorghum bicolor [XP_002453359], Triticum aestivum
/ wheat [AAZ67144], or Hordeum vulgare / barley [048604].
3. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
4 that comprises Asp or Asn, at position 418 of the amino acid sequence of
SEQ ID No. 4 (corresponding to position 421 of SEQ ID No. 2).
4. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
6 that comprises Asp or Asn at position 395 of the amino acid sequence of
SEQ ID No. 6 (corresponding to position 421 of SEQ ID No. 2).

119
5. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
8 that comprises Asp or Asn at position 412 of the amino acid sequence of
SEQ ID No. 8 (corresponding to position 421 of SEQ ID No. 2).
6. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
that comprises Asp or Asn at position 335 of the amino acid sequence of
SEQ ID No. 10 (corresponding to position 421 of SEQ ID No. 2).
7. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
14 that comprises Asp or Asn at position 327 of the amino acid sequence of
SEQ ID No. 14 (corresponding to position 421 of SEQ ID No. 2).
8. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
16 that comprises Asp or Asn at position 361 of the amino acid sequence of
SEQ ID No. 16 (corresponding to position 421 of SEQ ID No. 2).
9. The isolated nucleic acid of claim 1, wherein the amino acid sequence of
said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
18 that comprises Asp or Asn at position 381 of the amino acid sequence of
SEQ ID No. 18 (corresponding to position 421 of SEQ ID No. 2).
10. The isolated nucleic acid of claim 1, wherein the amino acid sequence
of said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
30 that comprises Asp or Asn at position 382 of the amino acid sequence of
SEQ ID No. 30 (corresponding to position 421 of SEQ ID No. 2).

120
11. The isolated nucleic acid of claim 1, wherein the amino acid sequence
of said
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
20 that comprises Asp at position 348 of the amino acid sequence of SEQ ID
No. 20 (corresponding to position 421 of SEQ ID No. 2).
12. The isolated nucleic acid of claim 1, wherein said the amino acid
sequence of
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
22 that comprises Asp or Asn at position 367 of the amino acid sequence of
SEQ ID No. 22 (corresponding to position 421 of SEQ ID No. 2).
13. The isolated nucleic acid of claim 1, wherein said the amino acid
sequence of
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
24 that comprises Asp or Asn at position 412 of the amino acid sequence of
SEQ ID No. 24 (corresponding to position 421 of SEQ ID No. 2).
14. The isolated nucleic acid of claim 1, wherein said the amino acid
sequence of
mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
26 that comprises Asp or Asn at position 408 of the amino acid sequence of
SEQ ID No. 26 (corresponding to position 421 of SEQ ID No. 2).
15. The nucleic acid of any one of claims 1 to 14, wherein said mutated
HPPD
protein increases the tolerance of a plant to at least one herbicide acting on

HPPD.
16. A protein encoded by the isolated nucleic acid of any one of claims 1
to 15.
17. A gene or a nucleic acid comprising a coding sequence comprising the
nucleic
acid of any one of claims 1 to 15 operably linked to a plant-expressible
promoter and optionally a transcription termination and polyadenylation
region.

121
18. A method of obtaining a mutated HPPD protein modulating the tolerance
of a
plant to at least one herbicide acting on HPPD, wherein said mutated HPPD
protein has HPPD activity, the method comprising
i) providing an HPPD protein;
ii) replacing the amino acid at position 421 of SEQ ID NO. 2 in said HPPD
protein so that the resulting amino acid sequence comprises Asp, Phe, or
Asn at the position corresponding to position 421 of the amino acid
sequence of SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one
herbicide acting on HPPD,
wherein an inhibition of the resulting protein of less or more than that
observed
with a reference HPPD protein is indicative that the resulting protein
modulates
the tolerance of a plant to said herbicide.
19. The method of claim 18, wherein said mutated HPPD protein increases the

tolerance of a plant to at least one HPPD inhibitor herbicide.
20. The nucleic acid of claim 15 or the method of claim 19, wherein the
herbicide
acting on HPPD is tembotrione.
21. A method of producing a transgenic plant comprising introducing into
said
plant the nucleic acid of any one of claims 1 to 15 and 20, operably linked to
a
plant expressible promoter or the chimeric gene of claim 17.
22. The method of claim 21, wherein the nucleic acid of claim 16 or a
nucleic acid
identified by the method of claim 18 or 19, both operably linked to a plant
expressible promoter, or the chimeric gene of claim 17 comprising the nucleic
acid of claim 15 is introduced into said plant.
23. A plant cell comprising the isolated nucleic acid of any one of claims
1 to 15 or
the chimeric gene of claim 17 in its genetic information.

122
24. A cell of a plant, a cell of a part thereof or a cell of plant tissue
comprising the
isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of
claim
17 in its genetic information.
25. A cell of a plant comprising the isolated nucleic acid of any one of
claims 1 to
15 or the chimeric gene of claim 17 in its genetic information obtained from
the
method of claim 21 or 22.
26. The cell of claim 24 or 25 which is a cell comprising the isolated
nucleic acid of
any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic
information of a plant selected from wheat, cotton, canola, rice, corn,
soybean,
and sorghum.
27. A cell of a seed of a plant comprising the isolated nucleic acid of any
one of
claims 1 to 15 or the chimeric gene of claim 17 in its genetic information as
defined in any one of claims 24 to 26.
28. A cell of progeny of a plant cell comprising the isolated nucleic acid
of any one
of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information
as
defined in any one of claims 23 to 26 or a cell of progeny of a seed cell as
defined in claim 27.
29. A method of modulating a plant's tolerance to at least one herbicide
acting on
HPPD or of obtaining a plant tolerant to an HPPD inhibitor herbicide
comprising introducing the isolated nucleic acid of any one of claims 1 to 15
operably linked to a plant expressible promoter or the chimeric gene of claim
17 into a plant's genome.
30. A method for controlling weeds comprising spraying at least one HPPD
inhibitor herbicide on or around a crop plant, wherein said crop plant
contains
the nucleic acid according to claim 15 operably linked to a plant expressible

123
promoter or the chimeric gene of claim 17 comprising the nucleic acid of claim

15.
31. Use of a chimeric gene of claim 17 or the nucleic acid of any one of
claims 1 to
15 operably linked to a plant expressible promoter for modulating the
tolerance
of a plant to at least one HPPD inhibitor herbicide applied for controlling
weeds.
32. The method of any one of claims 22, 29 or 30 or the use of claim 31,
wherein
the tolerance of a plant to at least one HPPD inhibitor herbicide is
increased.

Description

81576661
HPPD variants and methods of use
Description
The present invention relates to an isolated nucleic acid comprising a
nucleotide
sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein
has HPPD activity, wherein in said mutated HPPD protein at least one amino
acid
has been replaced so that the resulting amino acid sequence comprises at least
one
amino acid selected from certain amino acids at specific positions important
for
conferring an increased HPPD inhibitor tolerance. The present invention also
relates
to proteins encoded by the nucleic acid of the invention, to chimeric genes,
plant
cells comprising the nucleic acid of the invention operably linked to a plant-
expressible promoter and optionally a transcription termination and
polyadenylation
region, plants essentially consisting of the plant cells of the invention and
methods of
obtaining transgenic plants.
In this specification, a number of documents including patent applications and

manufacturer's manuals are cited.
HPPD (hydroxyphenylpyruvate dioxygenase) proteins are enzymes which catalyse
the reaction in which para-hydroxyphenylpyruvate (abbreviated herein as HPP),
a
tyrosine degradation product, is transformed into homogentisate (abbreviated
herein
as HG), the precursor in plants of tocopherol and plastoquinone (Crouch N.P.
et al.
(1997) Tetrahedron, 53, 20, 6993-7010, Fritze et al., (2004), Plant Physiology

134:1388-1400). Tocopherol acts as a membrane-associated antioxidant.
Plastoquinone, firstly acts as an electron carrier between PSII and the
cytochrome
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, .
2
b6/f complex and secondly, is a redox cofactor for phytoene desaturase, which
is
involved in the biosynthesis of carotenoids.
Up to now, more than 700 nucleic acid sequences from various organisms present
in
NCB! database were annotated as coding for a putative protein having an HPPD
domain. But for most of these sequences, it has not been proven that the
protein
would have an HPPD enzymatic activity either in an in vitro assay or an in in
planta
approach, nor that such HPPD protein can confer herbicide tolerance to HPPD
inhibitor herbicides when expressed in a plant. Several HPPD proteins and
their
primary sequences have been described in the state of the art, in particular
the
HPPDs of bacteria such as Pseudomonas (Ruetschi et al, Eur. J. Biochem., 205,
459-466, 1992, WO 96/38567), of plants such as Arabidopsis (WO 96/38567,
Genebank AF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa (WO
02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO 02/046387),
Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO 02/046387), Festuca
arundinacea (WO 02/046387), Setaria faberi (WO 02/046387), Eleusine indica (WO

02/046387), Sorghum (WO 02/046387), Coccicoides (Genebank COlTRP),of Coptis
japonica ONO 06/132270), Chlamydomonas reinhardtii (ES 2275365), or of
mammals such as mouse or pig.
Most plants synthesize tyrosine via arrogenate (Abou-Zeid et al. (1995),
Applied Env
Microb 41: 1298-1302; Bonner et al., (1995), Plant Cells Physiol. 36, 1013-
1022;
Byng et al., (1981), Phytochemistry 6:1289-1292; Connely and Conn (1986), Z.
Naturforsch 41c: 69-78; Gaines et al., (1982), Plants 156: 233-240). In these
plants,
the HPP is derived only from the degradation of tyrosine. On the other hand,
in
organisms such as the yeast Sacharomyces cerevisiae or the bacterium
Escherichia
coil, HPP is a tyrosine precursor, and it is synthesized by the action of an
enzyme,
prephenate dehydrogenase (hereinafter referred to as PDH), which converts
prephenate to HPP (Lingens et al., (1967) European J. Biochem 1:363-374;
Sampathkumar and Morrisson (1982), Bloch Biophys Acta 701: 204-211). In these
organisms, the production of HPP is therefore directly connected to the
aromatic

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amino acid biosynthetic pathway (shikimate pathway), and not to the tyrosine
degradation pathway.
Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency in
accessory
light-harvesting pigments and, most importantly, to destruction of chlorophyll
by UV-
radiation and reactive oxygen species (bleaching) due to the lack of photo
protection
normally provided by carotenoids (Norris et al. (1995), Plant Cell 7: 2139-
2149).
Bleaching of photosynthetically active tissues leads to growth inhibition and
plant
death.
At present, most commercially available HPPD inhibitor herbicides belong to
one of
these four chemical families:
1) the triketones, e.g. sulcotrione [i.e. 242-chloro-4-
(methylsulfonyl)benzoy1]-1,3-
cyclohexanedione], mesotrione [i.e.244-(methylsulfony1)-2-nitrobenzoy1]-1,3-
cyclohexanedione]; tembotrione [i.e.242-chloro-4-(methylsulfony1)-3-[(2,2,2,-
tri-
fluoroethoxy)methyl] benzoyI]-1,3-cyclo-hexanedione]; tefuryltrione [i.e. 242-
chloro-
4-(methylsulfony1)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoy1]-1,3
cyclohexaned ione]]; bicyclopyrone [i.e. 4-hydroxy-34[2-[(2-
methoxyethoxy)methy1]-6-
(trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one] ;
Benzobicyclon
[i.e. 3-(2-chloro-4-mesylbenzoy1)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one]
2) The diketonitriles, e.g. 2-cyano-3-cyclopropy1-1-(2-methylsulphony1-4-
trifluoromethylpheny1)-propane-1,3-dione and 2-cyano-1-[4-(methylsulphonyI)-2-
trifluoromethylpheny1]-3-(1-methylcyclopropyl)propane-1,3-dione;
3) the isoxazoles, e.g. isoxaflutole [i.e.(5-cyclopropy1-4-isoxazoly0[2-
(methylsulfony1)-
4-(trifluoromethyl)phenyllmethanone]. In plants, the isoxaflutole is rapidly
metabolized in DKN, a diketonitrile compound which exhibits the HPPD inhibitor

property; and
4) the pyrazolinates, e.g. topramezone [i.e.[3-(4,5-dihydro-3-isoxazoly1)-2-
methyl-4-
(methylsulfonyl) phenylli5-hydroxy-1-methy1-1H-pyrazol-4-yl)methanone], and

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pyrasulfotole [(5-hydroxy-1,3-dimethylpyrazol-4-y1(2-mesy1-4-
trifluaromethylphenyOmethanone]; pyrazofen [2-[4-(2,4-dichlorobenzoy1)-1,3-
dimethylpyrazol-5-yloxy]acetophenone].
These HPPD-inhibiting herbicides can be used against grass and/or broad leaf
weeds in crop plants that display metabolic tolerance, such as maize (Zea
mays) in
which they are rapidly degraded (Schulz et al., (1993). FEBS letters, 318, 162-
166;
Mitchell et al., (2001) Pest Management Science, Vol 57, 120-128; Garcia et
al.,
(2000) Biochem., 39, 7501-7507; Pa!lett et al., (2001) Pest Management
Science,
Vol 57, 133-142). In order to extend the scope of these HPPD-inhibiting
herbicides,
several efforts have been developed in order to confer to plants, particularly
plants
without or with an underperforming metabolic tolerance, a tolerance level
acceptable
under agronomic field conditions.
Besides the attempt of by-passing HPPD-mediated production of homogentisate
(US
6,812,010), overexpressing the sensitive enzyme so as to produce quantities of
the
target enzyme in the plant which are sufficient in relation to the herbicide
has been
performed (W096/38567). Overexpression of HPPD resulted in better pre-
emergence tolerance to the diketonitrile derivative (DKN) of isoxaflutole
(IFT), but
tolerance was not sufficient for tolerance to post-emergence treatment
(Matringe et
al., (2005), Pest Management Science 61: 269-276).
In WO 04/024928, the inventors have sought to increase the prenylquinone
biosynthesis (e.g., synthesis of plastoquinones, tocopherols) in the cells of
plants by
increasing the flux of the HPP precursor into the cells of these plants. This
has been
done by connecting the synthesis of said precursor to the "shikimate" pathway
by
overexpression of a PDH enzyme. They have also noted that the transformation
of
plants with a gene encoding a PDH enzyme makes it possible to increase the
tolerance of said plants to HPPD inhibitors.

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Another strategy was to mutate the HPPD in order to obtain a target enzyme
which,
while retaining its properties of catalysing the transformation of HPP into
homogentisate, is less sensitive to HPPD inhibitors than is the native HPPD
before
mutation.
5 This strategy has been successfully applied for the production of plants
tolerant to 2-
cyano-3-cyclopropy1-1-(2-rnethylsulphony1-4-trifluoromethylpheny1)-propane-1,3-

dione and to 2-cyano-144-(methylsulphony1)-2-trifluoromethylphenyl]-3-(1-
methylcyclopropyl)propane-1,3-dione (EP496630), two HPPD-inhibiting herbicides

belonging to the diketonitriles family (WO 99/24585). Pro215Leu, Gly336G1u,
Gly33611e, and more particularly Gly336Trp (positions of the mutated amino
acid are
indicated with reference to the Pseudomonas HPPD) were identified as mutations

which are responsible for an increased tolerance to pre-emergence treatment
with
these diketonitrile herbicides without causing an alteration of the activity
of the
enzyme.
More recently, introduction of a Pseudomonas HPPD gene into the plastid genome

of tobacco and soybean has shown to be more effective than nuclear
transformation,
conferring even tolerance to post-emergence application of isoxaflutole
(Dufourmantel et al., 2007, Plant Biotechnol J.5(1):118-33).
In the patent application WO 2009/144079, a nucleic acid sequence encoding a
mutated hydroxyphenylpyruvate dioxygenase (HPPD) at position 336 of the
Pseudomonas fluorescens HPPD protein and its use for obtaining plants which
are
tolerant to HPPD inhibitor herbicides is disclosed.
In WO 2002/046387, several domains of HPPD proteins originated from plants
have
been identified that may be relevant to confer tolerance to various HPPD
inhibitor
herbicides but no in planta nor biochemical data have been shown to confirm
the
impact of the as described domain functions.
In WO 2008/150473, the combination of two distinct tolerance mechanisms ¨ a
modified Avena sativa gene coding for a mutant HPPD enzyme and a CYP450

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Maize monooxygenase (nsf1 gene) ¨ was exemplified in order to obtain an
improved
tolerance to HPPD inhibitor herbicides, but no data have been disclosed
demonstrating the synergistic effects based on the combination of both
proteins.
US 2010/0197503 suggests a number of mutations at different positions within
or
close to the active site of the HPPD taken from Avena sativa and examined some
of
them for their inhibition by certain HPPD inhibitors such as sulcotrione in
vitro and in
planta.
.. Despite these successes obtained for the development of plants showing
tolerance
to several HPPD inhibitors herbicides described above, it is still necessary
to
develop and/or improve the tolerance of plants to newer or to several
different HPPD
inhibitors, particularly HPPD inhibitors belonging to the classes of the
triketones
(e.g.sulcotrione, mesotrione, tembotrione, benzobicyclon and bicyclopyrone)
and the
pyrazolinates (e.g., topramezone and pyrasulfotole).
Accordingly, the present invention relates to an isolated nucleic acid
comprising a
nucleotide sequence encoding a mutated HPPD protein, wherein said mutated
HPPD protein has HPPD activity,
wherein in said mutated HPPD protein at least one amino acid has been replaced
so
that the resulting amino acid sequence comprises at least one amino acid
selected
from:
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met
at a
position in an HPPD protein, said position corresponding to position 250 of
the amino
acid sequence of SEQ ID No. 2;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg,Ser Gly, His or Tyrõ at a
position in an
HPPD protein, said position corresponding to position 251 of the amino acid
sequence of SEQ ID No. 2;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Thr or Tyr at a
position in
an HPPD protein, said position corresponding to position 252 of the amino acid
sequence of SEQ ID No.;

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d) Ala, Phe, His, Gin, Val, Trp, lie, Leu or Met at a position in an HPPD
protein, said
position corresponding to position 253 of the amino acid sequence of SEQ ID
No. 2;
e) Leu, Val, or Met at a position in an HPPD protein, said position
corresponding to
position 265 of the amino acid sequence of SEQ ID No. 2;
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile, Lys or Met at a position in an HPPD
protein, said
position corresponding to position 268 of the amino acid sequence of SEQ ID
No. 2;
g) Ala, Thr or Val at a position in an HPPD protein, said position
corresponding to
position 269 of the amino acid sequence of SEQ ID No. 2;
h) Ala, Ile, Asn, Pro, Thr or Val at a position in an HPPD protein, said
position
corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i) Leu, Gln, Val, Ala, Phe, Gly, Met, Arg or Ser at a position in an HPPD
protein, said
position corresponding to position 293 of the amino acid sequence of SEQ ID
No. 2;
j) Ile, Met, Ala, Pro, Ser, Thr or Val at a position in an HPPD protein, said
position
corresponding to position 294 of the amino acid sequence of SEQ ID No. 2;
k) Gin, His or Asn at a position in an HPPD protein, said position
corresponding to
position 307 of the amino acid sequence of SEQ ID No. 2;
I) lie, Met, Asn or Leu at a position in an HPPD protein, said position
corresponding
to position 335 of the amino acid sequence of SEQ ID No. 2;
m) Leu or Met at a position in an HPPD protein, said position corresponding to
position 368 of the amino acid sequence of SEQ ID No. 2;
n) Phe or Tyr at a position in an HPPD protein, said position corresponding to

position 381 of the amino acid sequence of SEQ ID No. 2;
o) Phe or Ser at a position in an HPPD protein, said position corresponding to

position 392 of the amino acid sequence of SEQ ID No. 2;
p) Phe or Tyr at a position in an HPPD protein, said position corresponding to
position 419 of the amino acid sequence of SEQ ID No. 2;
q) Asp, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val
at a
position in an HPPD protein, said position corresponding to position 421 of
the amino
acid sequence of SEQ ID No. 2;
r) Ala, Gly, Met, Pro or Thr at a position in an HPPD protein, said position
corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;

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s) Ala, Phe, Ile or Val at a position in an HPPD protein, said position
corresponding
to position 424 of the amino acid sequence of SEQ ID No. 2;
t) Ile, Pro, Arg, Ser Ala, Gly, Lys, Asn or Gin at a position in an HPPD
protein, said
position corresponding to position 425 of the amino acid sequence of SEQ ID
No. 2;
u) Glu, Phe, Thr, Val Ala, Gin or Ser at a position in an HPPD protein, said
position
corresponding to position 426 of the amino acid sequence of SEQ ID No. 2;
v) Ile, Met, Gln, Val Phe or Leu at a position in an HPPD protein, said
position
corresponding to position 431 of the amino acid sequence of SEQ ID No. 2; and
w) at least one amino acid deletion or replacement at any one of positions
228, 248,
270, 271, 379 and/or 427.
Unless indicated otherwise, the specific definitions or specific features of
certain
embodiments can be introduced into any other embodiment of the present
invention.
According to the present invention, a "nucleic acid" is understood as being a
nucleotide sequence which can be of the DNA or RNA type, preferably of the DNA

type, and in particular double-stranded, whether it be of natural or synthetic
origin, in
particular a DNA sequence in which the codons which encode the HPPD according
to the invention have been optimized in accordance with the host organism in
which
it is to be expressed (e.g., by replacing codons with those codons more
preferred or
most preferred in codon usage tables of such host organism or the group to
which
such host organism belongs, compared to the original or source organism).
An "isolated nucleic acid/DNA/protein", as used in the present application,
refers to a
nucleic acid/DNA/protein which is not naturally-occurring (such as an
artificial or
synthetic DNA with a different nucleotide sequence than the naturally-
occurring DNA,
or a modified protein) or which is no longer in the natural environment
wherein it was
originally present, e.g., a DNA coding sequence associated with a heterologous

regulatory element (such as a bacterial coding sequence operably-linked to a
plant-
expressible promoter) in a chimeric gene, a DNA transferred into another host
cell,
such as a transgenic plant cell.

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The terminology relating to nucleic acid or protein "comprising" a certain
nucleotide
sequence or amino acid sequence, as used throughout the text, refers to a
nucleic
acid or protein including or containing at least the described sequence, so
that other
nucleotide or amino acid sequences can be included at the 5' (or N-terminal)
and/or
3' (or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker
protein,
(the nucleotide sequence of) a transit peptide, and/or a 5' leader sequence or
a 3'
trailer sequence. Similarly, use of the term "comprise", "comprising" or
"comprises"
throughout the text and the claims of this application should be understood to
imply
the inclusion of a stated integer or step or group of integers or steps but
not the
exclusion of any other integer or step or group of integers or steps. The term
"comprising" also includes the term "consisting of".
In accordance with the present invention, the term "mutated HPPD protein"
which is
interchangeably used with the term "mutant HPPD protein" denotes an HPPD
protein
having an amino acid sequence which does not occur in nature. As opposed to
the
term "isolated" referred to above, the term "mutated" cannot refer to the
environment
of the (amino acid or protein) sequence in question, such as it being isolated
from its
natural environment or being coupled to a heterologous (amino acid or protein)

sequence, but only refers to the amino acid sequence defining said mutated
HPPD
protein which cannot be found anywhere in nature but arose from a unmutated or
wild-type starting amino acid sequence. In other words, in arriving at the
nucleic acid
of the present invention encoding a mutated HPPD protein, a starting amino
acid
sequence of a naturally existing protein has to be taken and to be modified by
man
by replacing at least one amino acid as defined in the present application.
The sequence which encodes an original unmutated HPPD which will be mutated
according to the invention can be of any origin. In particular, it can be of
bacterial,
plant or animal origin. Advantageous examples which may be cited are bacteria
of
the Pseudomonas sp. type, for example Pseudomonas fluorescens, or otherwise
cyanobacteria of the Synechocystis genus. The sequence can also be of plant
origin,
in particular derived from dicotyledonous plants, umbelliferous plants, or
otherwise
monocotyledonous plants. Advantageous examples which may be cited are plants

CA 02808152 2013-03-05
. .
such as tobacco, Arabidopsis, Daucus carotta, Zea mays (corn), wheat, barley,
Avena sativa, wheat, Brachiaria platyphylla, Cenchrus echinatus, Lolium
rigidum,
Festuca arundinacea, Setaria faberi, Eleusine indica, and Sorghum. The coding
sequences, and the way of isolating and cloning them, are described in the
5 previously cited references. In a particular embodiment of the invention,
the HPPD is
from a bacterial origin, particularly from Pseudomonas sp., more particularly
from
Pseudomonas fluorescens, Rhodococcus sp., Blepharisma japonicum,
Synechococcus sp., Picrophilus torrid us, Kordia algicida or from a plant
origin,
particularly from Arabidopsis thaliana or Avena sativa. The HPPD to make the
10 mutation (s) in for the purpose of the invention, can be any naturally-
occurring HPPD,
or any active fragment thereof or any variant thereof wherein some amino acids
(1 to
10 amino acids) have been replaced, added or deleted for cloning purposes, to
make
a transit peptide fusion, and the like, which retains HPPD activity, i.e. the
property of
catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate.
The mutated HPPD protein according to the present invention has HPPD activity,
i.
e., as described above, catalyses the reaction in which para-
hydroxyphenylpyruvate
is transformed into homogentisate. Preferentially, the catalytic activity of
the isolated
mutated HPPD of the present invention, when tested in vitro, does not differ
from
that of the unmutated reference HPPD protein by more than 70%, preferably more
than 50%, more preferably more than 30%, even more preferably more than 20%
when assayed under identical conditions and in the absence of the HPPD
inhibitor
herbicides described above. The catalytic activity of an HPPD enzyme may be
defined by various methods well-known in the art. WO 2009/144079 describes
various suitable screening methods.
Initial screens may be performed with the nucleic acid encoding the mutated
HPPD
protein of the invention being expressed in bacteria.
Colorimetric Screening Test for Active HPPD Enzymes:
A YT-broth-type culture medium with 1% agarose, 5mM L-Tyrosine and 42mM
Succinate, which contains the selection agent for the vector pSE420
(Invitrogen,

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11
Karlsruhe, Germany) is poured into deep well plates. E.coli culture in the
exponentional growth phase which contains the vector pSE420-HPPDx (HPPDx
means any gene coding for a putative HPPD enzyme/protein) is applied to each
well.
After 16 hours at 37 C, the wells which do not contain the culture medium,
those
which have been seeded with an E. coil culture containing the empty vector
pSE420
are transparent, or those which have been seeded with an E. coli culture
containing
a vector pSE420-HPPDx containing a gene coding for an inactive HPPD are
transparent, while the weels seeded with an E. coli culture containing the
vector
pSE420-HPPDx coding for an active HPPD are brown. It has been previously
demonstrated that this test refelects the HPPD activity, whatever the orgin of
this
activity is, and allows the identification of HPPD activities (US 6,768,044),
i.e. at a
qualitative level.
Further and more elaborate screens may be carried out in plant cells or plants
expressing the mutated HPPD protein of the invention.
The same screenings may also be used when examining of whether a mutated
HPPD protein is capable of modulating, such as decreasing or increasing, the
tolerance of a plant to at least one HPPD herbicide inhibitor which will be
referred to
further below, with the difference that at least one of such an HPPD inhibitor
is
added. Examples of HPPD inhibitors to be used in those screenings include
tembotrione, mesotrione, pyrasulfotole, bicyclopyrone, topramezone and
sulcotrione.
A screening method which is simple to implement is to determine the dose of
HPPD
inhibitor which fully inhibits the original unmutated HPPD, and which is
lethal for the
cells which express this unmutated HPPD, and to subject the mutated cells to
this
predetermined dose, and thereafter to isolate the mutated cells which have
withstood
this lethal dose, and then to isolate and to clone the gene which encodes the
mutated HPPD.
Alternatively, at the quantitative level data like p150 (1)150-value means the
log value of
the concentration of inhibitor necessary to inhibit 50% of the enzyme activity
in molar
concentration) can be obtained by employing the isolated and purified HPPD

CA 02808152 2013-03-05
12
polypeptide, i.e. the mutated vs. the unmutated HPPD polypeptide and in
presence
or absence of the any respective HPPD inhibitor herbicide.
The terms "tolerance", "tolerant" or "less sensitive" denotes the lack of
susceptibility
of a plant expressing the mutated HPPD protein of the present invention to
substances, particularly herbicides, which inhibit HPPD proteins, optionally
in
comparison with the plant's own HPPD protein or with any known HPPD protein.
More specifically, said terms mean the relative levels of inherent tolerance
of the
HPPD screened according to a visible indicator phenotype of the strain or
plant
transformed with a nucleic acid comprising the gene coding for the respective
HPPD
protein in the presence of different concentrations of the various HPPD
inhibitors.
Dose responses and relative shifts in dose responses associated with these
indicator
phenotypes (formation of brown colour, growth inhibition, bleaching,
herbicidal effect
etc) are conveniently expressed in terms, for example, of GR50 (concentration
for
50% reduction of growth) or MIC (minimum inhibitory concentration) values
where
increases in values correspond to increases in inherent tolerance of the
expressed
HPPD, in the normal manner based upon plant damage, meristematic bleaching
symptoms etc. at a range of different concentrations of herbicides. These data
can
be expressed in terms of, for example, GR50 values derived from dose/response
curves having "dose" plotted on the x-axis and "percentage kill", "herbicidal
effect",
"numbers of emerging green plants" etc. plotted on the y-axis where increased
GR50
values correspond to increased levels of inherent tolerance of the expressed
HPPD.
Herbicides can suitably be applied pre-emergence or post emergence.
Likewise, tolerance level of the nucleic acid or gene encoding an HPPD protein
according to the invention, or the mutated HPPD protein of the invention is
screened
via transgenesis, regeneration, breeding and spray testing of a test plant
such as
tobacco, or a crop plant such as soybean or cotton. In line with the results
obtained
by such screening, such plants are at least 2-4 times more tolerant to HPPD
inhibitors like tembotrione, mesotrione, diketonitrile, and/or bicyclopyrone,
pyrasulfotole, than plants that do not contain any exogenous gene encoding an
HPPD protein, or than plants that contain a gene comprising an Arabidopsis
thaliana

CA 02808152 2013-03-05
13
HPPD-encoding DNA, under control of the same promoter as the nucleic acid
encoding the mutated HPPD protein of the invention. Accordingly, the term
"capable
of increasing the tolerance of a plant to at least one herbicide acting on
HPPD"
denotes a tolerance increased in a plant by at least the factor of 2,
alternatively at
least the factor of 3 or 4 or even 5 or 6 as compared to a plant only
expressing it's
endogenous HPPD or a plant expressing an Arabidopsis thaliana HPPD. In this
regard, the term "herbicide acting on HPPD" is not limited to substances which
are
known and/or used as herbicides but to any substances which inhibits the
catalytic
activity of HPPD proteins.
In an alternative embodiment of the nucleic acid encoding a mutated HPPD
polypeptide comprising at least one of the mutations as defined above, the
HPPD
protein comprises
a) a His at a position in an HPPD protein, said position corresponding to
position
226 of the amino acid sequence of SEQ ID No. 2;
b) a Ser at a position in an HPPD protein, said position corresponding to
position
267 of the amino acid sequence of SEQ ID No. 2;
c) an Asn at a position in an HPPD protein, said position corresponding to
position
282 of the amino acid sequence of SEQ ID No. 2;
d) a His at a position in an HPPD protein, said position corresponding to
position
308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr at a position in an HPPD protein, said position corresponding to
position
342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu at a position in an HPPD protein, said position corresponding to
position
394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly at a position in an HPPD protein, said position corresponding to
position
420 of the amino acid sequence of SEQ ID No. 2; and
h) an Asn at a position in an HPPD protein, said position corresponding to
position
423 of the amino acid sequence of SEQ ID No. 2
In the mutated HPPD protein encoded by the nucleic acid of the invention at
least
one amino acid has been deleted or replaced as defined above.

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14
The replacement or deletion can be effected in the nucleic acid sequence which

encodes the original unmutated, i. e. naturally occurring HPPD as defined
above by
any means which is appropriate for replacing, in the said sequence, the codon
which
encodes the amino acid to be replaced with the codon which corresponds to the
amino acid which is to replace it, or by deleting a codon, with the said
codons being
widely described in the literature and well known to the skilled person.
Several molecular biological methods can be used to achieve this replacement
or
deletion. A preferred method for preparing a mutated nucleic acid sequence
according to the invention and the corresponding protein comprises carrying
out site-
directed mutagenesis on codons encoding one or more amino acids which are
selected in advance. The methods for obtaining these site-directed mutations
are
well known to the skilled person and widely described in the literature (in
particular:
Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON,
IRL PRESS), or are methods for which it is possible to employ commercial kits
(for
example the U. S. E. mutagenesis kit from PHARMACIA). After the site-directed
mutagenesis, it is useful to select the cells which contain a mutated HPPD
which is
less sensitive to an HPPD inhibitor by using an appropriate screening aid.
Appropriate screening methods to achieve this have been described above.
In accordance with the present invention, the term "said position
corresponding to
position X", X being any number to be found in the respective context in the
present
application, does not only include the respective position in the SEQ ID No.
referred
to afterwards but also includes any sequence encoding an HPPD protein, where,
after alignment with the reference SEQ ID No., the respective position might
have a
different number but corresponds to that indicated for the reference SEQ ID
No.
Whereas HPPD sequences may be very diverse and may only show a low sequence
identity of about 30%, HPPD proteins are characterized by a common three
dimensional consensus structure which is achieved despite a low sequence
identity.
Due to specific positions being conserved within HPPD proteins, alignment of
HPPD
proteins can be effected by by applying various alignment tools in a senseful
manner.

CA 02808152 2013-03-05
. .
Methods of aligning nucleic acid or amino acid sequences and, accordingly,
determining the sequence identity of two or more sequences, are well-known in
the
art. They include performing mathematical algorithms such as the algorithm of
Myers
and Miller (1988) CABIOS 4:11-17 or the local alignment algorithm of Smith and
5 Waterman (1981) Adv. Appl. Math. 2:482-489; the global alignment
algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the algorithm of Karlin
and
Altschul (1990) Proc. Natl. Acad. Sci USA 872264 and that of Brutlag et al.
(Comp.
App. Biosci. 6:237-245 (1990 )).
Such algorithms can be implemented in computer programs including but not
limited
10 to CLUSTALX, ALIGN, GAP, BESTFIT, BLAST, FASTDB and FASTA.
For example, when using BESTFIT (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, WI 53711)or any other sequence alignment program to
determine whether a particular sequence is, for instance, 95% identical to a
15 reference sequence, the parameters are set, of course, such that the
percentage of
identity is calculated over the full length of the reference nucleotide
sequence and
that gaps in homology of up to 5% of the total number of nucleotides in the
reference
sequence are allowed.
The identity between a first sequence and a second sequence, also referred to
as a
global sequence alignment, is determined using the FASTDB computer program
based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245

(1990)). In a sequence alignment the query and subject sequences are both DNA
sequences. The result of said global sequence alignment is in percent
identity.
Preferred parameters used in a FASTDB alignment of DNA sequences to calculate
percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence, whichever is shorter.
The present invention is based on the results of a combination of a comparison
of
the amino acid sequences of HPPD proteins from various organisms and the
analysis of the substrate binding and inhibitor binding site of selected HPPD
proteins

CA 02808152 2013-03-05
16
using X-ray crystallography. Using this combined approach, it was possible to
determine key positions in HPPD proteins, where an amino acid can be replaced
with one of a defined set of other amino acids in order to modulate HPPD
catalytic
activity and the affinity to at least one HPPD herbicide inhibitor to a plant
expressing
the mutated HPPD protein.
Superposition of the 3D structure of HPPD from Arabidopsis thaliana (1TFZ)
(Yang
et al., 2004, Biochemistry 43, 10414-10423) with the 3D structures of HPPD
from
other species such as Pseudomonas fluorescens (1CJX) (Serre et al., 1999,
Structure Fold Des. 7, 977-988), Streptomyces avermitilis (1147) (Brownlee et
at.,
2004, Biochemistry 43, 6370-6377), Homo sapiens (3ISQ) (PDB ID: 31sq Pilka et
al,
Structural Genomics Consortium (SGC). Crystal structure of human 4-
Hydroxyphenylpyruvate dioxygenase), Rattus norvegicus (1SQ1) (Yang et al.,
2004,
Biochemistry 43, 10414-10423) shows that they have the same folding and
corresponding amino acids are at equivalent position in the 3D structure of
the
protein. Since the species with known 3D structures are very diverse in their
amino
acid sequence, it can be assumed that all HPPD sequences have the same basic
folding even though the overall sequence identity is low. The sequence and the
3D
structure of Arabidopsis thaliana has been used as reference structure in the
present
invention. Figure 1 shows the superposition of the structure of A.thaliana
HPPD with
the structure of (a) Pseudomonas fluorescens, (b) Streptomyces avermitilis,
(c)
Homo sapiens and (d) Rattus norvegicus. In order to define the binding site of
the
substrate and/or inhibitors, amino acids were selected which play a role in
catalysis
or inhibitor binding. This includes amino acids in the active site and amino
acids from
the C-terminal helix. The 3D arrangement is demonstrated in Figure 2 which
displays
the amino acids defined as binding site in case of (a) Arabidopsis thaliana,
(b)
Pseudomonas fluorescens, (c) Streptomyces avermitilis, (d) Homo sapiens and
(e)
Rattus norvegicus. The amino acid numbering of the Pseudomonas Fluorescens
structure (1cjx) was changed into the numbering according to SEQ ID No. 10.
The
36 amino acids defining the binding site including their position are listed
in Table 1
for (a) Arabidopsis thaliana, (b) Pseudomonas fluorescens, (c) Streptomyces
avermitilis, (d) Homo sapiens and (e) Rattus norvegicus.

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Table 1: Amino acids forming the binding site in A.thaliana, P.fluorescens,
S.avermitilis,
H.sapiens, R. norvegicus
Arabidopsis Pseudomonas Streptomyces Homo Rattus
thaliana fluorescens avermitilis
sapiens norvegicus
Amino Amino Amino Amino
Amino
Position Acid Position Acid Position Acid Position Acid Position Acid
226 H 162 H 187 H 183 H 183 H
228 V 164 T 189 V 185 V 185 V
248 H 186 R 211 T 207 H 207 H
250 F 188 A 213 M 209 F 209 F
251 A 189 R 214 K 210 W 210 W
252 E 190 Y 215 E 211 S 211 S
253 F 191 F 216 F 212 V 212 V
265 L 200 L 228 L 224 L 224 L
267 S 202 S 230 S 226 S 226 S
268 A 203 K 231 K 227 I 227 I
269 V 204 A 232 V 228 V 228 V
270 L 205 M 233 V 229 V 229 V
271 A 206 S 234 A 230 A 230 A
280 P 215 P 243 P 239 P 239 P
282 N 217 N 245 N 241 N 241 N
293 Q 226 Q 255 Q 251 Q 251 Q
294 I 227 I 256 I 252 I 252 I
307 Q 240 Q 269 Q 265 Q 265 Q
308 H 241 H 270 H 266 H 266 H
335 M 264 M 293 L 289 L 289 L
342 Y 271 Y 299 Y 295 Y 295 Y
368 L 295 L 323 L 323 L 323 L
379 Q 310 Q 334 Q 334 Q 334 Q
381 F 312 F 336 F 336 F 336 F
392 F 321 F 347 F 347 F 347 F
394 E 323 E 349 E 349 E 349 E
419 F 333 F 359 F 359 F 359 F
420 G 334 G 360 G 360 G 360 G
421 K 335 E 361 K 361 A 361 A
422 G 336 G 362 G 362 G 362 G
423 N 337 N 363 N 363 N 363 N
424 F 338 F 364 F 364 F 364 F
425 S 339 K 365 K 365 N 365 N
426 E 340 A 366 A 366 S 366 S
427 L 341 L 367 L 367 L 367 L
431 I 345 I 371 I 371 F 371 F

CA 02808152 2013-03-05
18
An exemplary alignment of HPPD proteins is given in Table 2a for the HPPD
proteins with known 3D structures. Table 2a gives the numbering of the amino
acids
of the Arabidopsis sequence and also the amino acids which are common within
these HPPD sequences, with these amino acids being designated by an asterisk.
On
the basis of such an alignment and from the definition of the Arabidopsis
amino acid
by its position and its nature, it is easy to identify the position of the
corresponding
amino acid in another HPPD sequence. Figure 2 shows that this can be done with

the alignment of sequences of different plant, mammalian and bacterial origin,

demonstrating that this method of alignment, which is well known to a skilled
person,
can be generalized to any other sequence. An alignment of different HPPD
sequences is also described in Patent Application WO 97/49816.
Table 2a: Alignment of HPPD sequences with known crystal structures i.e.
A.thaliana,
P.fluorescens, S.avermitilis, H.sapiens, R. norvegicus
Pos. 1TFZ Pos. 1= Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid
1
2
3
4
5
6 A
7 A
8 V
9
10 E
11
12
13
14 H - 1
15 2

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Pos. 1TFZ Pos. 1C,1X Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino
acid
16 D - 3 Q -
,
17 G - 4T -
18 A - 5 T -
,
19 A - 6.1-1 8 G 8 G
20 S - 7 H 9 A 9 P
21 S - 8 T 10 K 10 K
22 P - 9 P 11 P 11 P
23 G - - -
_
24 F - - -
25 K - - -
26 L - - -
27 V - - -
_
28 G - _ ...
, 29 F - - -
30 S - - -
_ _
31 K -- - -
,
- 32 F - -
_ _
33 V - - -
34 R - 10 D 12 E 12 E
35 K 2 A 11 T 13 R 13 R
36 N 3 D 12 A 14 G 14 G
,
37 P 4 L 13 R 15 R 15 R
38 K 5 Y 14 Q 16 F 16 F
39 S6 E'15 A 17 L 17 L
40 D 7 N 16 D 18 H 18 H
_
41 K 8 P 17 P -
42 F 9 M 18 F -
43 K 10 G 19 P -
44 V 11 L 20 V -

CA 02808152 2013-03-05
Pos. 1TFZ Pos. 1 CJX Pos. 1T47 Pos. 3ISQ
Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens
R. norvegicus amino acid
45 K 12 M 21 K
46 R 13 G 22 G
47 F 14 F 23 M 19 F 19 F
48 H 15 E 24 D 20 H 20 H
49 H 16 F 25 A 21 S 21 S
50 I 17 I 26 V 22 V 22 V
51 E 18 E 27 V 23 T 23 T
52 F 19 F 28 F 24 F 24 F x
53 W 20 A 29 A 25 W 25 W
54 C 21 S 30 V 26 V 26 V
55 G 22 P 31 G 27 G 27 G
56 D 23 T 32 N 28 N 28 N
57 A 24 P 33 A 29 A 29 A
58 T 25 G 34 K 30 K 30 K
59 N 26 T 35 Q 31 Q 31 Q
60 V 27 L 36 A 32 A 32 A
61 A 28 E 37 A 33 A 33 A
62 R 29 P 38 H 34 S 34 S
63 R 30 I 39 Y 35 F 35 F
64 F 31 F 40 Y 36 Y 36 Y
65 S 32 E 41 S 37 C 37 C
66 W 33 I 42 T 38 S 38 N
67 G 34 M 43 A 39 K 39 K
68 L 35 G 44 F - 40 M 40 M
69 G 36 F 45 G 41 G 41 G
70 M 37 T 46 M 42 F 42 F
71 R 38 K 47 Q 43 E 43 E
72 F 39 V 48 L 44 P 44 P
73 S 40 A 49 V 45 L 45 L

CA 02808152 2013-03-05
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Pos, 1TFZ Pos. 1CJK Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens
R. norvegicus amino acid
74 A 41 T 50 A 46 A 46 A
75 K 42 H 51 Y 47 Y 47 Y
76 S 43 R 52 S 48 R 48 K
77 D 44 S 53 G 49 G 49 G
78 L 45 K 54 P 50 L 50 L
79 S 46 N 55 E 51 E 51 E
80 T - 56 N 52 T 52 T
81 C - 57 G 53 G 53 G
82 N - 58 S 54 S 54 S
83 M - 59 R 55 R 55 R
84 V - 60 E 56 E 56 E
85 H - 61 T 57 V 57 V
86 A - 62 A 58 V 58 V
87 S 47 V 63 S 59 S 59 S
88 Y 48 H 64 Y 60 H 60 H
89 L 49 L 65 V 61 V 61 V
90 L 50 Y 66 L 62 I 62 I
91 T 51 R 67 T 63 K 63 K
92 S 52 Q 68 N 64 Q 64
93 G 53 G 69 G 65 G 65 G X
94 D 54 E 70 S 66 K 66 K
95 L 55 I 71 A 67 I 67 I
96 R 56 N 72 R 68 V 68 V
97 F 57 L 73 F 69 F 69 F
98 L 58 I 74 V 70 V 70 V
99 F 59 L 75 L 71 L 71 L
100 T 60 N 76 T 72 S 72 C
101 A 61 N 77 S 73 S 73 S
102 P 62 E 78 V 74 A 74 A

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22
Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
103 ' Y 63 P 79 I 75 L ' 75 L
104 S 64 N 80 K 76 N 76 N
105 P 65 S 81 P -
106 S 66 I 82 A -
107 L 67 A 83 T -
108 S 68 S 84 P 77 P 77 ' P
109 A - ' 85 W 78 W 78 W
110 G - 86 G 79 N 79 N
111 ' E - 87 ' H ' 80 K 80 K '
112 I - ' 88 F 81 E 81 E
113 K - 89 L 82 M 82 M
114 P - 90 A 83 G 83 ' G '
115 T - - -
116 T - . . , I
-
117 T - - -
118 A - - -
,
119 S - - -
120 I - - - ,
,
121 P - - -
122 S - - -
123 F - - -
124 D - - -
125 H - - -
,
,
126 G - - -
127 S - - -
128 C - - -
129 R - - -
130 S ' 69 Y 91 D 84 D 84 D
131 F 70 F 92 H 85 H ' 85 H

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23
Pos. 1TFZ Pos. 1 C,IX Pos. 1T47 Pos. 31S0
Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
132 F 71 A 93 V 86 L 86 L
133 S 72 A 94 A 87 V 87 V
134 S 73 E 95 E 88 K 88 K
135 H 74 H 96 H 89 H 89 H X
136 G 7= 5 G 97 G 90 G 90 G X
137 L 76 P 98 D 91 D 91 D
138 G 77 S 99 G 92 G - 92 G
139 V 78 V 100 V 93 V 93 V X
140 R 7= 9 C 101 V 94 K 94 K
141 A 80 G 102 D 95 D 95 D
142 V 81 M 103 L 96 I 96 I
143 A 82 A 104 A 97 A 97 A X
144 I 83 F 105 I 98 F 98 F
145 E 8= 4 R 106 E 99 E 99 E
146 V 85 V 107 V 100 V 100 V X
147 E 86 K 108 P 101 E 101 E
148 D 87 D 109 D 102 D 102 D X
149 A 88 S 110 A 103 C 103 C
150 E 89 Q 111 R 104 D 104 E
151 S 90 K 112 A 105 Y 105 H
152 A 91 A 113 A 106 I 106 I
153 F 92 Y 114 H 107 V 107 V
154 S 93 N 115 A 108 Q 108 Q
155 I 94 R 116 Y 109 K 109 K
156 S 95 A 117 A 110 A 110 A
157 V 96 L 118 I 111 R 111 R
158 A 97 E 119 E 112 E 112 E
159 N 98 L 120 H 113 R 113 R
160 G 99 G 121 G 114 G 114 G X

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24
Pos. 1TFZ, Pos. 1CJX Pos. 1147 Pos. 3ISQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
- 1= 61 - A 100 A 1= 22 A 115 A 115 A
162 I 101 Q 123 R 116 K 116 K
163 P - 124 S 117 I 117 I
164 S - 1= 25 - V 118 M 118 V
165 S - 126 A 119 R 119 R
- 1= 66 - P - 1= 27 E 120 E 120 E
167 P 102 P 128 P 121 P 121 P X
168 - I 103 I 129 Y 122 W 122 W
169 V 104 H 130 E 123 ¨ V 123 V
170 L 105 I 131 L 124 E 124 E
171 N 106 D 132 K 125 - Q 125 E
172 E 107 T 133 D 126 0 126 D
173 A 108 G 134 E 127 K 127 K
174 V 109 P 135 H 128 F 128 F
175 T 110 M 136 G 129 G 129 G
176 I 111 E 137 T 130 K 130 K
177 A 112 L 138 V 131 V 131 V
178 E 113 N 139 V 132 K 132 K
179 V 114 L 140 L 133 F 133 F
180 K 115 " P 141 A 134 A 134 A
181 L 116 A 142 A 135 V 135 V
1= 82 Y 117 I 143 I 136 L 136 L
183 G 118 K 144 A 137 Q 137 Q
- 1= 84 D 119 G 145 T 138 T 138
T
185 - V 120 I 146 Y 139 Y 139 Y
186 V 121 G 147 G 140 G 140 G
187 L 122 G 148 K 141 D 141 D
188 R 123 A 149 T 142 T 142 T
189 Y 124 P 150 R 143 T 143 1

CA 02808152 2013-03-05
= ,
Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
190 V 125 L 151 H 144 H 144 H
191 S 126 Y 152 T 145 T 145 T
192 Y 127 L 153 L 146 L 146 L
193 K 128 I 154 V 147 V 147 V
194 A 129 - D 155 D 148 E 148 E
195 E 130 R 156 R 149 K 149 K
196 D 131 F 157 T 150 M 150 I
197 T 132 G 158 G 151 N 151 N
198 E 133 E 159 Y 152 Y 152 Y
- 134 - G
199 K 135 S 160 0 153 I 153 T
200 S 136 S 161 G 154 G 154 G
201 E 137 - I 162 P 155 Q 155 R
202 F 138 Y 163 Y 1= 56 F 156 F
203 L 139 D 164 L 157 L 157 L
204 P 140 I 165 P 158 P 158 P
205 G 141 D 166 G 159 G 159 G
206 F 142 - F 167 Y 160 Y 160 F
207 E 143 V 168 V 1= 61 E 161 E
208 R 144 Y 169 A 162 A 162 A
209 V 145 L 170 A 1= 63 P 163 P
210 E 146 E 171 A 1= 64 A 164 T
211 D 147 G - 165 F 165
Y
- 166 M 166 K
- 167 D 167 D
- 172 P 168 P 168 T
212 A 148 V 173 I 169 L 169 L
213 S 149 E 174 V 170 L 170 L
214 S 150 - R 175 E 171 P 171 P

CA 02808152 2013-03-05
. ,
26
Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
215 F 151 N 176 P 172 K 172 K
216 P 152 P 177 P 173 L 173 L
217 L 153 V 178 A 174 P 174 P
218 D 154 G 179 H 175 K 175 S
219 Y 155 A 180 R 176 C 176 C
220 G 156 G 181 T 177 S 177 N
221 I 157 L 182 F 178 L 178 L
222 R 158 K 183 Q 179 E 179 E
223 R 159 V 184 A 180 M 180 I
224 L 160 I 185 I 181 I 181 I
225 D 161 D r 186 D 182 D 182 D x
226 H 162 H 187 H 183 H 183 H x
227 A 163 L 188 C 184 I 184 I
228 V 164 T 189 V 1= 85 V 185 V
229 G 165 H 190 G 186 G 186 G
230 N 166 N 191 N 187 N 187 N x
231 V 167 V 192 V 188 Q 188 Q
232 P 168 Y 193 E 189 P 189 P
- 169 R 194 L " 190 D 190 D
- 170 G 195 G 191 Q 191 Q
233 E 171 R 196 R 1= 92 E 192 E
234 L 172 M 197 M 193 M 193 M
235 G 173 V 198 N 194 V 194 E
236 P 174 Y 199 E 1= 95 S 195 S
237 A 175 W 200 W 196 A 196 A
238 L 176 A 201 V 197 S 197 S
239 T 177 N 202 G 198 E 198 E
240 Y 178 F 203 F 199 W 199 W
241 V 179 Y 204 Y 2= 00 Y 200 Y

CA 02808152 2013-03-05
=
27
Pos, 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
242 A 180 E 205 N 201 L 201 L
243 G 181 K 206 K 202 K 202 K
244 F 182 L 207 V 203 N 203 N
245 T 183 F 208 M 204 L 204 L
246 G 184 N 209 G 205 Q 205 Q
247 F 185 F 210 F 206 F 206 F x
248 H 186 R 211 T 207 H 207 H
249 Q 187 E 212 N 208 R 208 R
250 F 188 A 213 M 209 F 209 F
251 A 189 R 214 K 210 W 210 W
252 E 190 Y 215 E 211 S 211 S
253 F 191 F 216 F 212 V 212 V
254 T 192 D 217 V 213 D 213 D
255 A 193 I 218 G 214 D 214 D
256 D 194 K 219 D 215 T 215 T
257 D 195 G 220 D 216 Q 2= 16 Q
258 V 196 E 221 I 217 V 217 V
259 G - 222 A 218
H 218 H
260 T - 223 T 219
T 219 T
261 A - 224 E 220
E 220 E
262 E 197 Y 225 Y 221 Y 2= 21 Y
263 S 198 T 226 S 222 S 2= 22 S
264 G 199 G 227 A 223 S 223 S
265 L 200 L 228 L 224 L 224 L x
266 N 201 T 229 M 225 R 225 R
267 S 202 S 230 S 226 S 226 S x
268 A 203 K 231 K 227 I 227 I
269 V 204 A 232 V 228 V 228 V
270 L 205 M 233 V 229 V 229 V

CA 02808152 2013-03-05
,
28
Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid
271 A 206 S 234 A 230 A 230 A x
272 S 207 A 235 D 231 N 231 N
273 N 208 P 236 G 232 Y 232 Y
274 D 209 D 237 T 233 E 233 E
275 E 210 G 238 L 234 E 234 E
276 M 211 M 239 K 235 S 235 S
277 V 212 I 240 V 236 I 236 I
278 L 213 R 241 K 237 K 237 K
279 L 214 I 242 F 238 M 238 M
280 P 215 P 243 P 239 P 239 P x
281 I 216 L 244 I 240 I 240 I
282 N 217 N 245 N 241 N 241 N x
283 E 218 E 246 E 242 E 242 E x
284 P 219 E 247 P 243 P 243 P
285 V 220 S 248 A 244 A 244 A
286 H - 249 L 245 P 245 P
287 G - 250 A 246 G 246 G
288 T 221 S 251 K 247 K 247 R
289 K 222 K 252 K 248 K 248 K x
290 R 223 G
291 K 224 A 253 K 249 K 249 K
292 S 225 G 254 S 250 S 250 S
293 Q 226 Q 255 Q 251 Q 251 Q x
294 I 227 I 256 I 252 I 252 I x
295 Q 228 E 257 D 253 Q 253
296 T 229 E 258 E 254 E 254 E
297 Y 230 F 259 Y 255 Y 255 Y
298 L 231 L 260 L 256 V 256 V
299 E 232 M 261 E 257 D 257 D

CA 02808152 2013-03-05
. ,
29
Pos. 1TFZ Pos. 1C.JX Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common
A. thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
300 H 233 Q 262 F 258 Y 258 Y
301 N 234 F 263 Y 259 N 259 N
302 E 235 N 264 G 260 G 260 G
303 3 236 G 265 G 261 G 261 G x
304 A 237 E 266 A 262 A 262 A
305 G 238 G 267 G 263 G 263 G x
306 L 239 I 268 V 264 V 264 V
307 Q 240 Q 269 Q 265 Q 265 Q x
308 H 241 H 270 H 2= 66 H 266 H x
309 L 242 V 271 I 267 I 267 I
310 A 243 A 272 A 268 A 268 A x
311 L 244 F 273 L 269 L 269 L
312 M 245 L 274 N 270 K 270 R
313 S 246 T 275 T 271 T 271 T
314 E 247 D 276 G 272 E 272 E
315 D 248 D 277 D 273 D 273 D x
316 I 249 L 278 I 274 I 274 I
317 F 250 V 279 V 275 I 275 I
318 R 251 K 280 E 2= 76 T 276 T
319 T 252 T 281 T 2= 77 A 277 T
320 L 253 W 282 V 278 I 278 I
321 R 254 D 283 R 279 R 279 R
322 E 255 A 284 T 280 H 280 H
323 M 256 L 285 M 281 L 281 L
324 R 257 K 286 R 282 R 282 R
325 K 258 K 287 A 283 E 283 E
326 R 259 I 288 A 284 R 284 R
327 S
328 S

CA 02808152 2013-03-05
Pos. 1TFZ Pos. 1 C.IX Pos. 1T47 Pos. 3ISQ Pos. 1SQI
Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino
acid
329
330 G
331 G 260 G 289 G 285 G 285 G x
332 F 261 M 290 V 286 L 2= 86 M
333 D 262 R 291 Q 287 E 287 E
334 F 263 F 292 F 288 F 288 F x
335 M 264 M 293 L 289 L 289 L
336 P 265 T 294 D 290 S 290 A
337 S 266 A 295 T 291 V 291 V
338 P 267 P 296 P 292 P 292 P x
339 P 268 P
340 P 269 D 297 D 293 S 293 S
341 T 270 T 298 S 294 T 294 S
342 Y 271 Y 299 Y 295 Y 2= 95 Y x
343 Y 272 V 300 Y 296 Y 296 Y x
344 Q 273 E 301 D 297 K 297 R
345 N 274 M 302 T 298 Q 298 L
346 L 275 L 303 L 299 L 299 L x
347 K 276 E 304 - G 300 R 300 R
348 K 277 G 305 E 301 E 301 E
349 R 278 R 306 W 302 K 302 N
350 V 279 L 307 V 303 L 303 L
351 G 280 P 308 G 304 K 304 K
352 D 281 - D 309 D 305 T 305 T
353 V 282 H 310 T 306 A 306 S
354 L 283 G 311 R 307 K 307 K
355 S 284 E 312 V 308 I 3= 08 I
356 D 285 P 313 P 309 K 3= 09 Q
357 D 286 V 314 V 310 V 310 V

CA 02808152 2013-03-05
31
Pos. 1TFZ Pos. 1 CJX Pos. 1T47 Pos. 3ISQ Pos.
1SQI Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens
R. norvegicus amino acid
358 Q 287 D - 311 K 311 K
- 288 Q - 312 E 312 E
- 289 L - 313 N 313 N
359 I 290 Q - 3= 14 I 314 M
360 K 291 A 315 D 315 D 315 D
361 E 292 R 316 T 316 A 316 V
362 C 293 G 317 L 317 L 317 L
363 E 294 I 318 R 318 E 318 E
364 E 295 L 319 E 319 E 319 E
365 L 296 L 320 L 320 L 320 L x
366 G 297 D 321 K 321 K 321 K
367 I 298 G 322 I 322 I 322 I
368 L 299 S 323 L 323 L 323 L
369 V 300 S 324 A 324 V 324 V
370 D 301 V 325 D 325 D 325 D
371 R 302 E 326 R 326 Y 326 Y
372 D 303 G 327 D 327 D 327 D
373 D 304 D 328 E 328 E 328 E
374 Q 305 K 329 D 329 K 329 K
375 G 306 R 330 G 330 G 330 G
376 T 307 L 331 Y 331 Y 331 Y
377 L 308 L 332 L 32 L 332 L x
378 L 309 L 333 L 333 L 333 L x
379 Q 310 Q 334 Q 3= 34 Q 334 Q x
380 I 311 I 335 I 335 I 335 I x
381 F 312 F 336 F 336 F 336 F x
382 T 313 S 337 T 3= 37 T 337 T
383 K 314 E 338 K 338 K 338 K
384 P 315 T 339 P 339 P 339 P

CA 02808152 2013-03-05
32
Pos. I TFZ Pos. 1= Pos. 1147 Pos. 3ISQ Pos. 1 SQI Common
A. thaliana P. fluorescens S. avermitilis H. Sapiens
R. norvegicus amino acid
385 L 316 L 340 V 340 V 340 M
386 G 317 M 341 Q 341 Q 341
387 D 318 G 342 D 342 D 342 D
388 R - 343 R 343 R 3= 43 R
389 P - 344 P 344 P 344 P
390 T 319 P 345 T 345 T 345 T
391 I 320 V 346 V 346 L 346 L
392 F 321 F 347 F 347 F 347 F x
393 I 322 F 348 F 348 L 3= 48 L
394 E 323 E 349 E 349 E 349 E x
395 I 324 F 350 I 350 V 350 V
396 I 325 I 351 I 351 I 351 I x
397 Q 326 Q 352 E 352 0 352 Q
398 R 327 R 353 R 353 R 3= 53 R x
399 V 328 K 354 H 354 H 354 H
400 G
401 C
402 M
403 M
404 K
405 D
406 E
407 E
408 G
409 K
410 A
411
412 Q
413 S -

CA 02808152 2013-03-05
= =
33
Pos. 1TFZ Pos. 1= Pos. 1T47 Pos. 3ISQ Pos. 1SQ I Common
A thaliana P. fluorescens S. avermitilis
H. Sapiens R. norvegicus amino acid
414 G
415 - G 329 G 355 G 355 N 355 N
416 C 330 D 356 S 356 H 356 H
417 G 331 D 357 M 357 Q 357 Q
418 G 332 G 358 G 358 G 358 G x
419 F 333 F 359 F 359 F 359 F x
420 G 334 G - 360 G 360 G 360 G x
421 K 335 E 361 K 361 A 361 A
422 G 336 G 362 G 362 G 362 G x
423 N 337 N 363 N 363 N 363 N x
424 F 338 F 364 F 364 F 364 F x
425 S 339 K 365 K 365 N 365 N
426 E 340 .7- A - 366 A 366 S 366 S
427 - L 341 L 367 L 367 L 367 L x
428 F 342 F 368 F 368 F 368 F x
429 K 343 E 369 E 369 K 369 K
430 S 344 S 370 A 370 A 370 A
431 I 345 I 371 I 371 F 371 F
432 E 346 E 372 E 372 E 372 E x
433 E 347 R 373 R 373 E 373 E
434 Y 348 D 374 E 374 E 374 E
435 E 349 Q 375 Q 375 Q 375 Q
436 K 350 V 376 E 376 N 376 A
437 T 351 R 377 K 377 L 377 L
438 L 352 R 378 R 378 R 378 R
439 E 353 G 379 G 379 G 379 G
440 A 354 V 380 N 380 N 380 N
441 K 355 L 381 L 381 L 381 L
442 Q 356 A 382 T 382 T

CA 02808152 2013-03-05
34
Pos. 1TFZ
Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common
A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid
443 L 357 T 383 N 383 D
444 V 358 D 384 M 384 L
445 G 385 E 385 E
386 T 386 T
387 N 387 N
388 G 388 G
389 V 389 V
390 V 390 R
391 P 391 S
392 G 392 G
393 M 393 M
394 A 394
395 E 395
396 N 396
397 L 397
398 Y 398
399 F 399
400 Q 400
A sequence analysis of more than 700 HPPD sequences from public data bases
including sequences of HPPD proteins and predicted HPPD proteins such as from
plants, mammals, fungi and bacteria was performed using ClustalX. The
alignment
was corrected using the information of the available 3D structures. Identical
amino
acid sequences with different identifiers were included only once and some
sequences with obvious sequence errors were excluded. The alignment also
includes incomplete sequences. Table 2b shows the sequence alignment for a
representative set of HPPD proteins and includes sequences from plants,
bacteria,
mammals.

CA 02808152 2013-03-05
Table 2b: Alignment of a representative set of HPPD sequences
CLUSTAL X (1.81) multiple sequence alignment
5
RATTUS NORVEGICUS ---MTT ----------------------------------
YSNKGPKPE
HOMO-SAPIENS ---------------------------------------------------------- GAKPE
XENOPUS-LAEVIS -MTS ----------------------------------
YTDKGPKPD
BLEPHARISMA-JAPONICUS-----NT? ------------------------------------------
YDKQETRPD
10 MYCOSPHAERELLA-GRAMINICOLA ---MAPGALLVTSQNG ----------------------
RTSPLYDSDGYVPAPAALVVGG
ASPERGILLUS-FUMIGATUS ---MAPSAISTS ----------------------------
PPPTDRVSSS
MAGNAPORTHE-GRISEA ---MSPSAITESPRN -------------------------
SVVDHTSGLQVDSLAVQGP
CANDIDA-ALBICANS
PICHIA-STIPITIS ----LLKELPFLPTSS ------------------------
DPITEPDIDELLSDGHVNSKYP
15 RIODOCOCCUS-SP. ---MTIEQTLTD ----------------------------
KERLAGLDLGQLEQLVGLVEYDGTRD
RHODOCOCCUS-ERYTHROPOLIS ---MTVEQTLTD ----------------------------
DEQLAGLDLEQLRQLVGLVEYDSDHD
JANIBACTER-SP.-HTCC2649
MTDTTITNPTTDTASQPTPLDLTPUREANLNLEOLKOLVGLVEYDESKD
STREPTOMYCES-AVERMITILIS ---MTQTTHHTP ----------------------------
DTARQAD
KORDIA-ALGIC1DA MAAEIKNLKD ---------------------------
LQNTEYGLKKLFDEAED
20 LEEUWENHOEKIELLA-BLANDENSIS-ME ---MSKELK ----------------------------
SVDYGLEKIFDGAQD
GEMMATIMONAS-AURANTIACA ---MATLTT -------------------------------
PEIGTEQD
PICROPHILUS-TORRIDUS --------------------------------------------------
MYGKNLISELRE
ARABIDOPSIS-THALIANA --------------------------------------------------
MGHQNAAvSENQNHDDGAAS--SPGFKLvGFSKFVRKNPKSD
BRASSICA-RAPA-SUBSP.-PEKINENSI ----------------------------------------
MGHENAAVSENQHHDDAATTSASPGFKLVGFSKFVRKNPKSD
25 COPTIS-JAPONICA-VAR.-DISSECTA -------- MVPST -----------------------
ASNLKLVGHTNFVHNNPKSD
HEVEA-BRASILIENSIS ------------------ MGKENDSVPSS ---------------------
APGFKLLGFSNFVRTNPRSD
MEDICAGO-TRUNCATULA ----------------- MAIETETQTQT ---------------------
QTGFKLVGEKNEVRANPKSD
GLYCINE-MAX ------------------------- MCNEIQAQAQAQ --------------------
AQPGFKLVGFKNFVRTNPKSD
SOLANUM-LYCOPERSICUM ----MC--MGKETLS--TTDTTG -----------------
ATFKLVGFNNFIRANPRSD
30 SOLANUM-TUBEROSUM ----MG--MGKETLSTTTTDETG -----------------
ATFKLG-FNNFIRANPRSD
NICOTIANA-BENTHAMIANA ----MGKLETVTTTSATAADDSSEL ---------------
TTNFKLVGFKNFIRTNPRSD
DAUCUS-CAROTA ----MGKKQSEAEILSSNSSNTSP ----------------
ATFKLVGENNFVRANPKSD
SOLENOSTEMON-SCUTELLARIOIDES -MG --- QESTAAAAVVP ------------------
AEFKLVGHKNFVRSNPMSD
HORDEUM-VULGARE --------------------- MPPTPTTPAAT- GAAAAVTPEHARP ------
HRMVRFNPRSD
35 TRITICUM-AESTIVUM ---------------- MPPTPTTPAATGAGAAAAVTPEHARP ------
RRMVRFNPRSD
AVENA-SATIVA ----------------------------------------------------------
MPPTPATATGA---AAAAVTPEHAARS---EPRVVRVNPRSD
ORYZA-SATIVA ----------------------------------------------------------
MPPTPTPTATTGAVSAAAAAGENAGFRLVGHRRFVRANPRSD
SORGHUM-BICOLOR -------------------------------------------------------
MPPTPTTAAAT-GAAVAAASAEQAAFRLVGHRNFVRVNPRSD
ZEA-MAYS --------------------------------------------------------------
MPPTPTAAAAG-AAVAAASAAEQAAFRLVGHRNFVRFNPRSD
AB095005AOLUCIMARINUS ------------ MATVPS --------------------------
KRKLVGCANFVRSNPLSD
OTAURI ------------------------------ MTTSAS --------------------------
GRKLVGHANFVRCNPLSD
MICROMONAS-PUSILLA-CCMP1545 --------- MASSEANAAPA ---------------------
AKRHKLVGCKNFVFENNPMD
SYNECHOCOCCUS-SP. -----------------------------------------------------
MNPSIR
VIERIO-SP.-MED222 -----------------------------------------------------
MVDTYN
MARINOMONAS-SP.-MED121 ---------------------------------------------
MNTALKIDYSIN
PSEUDOMONAS-FLUORESCENS -----------------------------------------------
ADLYEN
SULFITOBACTER-SP.-NAS-14.1 --------------------------------------------
MGPFPHDAEKSKITDEN
OCEANICOLA-BATSENSIS-HTCC2597 -----------------------------------------
MGPFPHDAPKSEITDEN
BDELLOVISRIO-BACTERIOVORUS --------------------------------------------
MAQVTEKN
RATTUS NORVEGICUS
RGRELHFHSVTFWVGNAK--QAASFYCNKMGFEPLAYKGLETGSREVVSH
HOMO-SAPIENS
RGRELHFHSVTFWVGNAK--QAASFYCSKMGFEPLAYRGLETGSREVVSH
XENOPUS-LAEVIS
VGRYLAFDHITFYVGNAK--QAAAYYATRFGENPIAYRGLETGHRDVCTH
BLEPHARISMA-JAPONICUS
LGEFYGFHHVRFYVSNSE--QAASFYTSRFGFSPVAYEGLETGNQKFCTN
MYCOSPHAERELLA-GRAMINICOLA
EVNYRGYHHAEWWVGNAK--QVAQFYITRMGFEPVAHKGLETGSRFFASH
ASPERGILLUS-FUMIGATUS
LASYKGYDHVHWYVGNAK--QAASYYITRMGFKRIAYRGLETGCRSVCSH
MAGNAPORTHE-GRI SEA
FPSFEIGYDHVTWWVGNAK--QAASYYNTLEGMK1IAYRGLETGSRYFASY
CANDIDA-ALBICANS ------------------------------------------------------
MSKYLDLAMGEKEVAYKGLETGSKLIGAH
PICHIA-STIPITIS
TDGFIKFFSLKICSSNAK--QMSKYLQLAMDFKEIAYKGLENDSRLVGAH
RHODOCOCCUS-SP.
PFPVSGWDAVVWVVGNAT--QTAHYFQSAFGMTLVAYSGETTGNRDHHSF
RHODOCOCCUS-ERYTHROPOLIS
PFPVSGWDGLEWIVCNAT--QTSHFFQSAFGMELVAYSGPSTGNRDHHAF
JANIBACTER-SP.-HTCC2649
PFPVTGWDAIVETVGNAT--QAAAYYQGTWGMELVGYSGPENGNRDHKAF
STREPTOMYCES-AVERMITILIS
PFPVKGMDAVVFAVGNAK--QAAHYYSTAFGMQLVAYSGPENGSRETASY

CA 02808152 2013-03-05
36
KORDIA-ALGICIDA
FLPLLGTDYVELYVGNAK- -QSAHFYKTAFGFQSEAYAGLETGLTDRVSY
LEEUWENHOEKIELLA-BLANDENSIS -ME FLPLLGTDYVEFYVGNAK--
QAAHEYKTAFGFQSEAYSGLEIGKKDRVSY
GEMMATIMONAS-AURANTIACA
AFPINGTDYVEFYVGNAK--QASHYYRAAFGYSLVAYRGPETGVRDRASY
PICROPHILUS-TCRRIDUS
KEIFKRLHHVEFYVSSAK--TWSYFMNRGLGFKTVAYAGPETGIRDKISY
ARABIDOPSIS-THALIANA
KEKVKRFHHIEFWCGDAT--NVARRFSWGLGMRFSAKSDLSTGNMVHASY
BRASSICA-RAPA-SUBSP.-PEKINENSI KEKVKREHHIEFWCGDAT--
NVARRFSWGLGMRFSAKSDLSTGNMVHASY
COPTIS-JAPONICA-VAR.-DISSECTA
KFHVKKEHHIEFWSTDAT--NTARRFSWGLGMPMVAKSDLSIGNMVHASY
HEVEA-BRASILIENSIS
LEKVKREHHVEFWCTDAT--NTACRFSWGLGMPFVAKSDLSTGNVTHASY
MEDICAGO-TRUNCATULA
RENVKREHHVEFWCIDAT--NTARRFSHGLGMPIVAKSDLSIGNLTHASY
GLYCINE-MAX
RFQVNRFHHIEFWCIDAT--NASRRFSWGLGMPIVAKSDLSTGNQIHASY
SOLANUM-LYCOPERSICUM
FFSVKREHHIEFWCGDAT--NTSRRFSWSLGMPITAKSDLSTGNSVHASY
SOLANUM-TUBEROSUM
FFSVKREHHIEFWCGDAT--NTSRRFSWSLGMPITAKSDLSIGNSVHASY
NICOT1ANA-BENTHAMIANA
FFSVKREHHIEFWCGDAT- -NTSRRFSWSLGMPIAAKSDLSTGNSVHASY
DAUCUS-CAROTA
HFAVKREHKIEFWCGDAT--NTSRRFSWGLGMPLVAKSDLSIGNSVHASY
SOLENOSTEMON-SCUTELLARIOIDES HERVHRFEHVEFWCGDAT--
NTSRRFSWGLGMPLVAKSOLSIGNSAHASY
HORDEUM-VULGARE
RFKILSEHHVEFWCADAA--SAAGRFAFALGAPLAARSDLSTGNSAHASQ
TRITICUM-AESTIVUM
REHTLSEHHVEFWCADAA--SAAGRFAFALGAPLAARSDLSTGNSVHASQ
AVENA-SATIVA
RFPVLSEHHVELWCADAA--SAAGRFSFALGAPLAARSDLSTGNSAHASL
ORYZA-SATIVA
RFQALAFHHVELWCADAA--SAAGRFAFALGAPLAARSDLSIGNSAHASL
SORGHUM-BICOLOR
RFHTLAFHHVELWCADAA--SAAGRFSEGLGAPLAARSDLSIGNTAHASL
ZEA-MAYS
RFHTLAFHHVELWCADAA--SAAGRFSEGLGAPLAARSDLSTGNSAHASL
AB095005_OLUCIMARINUS
AFECEKEDHIEFWCGDAT--NAAAREGVGLGMGLRCKSDATIGNGTYASY
OTAURI
AFECVGFDHVEFWCGDAT--NAASRFGVGLGMSLRAKSDASIGNGIYASY
MICROMONAS-PUSILLA-CCMP1545
LETMRKEHHVEFWCROAT--TTAARFAVGLGMQLVAKSDLTTGNARYASY
SYNECHOCOCCUS-SP. --IVQGIHHLHFYLWDLP--RWREHFCRVWGFRVASDAGN -- TL
VIBRIO-SP.-MED222 PLGTDGFEFVEYTAVDHKGIEQLKALLVSLGFAEIAKHRSKE --
AW
MARINOMONAS-SP.-MED121 PLGTDGFEFVEYTAADEKGIADLKALFVSLGFTEVAKHRSKE --
VW
PSEUDOMONAS-FLUORESCENS PMGLMGFEFIEFASPTPG-- -TLEPIFEIMGFTKVATHRSKN
VH
SULFITOBACTER-SP.-NAS-14.1 PAGTDGFEFVEFASADPQ---ELRDLETRMGYAHVANHKTQK --
IE
OCEANICOLA-RATsENSIS-HTCC2597 PAGTDC,FERVEPARPDPQ---ELROLFSKMC,YELVGRHKSKD
VF.
BDELLOVIBRIO-BACTERIOVORUS PVGINGVDFIEYSGPDAH---FFEQVEKRYAFKEVGQVHGKN --
IK
RATTUS NORVEGICUS VIKQGK --IVFVLCSALNPWN ------------------
HOMO-SAPIENS VIKQGK--IVEVLSSALNPWN -------------------
XENOPUS-LAEVIS VVRQNN - -ATFVFQSPLNPGN -----------------
BLEPHARISMA-JAPONICUS VVRSNH--VVIAFTSALTPED -------------------
MYCOSPHAERELLA-GRAMINICOLA VVQNNG--VRFVFTSPVRSSA -------------------
ASPERGILLUS-FUMIGATUS VVRNGD --ITFILTSPLRS --------------------
MAGNAPORTHE-GRTSEA LVGKED--VRFVFTSPIRSHVH ------------------
CANDIDA-ALBICANS
VMQNGS--ITLEIINTLETIDDDNVLKFPFFQNDLNKFRNINHEYFLENF
PICHIA-STIPITIS
VIRNGD--VTLEIVNTLETVEDDNVLKEPYFEKDLKQFPQLNESKYLRDF
RHOD000CCUS-SP. VLESGA--VRFVIKGAVNPDSP ------------------
RHODOCOCCUS-ERYTHROPOLIS VLKSGA--VREVVKGAVDPASP ------------------
JANIBACTER-SP.-HTCC2649 VLKSGS --IRFVLKGAVSPDSP
STREPTOMYCES-AVERMITILIS VLTNGS --ARFVLTSVIKPATPW ----------------
KORDIA-ALGICIDA VLKQDK--IRLVLTTPLGKGGE ------------------
LEEUWENHOEKIELLA -BLANDENSIS -ME VLRQDK --IRLVLTSPLGSASP ----------------
GEMMATIMONAS-AURANTIACA LMQQGK--IRLVLTTSITADTP - ----------------
PICROPHILUS-TORRIDUS VMSQGT --ARISFTSSMNDDSY -----------------
ARABIDOPSIS-THALIANA LLTSGD--LRFLFTAPYSP---SLSAGEI -----------
BRASSICA-RAPA-SUBSP.-PEKINENSI LLTSGO--LRFLETAPYSP---SLSAGEN ---------
COPTIS -JAPONICA -VAR. -DISSECTA LLRSGE --LNELFTAPYSP - - -SIAGNT ----
HEVEA-BRASILIENSIS LLRSGD --LSELFTAPYSP---TIASMENF ---------
MEDICAGO-TRUNCATULA LLRSGD- -LNFLFSAAYSP - --SISLSS ---------
GLYCINE-MAX LLRSGD--LSFLFSAPYSP---SLSAGSS -----------
SOLANUM-LYCOPERSICUM LLRSVSGELQFVFTAPYSP---SISVPS ------------
SOLANUM-TUBEROSUM LLRSVSGELQFVFTAPYSP---SISVPS ------------
NICOTIANA-BENTHAMIANA LLRPVSGSLQELFTAPYSP---SISTPS ------------
DAUCUS-CAROTA LVRSAN- -LSFVFTAPYSP ---STITSSG ---------
SOLENOSTEMON-SCUTELLARIOIDES LLRSGE--LSFVFTAPYSP---SLAEPS ------------
HORDEUM-VULGARE LLRSGS- -LAFLETAPYAN -- GCDAA -----------
TRITICUM-AESTIVUM LLRSGN--LAFLETAPYAN -- GCDAA ------------
AVENA-SATIVA LLRSGA - -LAFLFTAPYAPPPQEAATAAA ---------
ORYZA-SATIVA LLRSAS--VAELFTAPYGGDHGVGADAAT -----------

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SORGHUM-BICOLOR LLRSGA--LAFLETAPYAB -- GADAA ------------
ZEA-MAYS LIRSGS--ESFLETAPYAH-----GADAA -----------
A3095005_OLUCIMARINUS AMKSND--LTFVFTAPYG ----- VESGGSR --------
OTAURI AMKSHD--LTFVFTAPYGDDERAVGCGGSS ----------
5 mICROMONAS-PUSILLA-CcmP1545 vLQSND--LRFvFSAPYDVPEGEENDDARS
SYNECHOCOCCUS-SP. ELEQGS--LRLRLSQPARAG --------------------
VIBRIO-SP.-MED222 LYRQGD--INFIVNEQPHS ---------------------
MARINOMONAS-SP.-MED121 LYROND--INFIVNSEPNS ---------------------
PSEUDOMONAS-FLUORESCENS LYRQGE--INLILNNEPNS ---------------------
SULFITOBACTER-SP.-NAS-14.1 LWQQGD--ITYVLNADPDS --------------------
OCEANICOLA-BATSENSIS-H1CO2597 LWQQCD--ITYIINAEPCT --
BDELLOVIBRIO-BACTERIOVORUS LFRQGD--INFILNCEPHT ---------------------
RATTUS NORVEGICUS
HOMO-SAPIENS
XENOPUS-LAEVIS
BLEPHARISMA-JAPONICUS
MYCOSPHAERELLA-GRAMINICOLA
ASPERGILLUS-FUMIGATUS
MAGNAPORTHE-GRISEA
CANDIDA-ALBICANS
KLTTDDLIFDFVNSRIESLSFKSDYLKFGKQFYNNIIRSDDYQESMAKVS
PICHIA-STIPITIS
KITTNDLVEDEVNSRIESFSVSPNABYFRRKLYNKIVSSRAFRNNMFDYN
RIODOCOcCUS-SP.
RHODOCOCCUS-ERYTHROPOLIS
JANIBACTER-SP.-HTCC2649
STREPTOMYCES-AVERMITILIS
KORDIA-ALGICIaA
LEEUWENBOEKIELLA-BLANDENSIS-ME -----------------------------------------
GEMmATIMONAS-AURANTIAcA
PICROPHILUS-TORRIDUS
ARABIDOPSIS-THALIANA
BRASSICA-RAPA-SUBSP.-PEKINENSI -----------------------------------------
COPTIS-JAPONICA-VAR.-DISSECTA
HEVEA-BRASILIENSIS
MEDICAGO-TRUNCATULA
GLYCINE-MAX
SOLANUM-LYCOPERSICUM
SOLANUM-TUBEROSUM
NICOTIANA-BENTHAMIANA
DAUCUS-CAROTA
SOLENOSTEMON-SCUTELLARIOIDES
HORDEUM-VULGARE
TRITICUM-AESTIVUM
AVENA-SATIVA
ORYZA-SATIVA
SORGHUM-BICOLOR
MA-MAYS
AB095005_OLUCIMARINUS
OTAURI
MICROMONAS-PUSILLA-CCMP1545
SYNECHOCOCCUS-SP.
VIBRIO-SP.-MED222
MARINOMONAS-SP.-MED121
PSEUDOMONAS-FLUORESCENS
SULFITOBACTER-SP.-NAS-14.1
OCEANICOLA-BATSENSIS-HTCC2597
BDELLOVIBRIO-BACTERIOVORUS
RATTUS NORVEGICUS ------------------------------------------------------
KEMGDHLVKHGDGVKDIAFEVEDCEHIVQK
HOMO- SAPIENS ----------------------------------------------------------
KEMGDHLVKHGDGVKDIAFEVEDCDYIVQK
KENOPUS-LAEVIS ---------------------------------------------------------
HPISDHVAmHGDGVKDVAFSVEDCRGIYKR
BLEPHARISMA-JAPONICUS --------------------------------------------------
NEVNRHVGKHSDGVODIAFSVSDARGMYEK
MYCOSPHAERELLA-GRAMINICOLA ------------------------------------------
RQTLKAAPLADQARLDEMYDHLDKHGDGVKDVAFEVDDVLAVYEN

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ASPERGILLUS-FUMIGATUS --------------------------------------------------
LDQVDREPPEEQELLKEIHAHLEKHGDGVKDVAFEVDSVDSVEYA
MAGNAPORTHE-GRISEA -----------------------------------------------------
LPEDEPISDEDRALLKEMHAHLEKHGDAVKDVCFEVDNVQGVYER
CANDIDA-ALBICANS
NEIVQTINNSEEIYNDMMECTLIQKFLKLHAEGVMDIAENVNDVDTIENR
PICHIA-STIPITIS
NLILNVINNSEVIYNDIMECTLIQKFLKTHGEGVMDISFLVEDVITIFDK
RHODOCOCCuS-5F. ------------------------------------------------------
LIDHHRThGDGVVDIALAVPDVDKCIAE
RHODOCOCCUS-ERYTHROPOLIS -----------------------------------------------
LIEHHSRHGDGIRDIALSVPDVDKCIAH
JANIBACTER-SP.-HTCC2649 ------------------------------------------------
LIAHHTKHGDGVVDISLEVPDVDKCIAQ
STREPTOMYCES-AVERMITILIS -----------------------------------------------
GHFLADHVAEHGDGVVDLAIEVPDARAAHAY
KORDIA-ALGICIDA --------------------------------------------------------
INEHIDLHGDGVKVVALWVEDATKAFEE
LEEUWENHOEKIELLA -BLANDENSIS -ME ---------------------------------
INEHIVKHGDGVKVVALWVEDARSAFEE
GEMMATIMONAS-AURANTIACA ------------------------------------------------
IAEHVHRHGDOvRDYALWVDDARLAYET
PICROPHILUS-TORRIDUS ---------------------------------------------------
ISNHVKKHGDGVKDIALEVDDLDEAKSL
ARABIDOPSIS-THALIANA ---------------------------------------------------
KPTTTASIPSFDHGSCRSFFSSHGLGVRAVAIEVEDAESAFSI
BRASSICA-RAPA-SUBSP.-PEKINENSI -----------------------------------------
PPITTASIPSFDHVTYRSFESSHGLGVRAVAVEVEDAEAAFSI
COPTIS-JAPONICA-VAR.-DISSECTA ---------------------------------------
LTHTASIPTYSHNLARLFASTHGLAVRAIAIEVQDAELAYNI
BEVEA-BRASILIENSIS -----------------------------------------------------
SHTATASIPTESHEACRNFSAKHGLGVRAIAlEVEDAEIAYNT
MEDICAGO-TRUNCATULA ----------------------------------------------------
PSSTAAIPTESASTCFSFSASHGLAVRAVAVEVEDAEVAFTT
GLYCINE-MAX ------------------------------------------------------------
AASSASIPSFDAATCLAFAAKHGEGVRAIALEVADAEAAFSA
SOLANUM-LYCOPERSICOM ---------------------------------------------------
TAGIPSFSTPTYRDFTAKHGLGVRAVALEVENAYLAFSA
SOLANUM-TUBEROSUM ---------------------------------------------------
TAGIPSFSTSTHRDFTAKHGLGVRAVALEVENAYLAFSA
NICOTIANA-BENTHAMIANA --------------------------------------------------
SAAIPSFSTSTHRSFAATHGLGVRAVALEVENAYTAFSA
DAUCUS-CAROTA ----------------------------------------------------------
SAAIPSFSASGFHSFAAKHGLAVRAIALEVADVAAAFEA
SOLENOSTEMON-SCUTELLARIOIDES -------------------------------------------
SASIPTFSFSDBRAFTSSHGLAVRAVAIQVDSASSAYSA
HORDEUM-VULGARE --------------------------------------------------------
TASLPSFSADAARRFSADHGIAVRSVALRVADAAEAFRA
TRITICUM-AESTIVUM ---------------------------------------------------
TASLPSFSADAARRFSADHGLAVRSIALRVADAAEAFRA
AVENA-SATIVA -----------------------------------------------------------
TASIPSFSADAARTFAAAHGLAVRSVGVRVADAAEAFRV
ORYZA-SATIVA -----------------------------------------------------------
TASIPSFSPGAARRFAADHGLAVHAVALRVADAADAFRA
SORGHUM-BICOLOR --------------------------------------------------------
TASLPSFSAAEARRFAADHGLAVRAVALRVADAEDAFRA
ZEA -MAYS --------------------------------------------------------------
TAALPSFSAAAARRFAADHGLAVRAVALRVADAEDAFR-
AB095005AOLUCIMARINUS ----------------------------------------------- CEAPHPC-
--HEGRAMMRFFEKHGLAARAVGVRVKDARAAYEE
OTAURI -----------------------------------------------------------------
VNVPHPG---NERGAMMRFFERHGLAARAVGLRVGDARAAYEE
MICROMONAS-PUSILLA-CCMP1545 --------------------------------------------
SMFEKSGVLSHDPSFMRTFCERHGLAVRAVCLLVDDAAVAFYT
SYNECHOCOCCUS-SP. ------------------------------------------------------
DEVDRHLQRDGPGVVDVALAVGEQELPALA
VIBRIO-SP.-MED222 ------------------------------------------------------
QAEAFAKVHGPSVCGMAFRVNEATAAMEQ
MARINOMONAS-SP.-MED121 ----------------------------------------------
QSEAFARIHGPSVCGMAERVKDASLAMQH
PSEUDOMONAS-FLUORESCENS ------------------------------------------------
IASYFAAEHGPSVCGMAFRVKDSQKAYNR
SULFITOBACTER-SP.-NAS-14.1 ---------------------------------------------
FAAGFVAEHGPCAPSMGWRVVDAQKALDH
OCEANICOLA-BATSENSIS-HICC2597 ------------------------------------------
HAATFIEEHGPCAPSMGWRVVDAQHAFDH
BDELLOVIBRIO-BACTERIOVORUS ---------------------------------------------
FATDFAKLHGPCVNATGFRVIDADQAFKT
RATTUS NORVEGICUS ARERGAKIVREPWVEED -----------------------
KFGKVKFAVLQTYG-DITHILVEK-- -
HOMO -SAPIENS ARERGAKIMREPWVEQD -- KFGKVKFAVLQTYG-DTTHTLVEK-
XENOPUS-LAEVIS AIERGAKSVREPWEESD -----------------------
EFGTVVMATIQTYG-DTTHTFVER---
BLEPHARISMA-JAPONICUS AIAKGCKSFREPQVLQD -----------------------
QFGSVIIASLQTYG-DTVHTLVQN---
MYCOSPHAERELLA-GRAMINICOLA AVANGAESVSSPHTDSC ----------------------
DEGDVISAAIKTYG-DTTHTFIQR---
ASPERGILLUS-FUMIGATUS ATNNGAKIVSQPRTLED -----------------------
DNGQVRVATIQTYG-ETTHILVER---
MAGNAPORTHE-GRISEA AVQQGAVSIAPPKTLSDK ----------------------
EHGSVTMAVIQTYG-DTTHTLLSR---
CANDIDA-ALBICANS AIKAGSGIIRLPKIISD -----------------------
ENGVVKLATISIPNSDIQHTLIEN---
PICHIA-STTPITIS AVAAGAGIIRLPKIISD -----------------------
CNGSVRLGTISIPKTDIQHTLIEN---
RHODOCOCCUS-SP. ARAQGATVLDEPHDVTD -----------------------
DHGTVRLAAIATYG-DTRHILVDR---
RHODOCOCCUS-ERYTHROPOLIS AITQGATVLSEPHDITD -----------------------
EHGTVRLASIATYG-ETRHTLVDR---
JANIBACTER-SP.-HTCC2649 AKAAGARVVQEAETVSD -----------------------
EFGSVRIGAIATYG-ETRHTLVQRTVD
STREPTOMYCES-AVERMITILIS AIEHGARSVAEPYELKD -----------------------
EHGTVVLAAIATYG-KTRHTLVDR---
KORDIA-ALGICIDA TTKRGAKPYMEPTKEED -----------------------
ENGYVIRSGIYTYG-ETVHVFVER-- -
LEEUWENHOEKIELLA-BLANDENSIS-ME TTKRGAKPFMEPEVEKD ------------------
EHGEVVRSGIYTYG-ETVHMFVER- - -
GEMMATIMONAS-AURANTIACA AIARGAIPIQEPQVYSD -----------------------
EHGEVVIAAIGTYG-DTIHSLVER---
PICROPHILUS-TORRIDDS IEKYGTKVS-KINEIKD -----------------------
GNGKIRTAEIKTYG-ETVHTLIET---
ARABIDOPSIS-THALIANA SVANGAIPSSPPIVLNE -----------------------
AVTIAEVKLYG-DVVLRYVSYKAE
BRASSICA-RAPA-SUBSP.-PEKINENSI SVSNGAVPSSPPIVIND ------------------------
AVTIAEVKLYG-DVVLRYVSYKVA
COPTIS-JAPONICA-VAR.-DISSECTA SVANGAKPSSSPIKLDE ----------------------
GVVLSEIQLYG-DVVLRYLSFKNT
HEVEA-BRASILIENSIS SVARGALPMGGPITLDN -----------------------
RAVVAEVHLYG-DVVLRYISYKNS
MEDICAGO-TRUNCATULA SVNLGAIPSSPPVILEN -----------------------
NVKLAEVHLYG-DVVLRYVSYNDL
GLYCINE-MAX SVAKGAEPASPPITLVDD ----------------------
RTGFAEVRLYG-DVVLRYVSYKDA
SOLANUM-LYCOPERSICUM SVARGAKPREEPVTIDE -----------------------
HVAVAEVHLYG-DVVLREVSLVKD
SOLANUM-TUBEROSUM SVSRGAKPRFEPVTIDE -----------------------
HVAVAEVHLYG-DVVLRFVSFVKD

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N/COTIANA-BENTHAMIANA SVSRGAKPMFEPVTIDG -----------------------
OVAMAEVHLYG-DVVLRFMS-LKD
DAUCUS-CAROTA SVARGARPASAPVELDD -----------------------
OAWLAEVELYG-DVVLRFVSFGRE
SOLENOSTEMON-SCUTELLARIOIDES AVSRaAKPVSPPVVLADC ----------------------
ETAIAEVHLYG-DTVLRFVSCGSG
HORDEUM-VULGARE SRARGARPAFAPVDLCR -----------------------
GFAFAEVELYG-DVVLRFVSHPDG
TRITICUM-AESTIVUM SVDGGARPAFSPVDLGR -----------------------
GEGFAEVELYG-DVVLRFVSHPDD
AVENA-SATIVA SVAGGARPAFAPADLGH -----------------------
GFGLAEVELYG-DVVLRFVSYPDE
ORYZA-SATIVA SVAAGARPAFQPADLGG -----------------------
GFGLAEVELYG-DVVLRFVSHPDG
SORGHUM-BICOLOR SVAAGARPAPEPVELGL -----------------------
GFRLAEVELYG-DVVLRYVSYPDD
ZEA-MAYS -------------------------------------------------------------------
GFRLAEVELYG-DVVLRYVSYPDG
AB095005_OLUCIMARINUS
AVKAGARGVLAPTVLTHTVDDGCAKGGQVIAEIELYG-DVVLREVNATDG
OTAURI
AMKRGARGVLEPTEMRHEKHDGCVMGTQIISEvELYG-DVVLRFVSRADG
MICROMONAS-PUSILLA-0CMP1545 SGOHGGR ---------------------------------
SPAFSSACDGFAR----VSEVELYG-DVVLRYYSFHAG
SYNECHOCOCCUS-SP. ELLRGRG ---------------------------------
AOLAWIP-AAAALCLHIPYG
VIBRIO-SP.-MED222 AFKGGGEEYK-TEIGP ------------------------
MELSIPAIYGIG-ESLLYFVDRYGK
MARINOMONAS-SP.-MED121 ALANGAKEFS-GNLGA ------------------------
MELKLPAVYGIG-ESTLYFIDRYGD
PSEUDOMONAS-FLUORESCENS ALELGAQPIH-IDTGP ------------------------
MELNLPAIKGIG-GAPLYLIDRFGE
SULFITOBACTER-SP.-NAS-14.1 AVSKGAEEYT-GAG --------------------------
KVLDVPAIKGIG-GSLIYFVDQYYD
OCEANICOLA-BATSENSIS-HICC2597 AVKNGATPYE-GDG --------------------------
KVMDVPAILGIG-GSLIYFIEDYYE
BDELLOVIBRIO-BACTERIOVORUS AVARGARPYEGNEHOK ------------------------
GATPFPAIYGIG-DSLIYFMDQKNQ
RATIOS NORVEGICUS -INYTG -- RFLPGFEAPTYKDT ----------------
LLPKLPSCNLEIIDHIVGNQ
HOMO-SAPIENS -MNYIG -- QFLPGYEAPAFMDP ----------------
LLPKLPKCSLEMIDHIVGNQ
XENOPUS-LAEVIS -TNYKAP--AHVFLPNYRA-CEVDP ---------------
INNVLPTVKLLNVDHVVGNQ
BLEPHARISmA-JAPONICUS -VDYTG -- PFLPGFRAITKDDP ----------------
LNSAFPOVNYD1I0HVvGNQ
MYCOSPRAERELLA-GRAMINICOLA -TTYTG -- PFLPGYRSCTIVDS ----------------
ANKFLPPVNLEAIDHCVGNQ
ASPERGILLUS-FUMIGATUS -GSYHG ----------------------------------
AFLPGYRMETGVEDP----ISQLLPGVHLNRIDHCVGNQ
MAGNAPORTHE-GRISEA -DNFRG ----------------------------------
TFLPGFRDVNRQPAA----YSALAP-VPLQRIDHCVGNQ
CANDIDA-ALBICANS -INYTG ----------------------------------
PFLPGESQPIYPLADYYQVQLNMMPPVNLTVLDHCVENY
PICHIA-STIPITIS -IDYTG ----------------------------------
PFLPNYSESVIQYNSKYYDOMQNIPTVSFQCIDHCVENY
RHODOCOCCUS-SP. -SHYTG -- PYLPGYTARTSGHT ----------------
FRDGAPERLFQALDHVVONV
RHODOCOCCUS-ERYTHROPOLIS -SRYTG -- PYLPGYVERTSSYR ----------------
KRDGAPKRIFQALDHVVGNV
JANIBACTER-SP.-HICC2649 GQTYSG -- PYLPGYVARSSSFV ----------------
KROGAPKRLFOALDHIVGNV
STREPTOMYCES-AVERMITILIS -TGYDG-----PYLPGYVAAAP ------------------
IVEPPAHRTFQAIDHCVGNV
KORDIA-ALGICIDA -KNYNG -- VFLPGYQRWES -------------------
HYNPEPVGLKFIDHMVGNV
LEEUWENHOEKIELLA-BLANDENSIS-ME -KNYNG --- QFLPGYRKWES --------------------
DYNPEPTGLKYIDHMVGNV
GEMMATIMONAS-AURANTIACA -RNYNG -- VFLPGFKAVTP -------------------
HYQPSDVGLKYIDHCVGNV
PICROPHILUS-TORRIDUS -GDYNG -- VFMPGYEESEI -------------------
NSKNTGIKKIDHIVGNV
ARABIDOPSIS-THALIANA DTE -- KSEFLPGFERVEDASSF ----------------
P-LDYGIRRLDHAVGNV
BRASSICA-RAPA-SUBSP.-PEKINENSI TV ----------- FLPRFETVDDTSSF ----------------
P-LDYGIRRLDHAVGNV
COPTIS-JAPONICA-VAR.-DISSECTA N----QSCP---FLPGFEEVGEVSSS -----------------
RGLDEGIRRLDHAVGNV
HEVEA-BRASILIENSIS NPNLNDSSPDSWFLPKFESVDEASSF --------------
P-LDYGIRRLDHAVGNV
MEDICAGO-TRUNCATULA NP---NQNPNLFFLPGFERVSDESSN --------------
SSLDFGIRRLDHAVGNV
GLYCINE-MAX APOAPHADPSRWFLPGFEAAASSSSF --------------
PELDYGIRRLDHAVGNV
SOLANUM-LYCOPERSICUM ADTL-------IFLPGFEAMDETSSF - ------------
KELDYGIHRLDHAVGNV
SOLANUM-TUBEROSUM EDSL --- IFLPGFEAMDETSSF ----------------
KELDYGIRRLDHAVGNV
NICOTIANA-BENTHAMIANA ADSL --- VFLPGFNAMDETASY - --------------
KELDYGIRRLDHAVGNV
DAUCUS-CAROTA KG ----- LFLPGFEAVEGTASF ----------------
PDLDYGIRRLDHAvGNV
SOLENOSTEMON-SCUTELLARIOIDES ADO -------------------------------------
WFLPGFEVVGDGVSC-------QELDYGIRRLDHAVGNV
HORDEUM-VULGARE TDVP ---- FLPGFEGVTNP -------------------
DAVDYGLTRFDHVVGNV
TRITICUM-AESTIVUM TDVP ---- FLPGFEGVSNP -------------------
DAVDYGLTRFDHVVGNV
AVENA-SATIVA TDLP ---- FLPGFERVSSP -------------------
GAVDYGLTRFDHVVGNV
ORYZA-SATIVA ADAP ---- FLPGFEGVSNP -------------------
GAVDYGLRRFDHVVGNV
SORGHUM-BICOLOR ADAS ---- FLPGFVGVISP -------------------
GAADYGLRREDHIvGNV
ZEA-MAYS AAGEP --- FLPGFEGVASP -------------------
GAADYGLSRFDHIVGNV
AB095005_OLUCIMARINUS ---------------- FDGDFLCNYSATRDAP -----------------
DVSYGLQRLDHAVGNV
OTAURI ---------------------------------- FDGDFLCNYEATRDVP -----------------
SVSYGLRRLDHAVGNV
MICROMONAS-PUSILLA-CCMP1545
EKNA----KPKTFLPGYEDVPLEPPHT------TPLTYGLQRLDHAVGNV
SYNECHOCOCCUS-SP. IRHS ---- LIPGPLDAAPAEAG ----------------
LFSHWDHVVLNV
VIBRIO-SP.-MED222 -QSIYD----VDFRFYDDAEQRMAEA --------------
NVGLYEIDHLTHNV
MAR1NOMONAS-SP.-MED121 -KSIYD----VDFNEYENYQEKMLSH --------------
QAGLYEVDHLTHNV
PSEUDOMONAS-FLUORESCENS GSSIYD----IDFVYLEGVERNPVG ---------------
AGLKVIDHLTHNV
SULFITOBACTER-SP.-NAS-14.1 -TSPYN----EEYDWIAQS--KPAG ---------------
VGFYYLDHLTHNV
OCEANICOLA-BATSENSIS-HICC2597 -TSPYN----AEFDWLAQS--KPRG ---------------
VGFYYLDHLTHNV
BDELLOVIBRIO-BACTERIOVORUS -DKLYN----EIFQVKPED-KAPVG ---------------
VGFTVVDHFTNNV
,

CA 02808152 2013-03-05
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RATTUS NORVEGICUS
PDQEMESASEWYLKNLQFHREWSVDDTQVHTEYSSLRSIVVANYEESIKM
HOMO-SAPIENS
PDQEMVSASEWYLKNLQFHRFWSVDDTQVHTEYSSLRSIVVANYEESIKM
XENOPUS-LAEVIS
PDDMMVPVAEWYEKMLMEHRFWSVDDTQMHTDYSAIRSIVVIDYDEVIKM
BLEPHARISMA-JAPONICUS
PCGDMTPTVEWYEKYLEFHRYWSADESVIHTDYSALRSVVVADWDEVIKM
5 MYCOSPHAERELLA-GRAMINICOLA
DWDEMSDACIDEYERCLGFHRFWSVDDEDICTEFSALKSIVMSSPNQVVICM
ASPERGILLUS-FUMIGATUS
DWDEMDKVCEYYEKALGEHRFWSVDDKQICTEYSALKSIVMASPNEVVKM
MAGNAPORTHE-GRI SEA
DWDDMRAACDFYERCLSEHRFWSVDDNQISTDFSALNSIVMASPNNVVKM
CANDIDA-ALBICANS
SWNQMMEQAKLYADMFGEHKYWSVDEDDISTGETALRSIVMSSSNGOIKM
PICHIA-STIPITIS
SWNQMMAQAKLYASLFGEHKYWSADDHDIATDNTALRSIVMASGNGKIKM
10 RHODOCOCCUS-SP.
ELGKMDHWVDFYNRVMGFTNMAEFVGEDIATDYSALMSKVVSNGNHRVKF
RHODOCOCCUS-ERYTHROPOLIS
ELCKMDQWVDFYNRVMGETNMAEFVGGDIATDYSALMSKVVSSONHRVKF
JANIBACTER-SP.-HTCC2649
ELGKMDEWVSFYNRVMGFVNMAEFVGDDIATDYSALMSKVVANGNHRVKF
STREPTOMYCES-AVERMITILIS
ELGRMNEWVGFYNKVMGETNMKEFVCDDIATEYSALMSKVVADGILKVKF
KORDIA-ALGICIDA
GWGEMKEWCEFYAKVMGFAQIISFTDDDISTDFTALMSKVMSNGNGRIKF
15 LEEUWENHOEKIELLA-BLANDENSIS-ME
GWGEMNTWVKWYEDVMGEVNFLTEDDKQITTEYSALMSKVMSNGNGRIKF
GEMMATIMONAS-AURANTIACA
ELGKMNQWVGYYADVLGERNLITEDDTDINTEYSSLMSKVMANGNDRIKF
PICROPHILUS-TORRIDUS YEGEMDSWVNFYIEKLGFEHLITEDDKDIRTDYSALRSKVVKY-
NDDIVF
ARABIDOPSIS-THALIANA P--
ELGPALTYVAGFTGEHQFAEFTADDVGTAESGLNSAVLASNDEMVLL
BRASSICA-RAPA-SUBSP.-PEKINENSI P--
ELGPALTYLSRLTGEHOFAEFTADDVGTAESGLNSAVLANNDETVLL
20 COPTIS-JAPONICA-VAR.-DISSECTA P--
NLAEAIGYLKEFTGEHEFAEFTAEDVGTTESGLNSIVLASNDEMVLL
HEVEA-BRASILIENSIS P--
ELAPAVSYVKEFTGFHEFAEFTAEDVGTSESGLNSLVLANNEDTVLL
MEDICAGO-TRUNCATULA P--
ELSSAVKYVKQFTGFHEFAEFTAEDVGTSESGLNSVVLANNEETVLL
GLYCINE -MAX P--
ELAPAVRYLKGFSGFHEFAEFTAEDVGTSESGLNSVVLANNSETVLL
SOLANUM-LYCOPERSICUM P--
ELGPVVDYIKAFTGEHEFAEFTAEDVGTAESGLNSVVLANNDETVLL
25 SOLANUM-TUBEROSUM P--
ELGPVVDYIKEFTGEHEFAEFTAEDVGTAESGLNSVVLANNDETVLL
NICOTIANA-BENTHAMIANA P--
ELGPAVDYIKRFTGFHEFAEFTSEDVGTAESGINSMVVANNDETVLL
DAUCUS-CAROTA T--
ELGPVVEYIKGFTGEHEFAEFTAEDVGTLESGLNSVVLANNEEMVLL
SOLENOSTEMON-SCUTELLARIOI DES P--
KLEPVVDYLKKFTGEHEFAEFTAEDVGTAESGLNSVVLANNNENVLF
HORDEUM-VULGARE P--
ELAPAAAYIAGFTGEHEFAEFTAEDVGTTESGLNSVVLANNSEGVLL
30 TRITICUM-AESTIVUM P--
ELAPAAAYVACFAGFREFAEFTTEDVCTAESCLNsmVLANNSECVLL
AVENA-SATIVA P--
EMAPVIDYMKGFLGEHEFAEFTAEDVGTTESGLNSVVLANNSEAVLL
ORYZA-SATIVA P--
ELAPVAAYISGFTGEHEFAEFTAEDVCTAESGLNSVVLANNAETVLL
SORGHUM-BICOLOR P--
ELAPAAAYFAGFTGEHEFAEFTAEDVGTTESGLNSMVLANNAENVLL
ZEA-MAYS P--
ELAPAAAYFAGFTGFHEFAEFTTEDVGTAESGLNSMVLANNSENVLL
35 AB095005OLUCIMARINUS H--
DLIETVDYITKVTGEHEFAEFTAEDIGTIDSGLNSMVLANNNEYVLL
OTAURI H--
NLLETVDYIMKITGEHEFAEFTAEDIGTIDSGLNSMVLANNNEYVLL
MICROMONAS-PUSILLA-CCMP1545 P--
NLLETVDYITAMTGMHEFAEFTAEDVGTVDSGLNSMVLANDDEMILL
SYNECHOCOCCUS-SP. EQGSLQAAADWYGRVLGWRRLYRYS -
IGTATSGLESVVVGDPEAGIQW
VIBRIO-SP. -MED222 KOGNMDVWSGEYERLGNFREIRYFDIEGKLIG---
LVSRAMTSPCGKIRI
40 MARINOMONAS-SP.-MED121 MRGNMDHWAGEYENIGNFREIRYFDIEGKLTG---
LVSRAMTSPCGKIRI
PSEUDOMONAS-FLUORESCENS YRGRMVYWANFYEKLENFREARYFDIKGEYTG---
LTSKAMSAPDGMIRI
SULFITOBACTER-SP.-NAS-14.1 FKGNMDVWFKFYGDLFNFREIRFFDIEGKFTG- - -
LTSRALTSPCGRIRI
OCEANICOLA-BATSENSIS-HTCC2597 FKGNMDTWFRFYGDLFNFREIRFFDIQGKYTG---
LFSRALTSPCGRIRI
BDELLOVIBRIO-BACTERIOVORUS PKGEMDKWQHFYEDIFGEYEAKYFDIRGSKTG---
LLSRAMRSPCGKFSV
RATTUS NORVEGICUS PINEPAPGRK -
KSQIQEYVDYNGGAGVQHIALRTEDIITTIRHLRER -
HOMO-S-APIENS PINEPAPGKK-
KSQIQEYVDYNGGAGVQHIALKTEDIITAIRHLRER---
xENOPus-LAEvIs PINEPAPOKK-
KSQIQEFVEYYCGAOVQHIALRTDDILRDVSAMRAR - -
BLEPRARISMA-JAPONICUS PINEPADGLR-KSQIQEYVEYYGGAGVQHIALKVNDIISVISTLRAR- - -
MYCOSPHAERELLA-GRAMINICOLA PINEPAHGKK-
KSQIEEYVDEYNGPGVQHIALRTPNIIEAVSNLRSR---
ASPERGILLUS-FUMIGATUS PINEPAKGKK-
QSQIEEYVDEYNGAGVQHIALLTDDIIRDITNLKAR---
MAGNAPORTHE-GRISEA PINEPAKGKK -
RSQIEEYVTFNSGAGVQHIALLTSDIITTVEAMRSR -
CANDIDA-ALBICANS PINEPVKSIM-
KGQIEEENDENGGPGIQHIAFRTNNIIETVMALMOR---
PICHIA-STIPITIS PINEPVKSKM-RGQIEEFHDENGGPGVQHIALRTNDIIDTVCALLAR---

RHODOCOCCUS-SP. PLNEPALAKK-
RSOIDEYLDFYRGPGAQHLALATNDILTAVDOLTAE---
RHODOCOCCUS-ERYTHROPOLIS PLNEPAIAKK-
RSQIDEYLEFYQGPGAQHLALATNDILGAVDALVDE---
JANIBACTER-SP.-HTCC2649 PLNEPAIAKK -
RSQIDEYLDFYQGPGAQHLAVATNDILRSVDELRKE-- -
STREPTOMYCES-AVERMITILIS PINEPALAKK-
KSQIDEYLEFYGGAGVQHIALNTGDIVETVRTMRAA---
KORDIA-ALGICIDA PINEPAEGKK-KSQIEEYLDFYNGSGVQHIAVATDNIIDTVSQMRER---

LEEUWENHOEKIELLA-BLANDENSIS-ME PINEPAEGIK-KSQIEEYLDFYEGPGVQHLAVATDDIVKIVAALKAR-
--
GEMMATIMONAS-AURANTIACA PINEPASOKK-
KSQIEEYLDFYGGPGAQHLALATDDILATVTALRDR-- -
PTCROPHILUS-TORRIDUS PINEPAKGLR-
KSQIEEYLDYYRSEGVOHIALLTDDIIKTVSMMEEN---
ARABIDOPSIS-THALIANA
PINEPVHGTKRKSQIQTYLEHNEGAGLQHLALMSEDIERTLREMRERSS-
BRASSICA-RAPA-SUBSP.-PEKINENSI
PVNEPVHGTKRKSQIQTYLEHNEGAGVQHLALMSEDIFRTLREMRKRSG-

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COPTIS-JAPONICA-VAR.-DISSECTA
PMNERVYGTKRKSQIQTYLEHNEGAGVQHLALVSEDIFTTLREMRRRSG-
HEVEA-BRASILIENSIS
PLNEPVEGTKRKSQIQTYLEHNEGAGLQHLALVSEDIFKTLREMRRRSG-
MEDICAGO-TRUNCATULA
PMNEPVYGTKRKSQIETYLEHNEGAGLQHLALMSADIFRTLREMRKRSG-
GLYCINE-MAX
PLNEPVYGTKRKSQIETYLEHNEGAGVQHLALVTHDIFTTLREMRKRSF-
SOLANUM-LYCOPERSICUM
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIETTLREMWKRSG-
SOLANUM-TUBEROSUM
PMNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIERTLREMRKRSG-
NICOTIANA-BENTHAMIANA
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIFFTLKEMRKRSG-
DAUCUS-CAROTA
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVSEDIFRTLREMRKRSC-
SOLENOSTEMON-SCUTELLARIOIDES
PLNEPVYGTKRKSQIQTYLDHNEGAGVQHLALITEDIFRTLREMRKRSE-
HORDEUM-VULGARE
PLNEPVHGTKRRSQIQTFLEHHGGPGVQHIAVASSDVLATLRKMRARSA-
TRITICUM-AESTIVUM
PLNEPVHGTKRRSQIQTFLEHHGGSGVOHIAVASSDVLRILREMRARSA-
AVENA-SATIVA
PLNEPVHGTKRRSQIQTYLEYRGGPGVQHIALASNOVERTLREMRARTP-
ORYZA-SATIVA
PLNEPVHGTKRRSQIQTYLDHHGGPGVQHIALASDDVLGTEREMRARSA-
SORGHUM-BICOLOR
PLNEPVHGTKRRSQIQTYLDHHGGPGVQHMALASDDVERTLREMQARSA-
ZEA-MAYS
PLNEPVHGTKRRSQIQTFLDHHGGPGVQHMALASDDVLRTLREMQARSA-
AB095005_OLUCIMARINUS
PVNEPTEGTKRKSQIQTYLEQNNGPGLQHLALKTDDIFATVREMRKYSHL
OTAURI
PVNEPTEGTKRKSQ1QTYLEQNNGPGLOHLALKTDDIFTTVREMRKYSHM
MICROMONAS-PUSILLA-CCMP1545
PVNEPTEGTKRKSQIQTYLECNNGAGLQHLALKSDDVFATVREMRKHGGG
SYNECHOCOCCUS-SP. AINEPTCAAS---
QIQEFLHAHGGPGIQHAALHSSDIVASLRRER
-- VIBRIO-SP.-MED222 PINE-SSDDK--
SQIEEFIREYNGEGIQHIALATDDIYKTVKTLRDR MARINOMONAS-SP.-MED121 PINE-SSDDK--
SQIEEFLNQYNGEGIQHIAMSSSDIYETVRQLKAG PSEUDOMONAS-FLUORESCENS PLNEESSKGA--
GQIEEFLMQFNGEGIQHVAFLTDDLVKTWDALKKI SULFITOBACTER-SP.-NAS-14.1 PINE-
DRDEK--GQIVAYLKKYNGEGIQHIAVGARNIYDATDAIADN OCEANICOLA-BATSENSIS-HTCC2597
PINE-DRGET--GQIVAYLKKYNGEGIQHIAVGARDIYAATDAIAEN 25 BDELLOVIBRIO-
BACTERIOVORUS PINE-PTEEK--SQIQEYLDEYKGSGIQHIALLTHDINYSLESLKNS RATTUS
NORVEGICUS --GMEFLAVP-SSYYRLL RENLKTSK -IQVKEN MDV
HOMO-SAPIENS --GLEFLSVP-STYYKQL ---------------
REKLKTAK -IKVKEN IDA
XENOPUS-LAEVIS --GLEFLTIP-RTYYKNL ---------------
RARLSMSK -VQVEED LAE
BLEPHARISMA-JAPONICUS --GVEFLEVP-PKYYDSL --------------- RKRLAHSA -
VQIEED LKR
MYCOSPHAERELLA-GRAMINICOLA --GVEFISVP-UTYYENM ---------------
RLRLKAAG -MKLEES FDI
ASPERGILLUS-FUMIGATUS --GVEFIKVP-DTYYEDI ---------------
KVRLKKAG -LTLHED YET
MAGNAPORTHE-GRI SEA --GVEFIEVP-HTYYDTM ---------------
RRRLKTEKRDWELQED FDR
CANDIDA-ALBICANS --GVEFNHTS-ENYYNNL ---------------
KQRLNNDG -IKLYED FDT
PICHIA-STIPITIS --GIEFNTAS-DKYYTNL --------------- ERLLREDD -
VALFED FDT
RHODOCOCCUS-SP. --GVEFLATP-DSYYEDP --------------- ELRARIG-
NVRAP IAE
RHODOCOCCUS-ERYTHROPOLIS --GIEFLSTP-ASYYEDP --------------- ELRARIG-
EVRVP IEE
JANIBACTER-SP.-HICC2649 --GVEFLDTP-DAYYDDP --------------- EMRARIG-
EVRVP IEE
STREPTOMYCES-AVERMITILIS --GVQFLDTP-DSYYDT ---------------- LGEWVG-
DTRVP VDT
KORDIA-ALGICIDA --GVEFLYVP-DTYYDD ---------------- LLERVG-
DIDED VEE
LEEUWENHOEKIELLA-BLANDENSIS-ME --GVEFLPPPPQAYYDD ---------------------
IPRRLGAMMDTMKED LNK
GEMMATIMONAS-AURANTIACA --GVEFLSVP-TSYYED ---------------- LQERVG-
KIDEK LEE
PICROPHILUS-TORRIDUS --GIEFLKTP-GSYYES ---------------- LSSRIG-
SIDED LNE
ARABIDOPSIS-THALIANA IGGFDEMPSPPPTYYQN -----------------------
LKKRVG---DVLSDD----QIKE
BRASSICA-RAPA-SUBSP.-PEKINENSI VGGFDFMPSPPPTYYKN -------------------------
LKNRVG---DVLSEE----QIEE
COPTIS-JAPONICA-VAR.-DISSEOTA VGGFEFMPSPPPTYYKN -----------------------
LKNRAG---DVLSDE----Q1KE
HEVEA-BRASILIENSIS VGGFDEMPSPPPTYYRN -----------------------
LKNRVG---DVLIDE----QIKE
MEDICAGO-TRUNCATULA VGGFEFMPSPPVTYYRN -----------------------
LKNRVG---DVLSDE----QIKE
GLYCINE-MAX LGGFEEMPSFPFTYYAN -----------------------
LHNRAA---DVLIVD----QIKQ
SOLANUM-LYCOPERSICUM VGGFEEMPAPPPTYYKN ----------------------- -
LRSRAG---DVLSDE----QIQA
SOLANUM-TUBEROSUM VGGFEEMPUPPTYYKN ------------------------
LKSRAG---DVLSDE----QIQA
NICOTIANA-BENTHAMIANA VGGFEFMPSPPPTYYKN -----------------------
LKNRAG---DVLIDE----QIQA
DAUCUS-CAROTA LGGFEEMPSPPPTYYKN -----------------------
LKNRVG---DVLSDE----QIKE
SOLENOSTEMON-SCUTELLARIOIDES VGGFEFMPSPPPTYYRN -----------------------
LKSRAG---DVLSDE----QIEE
HORDEUM-VULGARE MGGFDFLPPPLPKYYEG -----------------------
VRRLAG---DVLSEA----QIKE
TRITICUM-AESTIVUM MGGFDFLPPRCRKYYEG -----------------------
VRRIAG---DVLSEA----QIKE
AVENA-SATIVA MGGFEFMAPPQAKYYEG -----------------------
VRRIAG---OVLSEE----QIKE
ORYZA-SATIVA MGGFEFLAPPPPNYYDG -----------------------
VRRRAG---DVLSEE----QINE
SORGHUM-BICOLOR MGGFEFMAPPAPEYYDG -----------------------
VRRRAG---DVLTEA----QIKE
ZEA-MAYS MGGFEFMAPPTSDYYDG -----------------------
VRRRAG---DVLTEA----QIKE
AB095005_OLUCIMARINUS RGGFDFQAPASDDYYKQ -----------------------
LKAKIG---DALNDE----QYAL
OTAURI HGGFDFQAPASDDYYKH -----------------------
LKEKIG---DALTDE----QYAL
MICROMONAS-PUSILLA-CCMP1545 RGGFEFQKPASADYYAN -----------------------
LKARVGE--DALTER----QFKE
SYNECHOCOCCUS-SP. QGGVDFLQVAP-QYYTS -----------------------
LERELGL--ALRSALGQAISWQD
VIBRIO-SP.-MED222 --GMDFMPTP-DTYYEKVDDRVKGHG- ------ ED --- TDL

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. =
42
MARINOMONAS-SP.-MED121 --GLKFMSTP-DTYYAKVNDRVVGHG ----- ED -----
LEK
PSEUDOMONAS-FLUORESCENS --GMRFMTAPPDTYYEMIEGRLPDHG ----- EP -----
VDQ
SULFITOBACTER-SP.-NAS-14.1 --GLKEMPGPPETYYKMSKURVTGHQ ----- EP -----
LDR
OCEANICOLA-BATSENSIS-HTCC2597 --GVTYMPGPPDAYYDMSHDRVKDHG ----- EP -----
IDR
BDELLOVIbRIO-BACTEKIOVORUS --EIQFLTPFPHSYYEMIPERVPGVT ----- ED -----
1SK
RATTUS NORVEGICUS LEELKILVDYD -----------------------------
EKGYLLQIFTKPMQDRPTLFLEVI
HOMO-SAPIENS LEELKILVDYD -----------------------------
EKGYLLOIFTKPVQDRPTLFLEVI
XENOPUS-LAEVIS IEKLSILVDFD -----------------------------
EEGYLLQIFTKPLEDRPTLFIEII
BLEPHARISMA-JAPONICUS IEDLHILVDFD -----------------------------
DRGYLLQIFTKPVEDRPTLFYEII
MYCOSPHAERELLA-GRAMINICOLA IQKLNILIDFD -----------------------------
EGGYLLQLFTKPLMDRPTVFIEII
ASPERGILLUS-FUMIGATUS IRSLDILIDFD -----------------------------
EGGYLLQLFTKHLMDRPTVFIEII
MAGNAPORTHE-GRISEA LVRNNILIDYD -----------------------------
EGGYLLQIFTRPUMDRPTVFIEII
CANDIDA-ALBICANS
LRSLNILIDYDPSTKPKPKSKRKRNNKCNYLLQIFSKPLHDRPTLFIEII
PICHIA-STIPITIS LRKLNILVDYDISTR -------------------------
NKKTGICNYLLQIFTKPLHDRPTLFIEII
RHODOCOCCUS-SP. LQKRGILVDRD -----------------------------
EDGYLLQIFTKPLVDRPTVFFELI
RHODOCOCCUS-ERYTHROPOLIS LQKRGILVDRD -----------------------------
EDGYLLQIFTKPIGDRPTVFFEII
JANIBACTER-SP.-HTCC2549 LKSRKILVDRD -----------------------------
EDGYLLQIFTKPLGDRPTVFFEII
STREPTOMYCES-AVERMITILIS LRELKILADRD -----------------------------
EDGYLLQIFTKPVQDRPTVFFEII
KORDIA-ALGICIDA LKKHGILIDRD -----------------------------
EEGYLLQLFTKTIVDRPTMFFEVI
LEEUWENHOEKIELLA-BLANDENSIS-ME LQELSILVDAD ----------------------------------
EEGYLLQIFTKPLQDRPTLFFEII
GEMMATIMONAS-AURANTIACA LAALGILVDRD -----------------------------
PDGYLLQIFTKPVEDRPTLFFEII
PICROPHILUS-TORRIDUS IEKHNILVDRD -----------------------------
ENGYLLQIFTKPVTDRPTFFFEVI
ARABIDOPSIS-THALIANA CEELGILVDR ------------------------------
DDQGTLLQIFTKPLGDRPTIFIEII
BRASSICA-RAPA-SUBSP.-PEKINENSI CEELGILVDR --------------------------------
DDQGTLLQIFTKPLGDRPTIFIEII
COPTIS-JAPONICA-VAR.-DISSECTA CEELGILVDR ------------------------------
DAQGILLQIFTKPVGDRPTIFVEII
HEVEA-BRASILIENSIS CEELGILVDR ------------------------------
DDQGILLQIFTKPVGDRPTIFIEII
MEDICAGO-TRUNCATULA CEELGILVDR ------------------------------
DDQGILLQIFTKPIGDRPTIFIEII
GLYCINE-MAX CEELGILVDR ------------------------------
DDQGTLLQIFTKPVGDRPTIFIXII
SOLANUM-LYCOPERSICUM
CEELGILVDR --- --- --- DDQGTLLQIFTKPVGDRPTIFIEII
SOLANUM-TUBEROSUM CEDLGILVDR ------------------------------
DDQGTLLQIFTKPVGDRPTIFIEII
NICOTIANA-BENTHAMIANA CEDLGILVDR ------------------------------
DDQGTLLQIFTKPVGDRPTIF1EII
DACCUS-CAROTA CEDLGILVDR ------------------------------
DDQGILLQIFTKPVGDRPTLFIEII
SOLENOSTEMON-SCUTELLARIOIDES CEKLGILIDR ------------------------------
DDQGILLQIFTKPVGDRPTIFIEII
HORDEUM-VULGARE CQELGVLVDR ------------------------------
DDQGVILOIFTKPVGDRPTLFLEMI
TRITICUM-AESTIVUM CQELGVLVDR ------------------------------
DDOGVILOIFTKPVGDRPTLFLEMI
AVENA-SATIVA CQELGVLVDR ------------------------------
DDQGVILQIFTKPVGDRPIFFLEMI
ORYZA-SATIVA CQELGVLVDR ------------------------------
DDQGVILQIFTKPVGDRPTFFLEMI
SORGHUM-BICOLOR CQELGVLVDR ------------------------------
DDOGVILOIFTKPVGDRPTLFLEII
ZEA-MAYS CQELGVLVDR ------------------------------
DDQGVLLQIFTKPVGDRPTLFLEII
AB095005_OLUCIMARINUS VEELGLLVDK ------------------------------
DDQGVLIQVFTKPVGDRPTLFLEII
OTAURI VEELGLLVDK ------------------------------
DDQGVLIQVFTKPVGDRPTLFLEII
MICROMONAS-PUS1LLA-CCMP1545 VEELGLLVDR ------------------------------
DDQGVLVQIFTKPLGDRPTVFIEII
SYNECHOCOCCUS-SP. LVEWILLDATLPASDG ------------------------
QDRPLLLQTFTQPLFGRPTFFFEVI
VIBRIO-SP.-MED222 LRDLRVLIDGAPTKDG ------------------------
ILLQIFTQTVIG--PVFFEII
MARINOMONAS-SP.-MED121 LQDLNILIDGAPLKDG ------------------------
ILLQIFTDTVIG--PVFFEII
PSEUDOMONAS-FLUORESCENS LQARGILLDGSSVEGDK -----------------------
RLLLQIFSETLMG--PVFFEFI
SULFITOBACTER-SP.-NAS-14.1 MKKHGILIDGEGVVDGGE ----------------------
TRILLQIFSKTVIG--PIFFEFI
ocEANIcoLA-BATsENsis-HTcC2597 mKKHGILIDGEGvvDGGE ----------------------
TRILLQIFSKTVIG--PIFFEFI
BDELLOVIBRIO-BACTERIOVORUS LEKNAILVDGD -KTG ----------------------
KYLLQIFTKNTFG--PIFYELI
RATTUS NORVEGICUS QRHN ------------------------------------
HOGFGAGNENSLFKAFEEEQAIRGN
HOMO-SAPIENS QRHN ------------------------------------
HQGFGAGNFNSLFKAFEEEQNLRGN
XENOPUS-LAEVIS QRHN ------------------------------------
HQGFGAGNFKALFESIEMEQAIRGN
BLEPHARISMA-JAPONICUS QRHN ------------------------------------
NNGFGIGNFKALFESLEQEQERRGN
MYCOSPHAERELLA-GRAMINICOLA QRNN ------------------------------------
FDGFGAGNFKSIFEAIEREQDLRGN
ASPERGILLUS-FUMIGATUS QRHN ------------------------------------
FSGFGAGNEKSIFEAIEREQAIRGN
MAGNAPORTHE-GRI SEA QRNE ------------------------------------
FDGFGAGNFKSLFEAIEREQAERGN
CANDIDA-ALBICANS ORHE ------------------------------------
HNGFGKGTFKGLFESIEEOQKLRGT
PICHIA-STIPITIS QRHH ------------------------------------
HNGFGKGTFKGLFETIEEQQRIRGT
RHODOCOCCUS-SP. ERHG ------------------------------------
SIGFGIGNFKALFEAIEREQAARGN
RHODOCOCCUS-ERYTHROPOLIS ERHG ------------------------------------
SLGEGIGNFKALFEAIEREQAARGN
JANTBACTER-SP.-HTCC2649 ERHG ------------------------------------
SLGFGKGNFKALFESIEREQDARGN
STREPTOMYCES-AVERMITILIS ERHG ------------------------------------
SMGEGKGNFKALFEAIEREQEKRGN
KORDIA-ALGICIDA QRKG ------------------------------------
AQSFGVGNFKALFEAIEREQAARGT

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LEEUWENHOEKIELLA-BLANDENSIS-ME QRMG -------------------------------------
AKGFGAGNFKALFESIEREQAQRGT
GEMMATIMONAS-AURANTIACA QRKG ------------------------------------
ATSFGKGNFRALFEAIEREQELRGN
PICROPHILUS-TORRIDCS QRKG ------------------------------------
ARSEGNGNEKALFEATEREQAKRGN
ARABIDOPSIS-THALIANA QRVGCMMKDEEGKA --
YQSGGCGGFGKGNFSELFKSIEEYEKTLEA
bRA561CA-RAPA-SUBSP.-FEKINENSI QKIGCMKKDEEGRV --
YQSGGGGGFGKGNFSELKKSIEEYEKTLEA
COPTIS-JAPONICA-VAR.-DISSECTA QRLGCMLKDEEGKT --
YOKAGCGGEGKGNESELFKSIEEYEKTLEA
HEVEA-BRASILIENSIS QRVGCMIKDETGKE --
YQKGGCGGEGKGNESELFKSIEEYEKTLEA
MEDICAGO-TRUNCATULA QRVGCMLKDEEGKE --
YOKGGCGGEGKGNESELFKSIEEYEKTLET
GLYCINE-MAX QRIGCMVEDEEGKV --
YQKGACGGEGKGNESELFKSIEEYEKTLEA
SOLANUM-LYCOPERSICUM QRIGCMLKDEKGQV -- YOKGGCGGEGKGNESELFRSIEEYEKMLEA
SOLANUM-TUBEROSUM QRIGCMLKDENGQV --
YQKGGCGGFGKGNFSELFRSIEEYEKMLEA
NICOTIANA-BENTHAMIANA QRIGCMLKDEKGQV -- YQKGGCGGEGKGNEWELFRSIEEYEKTL-
-
DAUCUS-CAROTA QRVGCMLKDDAGQM --
YQKGGCGGEGKGNESELFKSIEEYEKTLEA
SOLENOSTEMON-SCUTELLARIOIDES QRVGCMMKDEEGKM --
YQKGGCGGFGKGNFSELFKSIEEYEKMLES
HORDEUM-VULGARE QRIGCMEKDERGEE -- YQKGGCGGEGKGNESELFKSIEDYEKSLEA
TRITICUM-AESTIVUM QRIGCMEKDERGEE --
YQKGGCGGEGKGNESELEKSIEDYEKSLEA
AVENA-SATIVA QRIGCMEKDEVGQE --
YQKGGCGGEGKGNESELFKSIEDYEKSLEV
ORYZA-SATIVA QRIGCMEKDESGQE --
YQKGGCGGFGKGNFSELFKSIEEYEKSLEA
SORGHUM-BICOLOR QRIGCMEKDEKGQE --
YQKGGCGGEGKGNESQLEKSIEDYEKSLEA
ZEA-MAYS QRIGCMEKDEKGQE -- YQKGGCGGFGKGNFSQLFKSIEDYEKSLEA
AB095005_OLUCIMARINUS QRIGCMRRKADSES --------------------------
FEQAAGCGGEGKGNESELFKSIEAYEATLQI
OTAURI QRVGCMRKKADTDE --------------------------
LEQVAGCGGFGKGNFSELFKSIEAYEKTLNI
MICROMONAS-PUSILLA-CCMP1545
QRIGCLREVKSADANAPPRIEQAGGCGGEGKGNESELFKSIENYERTLKM
SYNEOHOCOOCUS-SP. QRLG--------------------
GATGEGEANFQALFEALERQQRQRHQ
VlBRIO-SP.-MED222 QRKG ------------------------------------
NEGFGEGNFKALFESIEEDQIRRGV
MARINOMONAS-SP.-MED121 QRKG ------------------------------------
NEGFGEGNFKALFESIEEDQIRRGV
PSEUDOMONAS-FLUORESCENS QRKG ------------------------------------
DDGFGEGNFKALFESIERDQVRRGV
SULFITOBACTER-SP.-NAS-14.1 ERKG ------------------------------------
DDGFGEGNFKALFESIEQEQIDSGE
OCEANICOLA-BATSENSIS-HICC2597 QRKG -----------------------------------
DDGFGEGNFKALFESIEQEQIDNGE
BDELLOVIDRIO-BACTERIOVORUS QRKG -----------------------------
HDGFGDGNFQALFRAIERDQRERGY
RATTUS NORVEGICUS LTDLETNGVRSGM --
HOMO-SAPIENS LTNMETNGVVPGMAENLYFQ
XENOPUS-LAEVIS
BLEPHARISMA-JAPONICUS LI -------------
MYCOSPHAERELLA-GRAMINICOLA
ASPERGILLUS-FUMIGATUS LV -------------
MAGNAPORTHE-GRI SEA
CANDIDA-ALBICANS FVKSQNN --------
PICHIA-STIPITIS LVQVDEDDDSQQST - --
RHODOCOCCUS-SP.
RHODOCOCCUS-ERYTHROPOLIS
JANIBACTER-SP.-HTCC2649
STREPTOMYCES-AVERMITILIS
KORDIA-ALGICIDA
LEEUWENHOEKIELLA-BLANDENSIS-ME L --------
GEMMATIMONAS-AURANTIACA
PICROPHILuS-ToRRIDus I. -------------
ARABIDOPSIS-THALIANA KQLVG ----------
BRASSICA-RAPA-SUBSP.-PEKINENSI KQLVG -----------
COPTIS-JAPONICA-VAR.-DISSECTA KANVVAA -------
HEVEA-BRASILIENSIS KRNAEAR --------
MEDICAGO-TRUNCATULA RRTA -----------
GLYCINE-MAX KRTA -----------
SOLANUM-LYCOPERSICUM KHVNQVAAVE -----
SOLANUM-TUBEROSUM KBVNQVAAA ------
NICOTIANA-BENTHAMIANA
DAUCUS-CAROTA KQITGSAAA ------
SOLENOSTEMON-SCUTELLARIOIDES KLVTKTAMA ------
HORDEUM-VULGARE KQSAAVQGS ------
TRITICUM-AESTIVUM KQSAAVQGS ------
AvENA-SATIVA KQSVvAQKS ------
ORYZA-SATIVA KQAPTVQGS ------
SORGHUM-BICOLOR KQAAAAQGS ------

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ZEA-MAYS KQAAAAAAAQGS --
AB095005_OLUCIMARINUS
OTAURI
MICROMONAS-PUSILLA-CCMP1545
SYNECHOCOCCUS-SP. ALT? ----------
VIBRIO-SP.-MED222 LDDA ----------
MARINOMONAS-SP.-MED121 LNDA ----------
PSEUDOMONAS-FLUORESCENS LATD ----------
SULFITOBACTER-SP.-NAS-14.1 LSEA ----------
OCEANICOLA-BAISENSIS-HTCC2597 IAAE ---------
BDELLOVIBRIO-BACTERTOVORUS LT ------------
The overall sequence identity between individual full length HPPD sequences is
in
general quite low and is shown for the representative HHPD proteins in Table
3.
Table 4a shows the sequence alignment of the binding pocket. In contrast, the
sequence identity of the 36 amino acids forming the binding site is
significantly
higher which is shown for the representative HPPD proteins in Table 4b. In
particular,
the amino acids at 8 positions are strictly conserved in all species and
illustrate that
these amino acids have a key role (e.g. His226, His308, Glu394 binding the
iron
required for catalysis). These positions, with reference to the HPPD from
Arabidopsis
(SEQ ID No. 2) are His226, Ser267, Asn282, His308, Tyr342, Glu394, Gly420,
Asn423 (Table 5a). A mutation of any of the amino acids at either of these
positions
will most likely lead to an inactive protein. The variability at other
positions within the
binding site is higher. Table 5b shows the 28 variable positions in the
binding site
and the amino acids which were identified at these positions using the
sequence
alignment. Some positions have only limited variability which reflects their
role in the
3D environment. An example for this represents the position 269. All HPPD
proteins
have at this position either a Val, Ala or Thr. Looking at the 3D structures
it seems
that at this position a small apolar amino acid is required and a mutation to
a polar
amino acid such as Arg, His or Lys will disturb the protein structure locally.
Another
example represents position 379. Most sequences have a glutamine at position
379.
However, there are also some bacterial sequences which have a histidine at
this
position. Looking at the 30 structure, it seems that only few amino acids are
tolerated at this position. GIn379 in A.thaliana stabilizes via its H-bond
donor the side
chain conformation of the strictly conserved Glu394 which in turn interacts
with the
catalytic iron. In addition, with its H-bond acceptors GIn379 stabilizes the
side chain
conformation of the strictly conserved Asn423 which in turn interacts with the
strictly
conserved Tyr342. Only glutamine, asparagine and histidine have an H-bond
donor

CA 02808152 2013-03-05
. .
=
and acceptor required for the stabilization of this particular 3D arrangement
which
very likely play a key role in the interaction of the C-terminal helix with
the core of the
HPPD protein. Limited variability is also seen at position 381 with either a
phenylalanine or a tyrosine in all HPPD sequences. The aromatic ring
stabilizes the
5 binding of HPPD inhibitors and very likely also the binding of the
substrate to the
HPPD binding. However, the presence of the additional hydroxyl group in
tyrosine
compared to phenylalanine does not disturb the catalytic activity. The third
category
of positions includes those positions which display a very high natural
variability.
These positions may not be crucial for substrate binding and catalysis but
influence
10 inhibitor binding. These positions include adjacent positions in strand
248 to 255 and
positions in the C-terminal helix 419-427. It can be assumed that the
interaction of
this particular strand and the C-terminal helix with the core of the protein
plays a
crucial role in inhibitor binding. Table 5b includes for each variable
position those
amino acids which have been identified in the sequence alignments using all
known
15 HPPD sequences.

Table 3: Pairwise sequence identity of HPPD sequences from the representative
set of HPPD proteins
RATTUS_NORYEOICUS 103 90 62 55 52 63 50 44 41 42 39 41 45 45 40 48 45 37 37
36 35 37 37 38 36 38 38 37 35 35 35 35 36 35 34 34 32 31 29 28 27 24 24 33
H0640-SAPIENS 93 100 63 55 53 55 51 42 41 42 40 42 46 46 49 48 45 38 37 37
37 37 36 37 37 38 37 37 35 35 35 35 35 35 34 35 34 31 29 29 27 25 25 31
XENOBJS-LAE\40 62 63 100 60 54 57 52 44 44 43 42 43 49 48 61 49 45 37 37 36
34 34 34 38 38 38 38 37 36 38 36 3535 36 38 36 35 30 31 32 31 28 25 31
BLEPH8RI6MA-JAP0I4CUS 55 55 130 103 49 49 47 41 40 42 41 41 45 42 4e 48 45
35 35 35 35 34 36 35 35 35 36 35 35 34 36 35 35 39 34 34 32 31 33 31 26 25 26
29
MYCOSPH55RELLA-GRAMINICO1A 52 53 54 49 100 65 61 45 40 44 43 41 46 48 47 48
44 34 34 34 32 32 33 34 33 33 33 34 31 30 32 32 32 31 33 33 31 30 33 34 32 29
28 32
ASPEROILLUS-FU410ATUS 53 55 57 49 es 100 63 45 43 43 42 44 46 49 51 49 46
33 33 32 31 32 32 33 33 33 34 35 32 31 32 32 33 32 32 33 30 29 35 34 32 27 28
33
M5O44AP0RTHE-0RI56A 50 61 52 47 81 63 100 42 40 41 40 40 48 45 45 47 41 33
34 34 32 32 32 33 33 34 33 35 32 31 33 32 32 32 31 33 33 30 33 30 28 26 26 31
CANDIDA-AUB1C4746 44 42 44 41 45 45 42 100 66 37 37 37 38 41 41 43 38 26 28
28 27 29 28 28 28 29 28 28 28 28 27 28 28 29 26 26 25 27 33 32 33 28 30 31
41 41 44 40 40 43 40 86 100 36 X 36 37 37 36 39 38 26 26 28 25 27 26 28 25 27
26 28 28 26 26 23 27 28 35 24 25 29 32 29 30 25 26 32
RH00000CCUS-SP. 42 42 43 42 44 43 41 37 38 100 135 77 62 48 49 55 96 34 34
36 35 35 36 36 35 35 36 35 35 34 37 35 37 38 36 M 34 34 30 31 32 29 29 32
R11ODOCOCCUS-E14Y0HROPOUS 33 40 42 41 43 42 40 37 36 85 1E0 75 60 47 49 55
46 35 35 36 34 36 36 36 36 35 37 39 34 33 36 34 36 35 35 35 34 32 30 30 31 30
29 31
JAMBACTER-SP -HTCC2349 41 42 43 41 41 44 40 37 36 77 75 100 53 47 49 53 44
34 35 34 34 34 36 36 35 35 35 37 32 32 33 33 34 34 36 36 33 32 31 30 31 29 29
32
STREPTOMYCES-AVERM775.16 45 48 49 45 46 45 46 30 37 62 60 63 100 50 53 58
48 38 38 36 37 37 26 37 37 37 38 39 35 34 35 35 38 36 38 38 35 34 34 33 32 32
30 33
K091215-ALO1C1DA 45 46 48 42 48 49 45 41 37 48 47 47 50 1120 73 60 s3 35 35
34 32 34 32 34 35 34 34 36 34 34 34 36 34 34 34 35 34 31 37 35 34 33 34 35
LEELANEN10ER1E1LA-BLANDENSIS-ME 48 49 51 46 47 61 45 41 38 49 49 49 53 73 100
63 51 315 38 36 36 37 37 39 38 30 37 39 38 37 37 33 38 39 38 38 38 34 37 38 36
32 33 35
GEMMATIMONAS=AURANTIACA
98 445 49 48 48 49 47 43 39 55 55 53 58 60
83 100 53 39 39 39 39 39 39 39 39 38 40 43 39 37 38 38 39 39 40 40 38 38 37 35
36 34 33 38 0
P1CROPHLUS.TORRIDUS
45 45 45 45 44 46 41 38 38 46 46 44 46 53
51 53 103 35 35 35 36 35 35 36 36 33 38 37 32 32 32 33 33 34 32 33 33 33 33 33
33 29 29 34 03
0
ARA86)OPSIS-Tli5LIAMA
37 36 37 35 34 33 33 28 28 34 35 34 38 35
38 39 35 100 90 75 78 75 73 72 72 70 73 71 CO 60 61 81 63 63 55 56 57 31 28 29
25 27 27 30 CO
8RASSICA3RAPA=SUBSP -PEHINENSI 37 37 37 35 34 33 34 28 26 34 35 35 38 35 36
39 35 90 100 75 76 76 75 74 74 73 73 72 60 60 61 61 62 63 57 57 58 31 26 28 26
27 27 29
ODPT1S-JAPONICANAR -D1SSECTA 36 37 36 35 34 32 34 2B 28 35
34 38 34 36 39 35 75 75 100 78 74 71 73 73
72 73 72 61 61 82 62 64 64 55 S6 56 29 29 31 29 28 28 31 1N3
HEVEA-BRASILIENSIS
36 37 34 35 32 31 32 27 25 35 34 34 37 32
36 39 36 78 76 78 100 78 74 75 75 74 74 72 62 62 62 53 64 64 56 57 57 29 28 29
27 27 27 30 N,
MEDICA00-TRUI4ATULA
37 37 34 34 32 32 32 29 27 35 36 34 37 34
37 39 35 75 76 74 78 100 76 74 75 74 75 74 132 82 63 53 65 65 55 56 59 29 26
30 27 27 27 29 .. 0
OLYCINE-MAX 37 38 34 16 33 32 33 28 26 36 36 36 38 32 37 39 35 73 75 71 74
76 100 75 74 74 75 73 62 62 62 63 68 67 54 55 56 29 30 30 25 27 26 31
S0LANUM-LYc04'ERSicu4 38 37 X 35 34 33 33 28 26 36 36 36 37 34 39 39 35 72
74 73 75 74 75 100 96 87 77 76 63 62 62 85 64 64 56 57 57 39 25 29 26 27 26 30
Lo
SOLANUM=TUBEROSUM 35 37 36 35 33 33 33 28 25 35 36 35 37 35 35 39 36 72 74
73 75 75 74 96 100 88 79 76 62 61 62 64 62 63 56 57 57 30 29 29 26 27 26 33
oI
HCOTIANA.BENTHAXANA X 38 301 35 33 33 34 29 27 35 35 35 37 34 38 38 36 70
73 72 74 74 74 07 88 133 76 75 El 60 61 133 84 64 55 56 56 31 31 30 27 27 26
30
DAUCUS-CARO14 36 37 38 36 33 34 33 25 26 36 37 35 33 34 37 40 36 73 73 73
74 75 75 77 78 76 100 75 63 62 64 65 65 65 56 59 59 29 29 30 27 27 26 29
SOLENOSTEMONSCUTELLARJOIDES 37 37 37 35 34 35 35 28 25 38 39 37 39 36 38 43
37 71 72 72 72 74 73 76 76 75 76 100 62 60 62 64 64 65 58 59 57 30 3D 31 27 28
27 30
HORDELIKVULOARE 35 35 36 35 31 32 32 28 26 35 34 32 35 34 36 39 32 60 60 61
62 62 62 53 82 51 63 62 100 95 83 63 83 83 54 55 54 33 30 33 27 27 26 33
TRITICUM-AESTIVUM 35 35 36 34 30 31 31 28 26 34 33 32 34 34 37 37 32 80 60
61 82 62 62 52 61 60 62 60 95 100 113 62 82 52 53 54 54 32 30 30 26 28 26 30
6VENA-5ATIVA 35 35 36 36 32 12 33 27 28 37 36 33 35 34 37 38 32 61 61 62 62
63 62 52 62 81 64 62 83 83 100 81 80 79 54 53 54 30 32 29 27 27 28 31
ORY2A-SATIVA 35 35 36 35 32 32 32 28 26 36 34 33 35 35 36 38 33 151 61 62
63 63 63 85 64 83 65 64 63 82 81 100 84 83 54 se 55 32 29 29 27 26 28 31
SOROHUMZICOLOR 36 35 35 35 32 33 32 28 27 37 36 34 33 34 38 39 33 53 62 64
64 65 66 64 62 54 65 64 63 82 80 84 100 93 54 56 56 30 29 30 27 26 27 29
2EA-MAYS 35 35 36 34 31 32 32 29 26 38 35 34 36 34 39 39 34 63 63 64 64
65 67 84 63 84 65 65 83 82 79 63 93 100 55 57 57 32 33 31 27 26 26 29
$809500530LUCIMARINUS 34 34 36 34 33 32 34 26 25 36 35 36 38 34 313 40 32
SS 57 SS 58 55 64 56 SS 55 SO 58 54 53 54 54 54 55 100 84 63 26 30 27 25 25 27
27
OTAUR1 34 35 36 34 33 33 33 26 24 36 35 36 38 35 36 40 33 56 57 56 57
56 55 57 57 56 59 59 55 54 53 55 56 57 54 103 62 2B 31 29 25 26 27 27
MICR0MONAS-4USLLA-CCM611545 32 34 35 32 31 31 33 26 25 34 34 33 35 34 36 38
33 57 58 58 57 69 56 S7 57 56 59 57 54 54 54 SS 56 57 63 62 100 29 27 28 24 25
25 27
NECHOCOCCU653. 31 31 30 31 30 29 30 27 26 34 32 32 34 31 34 36 33 31 31 29
28 29 29 30 30 31 29 30 33 32 30 32 30 32 ze 2e 29 100 25 27 26 23 24 ze
VBR6)-SP A1ED222 29 29 3i 30 33 35 33 33 32 30 33 31 34 37 37 37 33 28 2B
29 28 29 30 28 29 31 78 30 30 30 30 29 29 30 30 31 27 26 100 74 53 53 50 46
MARNOM0NAS-SP:5ED121 28 29 32 31 34 34 30 32 29 31 X 30 33 35 38 35 33 28
28 31 29 3o 3o 29 29 30 30 31 30 30 29 29 30 31 27 23 28 27 74 1M 56 51 49 44
PSECIJM62N0ASE-FLU0RESCENCE 27 27 31 26 32 32 29 33 30 32 31 31 32 34 38 35
33 25 26 29 27 27 26 28 26 27 27 27 27 26 27 27 27 27 25 25 24 28 63 56 100 48
SO 43
SULF1TOBACTER-SP -NASA 41 24 25 28 26 29 27 26 28 25 29 30 29 32 33 32 34
29 27 27 28 27 27 27 27 27 27 27 28 27 28 27 25 26 26 25 26 25 23 53 51 46 100
61 45
OCEAI4COLA-8ATSENSIS-HICC2597 24 25 25 26 28 25 28 30 26 29 29 29 30 34 33 33
29 27 27 28 27 27 26 26 26 26 26 27 26 28 28 29 27 26 27 27 25 24 50 49 50 51
100 45
MELLOVIBRIO-BACT3KFUV0RUS 553131293233 31 31 32 32 31 32 33 35 36 38 34 30
29 31 30 29 31 30 30 30 29 30 30 30 31 31 29 29 27 27 27 28 48 44 43 45 45
11:0

CA 02808152 2013-03-05
=
47
Table 4a: Sequence alignment of amino acids forming the binding pocket in the
representative set of HPPD sequences
HOMO-SAPIENS
HVHFWSVLSIVVAPNOIOHLYL0FFEFGAGNFNSLF
RATTUS NORVEGICUS
HVHFWSVLSIVVAPNQIQHLYLQFFEFGAGNFNSLF
XENOPU-LAEVIS
HVHFWSVLSIVVTPNOIOHLYLQFFEFGAGNFKALI
ASPERGILLUS-FUMIGATUS
HVHFWSVLSIVMAPNQIQHIYLQFFEFGAGNEKSLI
MAGNAPORTHE-GRISEA
HVHFWSVLSIVMAPNQIQHIYLQFFEFGAGNFKSLI
MYCOSPHAERELLA-GRAMINIOOLA
HVHFWSVLSIVMSPNQIQHIYLQFFEFGAGNFKSLI
CANDIDA-ALHICANS
HVHYWSVLSIVMSPNQIQHNYLQFFEFGKGTFKGLI
PICHIA-STIPITIS
HVHYWSALSIVMAPNQIQHNYLQFFEFGKGTFKGLI
ARABIDOPSIS-THALIANA
HVHFAEFLSAVLAPNQIQHMYLQFFEFGKGNFSELI
BRASSICA-RAPA-SUBSP.-PEKINENSI
HVHFAEFLSAVLAPNQIQHMYLQFFEFGKGNFSELI
HEVEA-BRASILIENSIS
HVHFAEFLSLVLAPNQIQHMYLQFFEFGKGNFSELI
COPTIS-JAPONICA-VAR.-DISSECTA
HVHFAEFLSIVLAPNQIQHMYLQFFEFGKGNFSELI
SOLENOSTEMON-SCUTELLARIOIDES
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
AVENA-SATIVA
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
DAUCUS-CAROTA
HVHFAEFLSVVLAPNOIQHMYLQFFEFGKGNFSELI
SOLANUM-TUBEROSUM
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
SOLANUM-LYCOPERSICUM
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
MEDICAGO-TRUNCATULA
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
GLYCINE-MAX
HVHFAEFLSVVLAPNQIQHMYLQFFXFGKGNFSELI
HORDEUM-VULGARE
HVBFAEFLSVVLAPNOIOHLYLQFFEFGKGNESELI
ORYZA-SATIVA
HVHFAEFLSVVLAPNQIQHLYLQFFEFGKGNFSELI
SORGHUM-BICOLOR
HVHFAEFLSMVLAPNQIQHMYLQFFEFGKGNFSQLI
ZEA-MAYS
HVHFAEFLSMVLAPNQIQHMYLQFFEFGKGNFSQLI
AB095005 OLUCIMARINUS
HVHFAEFLSMVLAPNQIQHQYLOFFEFGKGNESELI
OTAURI
HVHFAEFLSMVLAPNQIQHQYLQFFEFGKGNFSELI
MICROMONAS-PUSILLA-COMP1545
HVHFAEFLSMVLAPNQIQHQYLQFFEFGKGNFSELI
TRITICUM-AESTIVUM
HVHFAEFLSMVLAPNQIQHLYLQFFEFGKGNFSELI
NICOTIANA-BENTHAMIANA
HVHFAEFLSMVVAPNQIQHMYLQFFEFGKGNFWELI
RHODOCOCCUS-SP.
HVTMAEFLSKVVSPNQIQHLYLQFFEFGIGNFKALI
RHODOCOCCUS-ERYTHROPOLIS
HVIMAEFLSKVVSPNQ1QHLYLQ1FEFGLGNFKALI
STREPTOMYCES-AVERMITILIS
HVTMKEFLSKVVAPNQIQHLYLOFFEFGKGNFKALI
JANIBACTER-SP.-HTCC2649
HVVMAEFLSKVVAPNQIQHLYLQFFEFGKGNFKALI
KORDIA-ALGICIDA
HVAIISFLSKVMSPNQIQHLYLQFFEFGVGNFKALI
LEEUWENHOEKIELLA-BLANDENSIS-ME
HVVFLTFLSKVMSPNOIQHLYLQFFEFGAGNFKALI
PICROPHILUS-TORRIDUS
HVELITFLSKVVKPNQIQHLYLQFFEFGNGNFKALI
GEMMATIMONAS-AURANTIACA
HVRLITFLSKVMAPNQIQHLYLQFFEFGKGNFRALI
VIBRIO-SP.-MED222
HTRIRYFLSRAMTPNQIQHMYLQFFEFGEGNFKALI
MARINOMONAS-SP.-MED121
HTRIRYFLSRAMTPNQIQHMYLQFFEFGEGNFKALI
SULFITOBACTER-SP.-NAS-14.1
HTRIRFFLSRALTPNQIQHMYLQFFEFGEGNFKALI
OCEANICOLA-BATSENSIS-HTCC2597
HTRIRFFLSRALTPNQIQHMYLQFFEFGEGNFKALI
PSEUDOMONAS-FLUORESCENS
HTRARYFLSKAMSPNQIQHMYLQFFEFGEGNFKALI
BDELLOVIBRIO-BACTERIOVORUS
HTYAKYFLSRAMRPNQIQHLYLQFFEFGDGNFQALI
BLEPHARISMA-JAPONICUS
HVHYWSALSVVVAPNOIQHLYLQFFEFGIGNFKALL
SYNECHOCOCCUS-SP.
HVRLYRYLSVVVGANQIQHLYLQFFEFGEANFQALL

Table 4b: Pairwise sequence identity of amino acids forming the binding site
HPPD in the representative set
919940-SARENS 103 100 89 89 B9 86 75 75 72 72 72 75 72 72 72 72 72 72 69 75
75 72 72 72 72 72 75 75 69 69 72 72 69 72 69 69 58 58 58 58 58 61 81 64
RAT1US_N0RVE-4369US 103 100 89 69 89 86 75 75 72 72 72 75 72 72 72 72 72 72
69 75 75 72 72 72 72 72 75 75 69 69 72 72 69 72 69 69 58 58 58 58 53 61 81 64
XEMOPUS-LAEVIS 69 89 100 99 89 89 81 78 72 72 72 75 72 72 72 72 72 72 69 75
75 72 72 72 72 72 75 75 78 76 79 78 78 81 78 72 69 69 69 69 87 67 83 67
ASPER0ILLUS-91.1910A7US 69 89 89 100 100 97 83 63 75 75 75 76 75 75 75 75
75 75 72 75 75 75 75 75 75 75 75 75 69 89 72 72 75 78 69 72 67 87 84 64 67 64
78 58
MAOMAP0RTHE-0RISEA 89 89 89 100 100 97 83 83 75 75 75 78 75 75 75 75 75 75
72 75 75 75 75 75 75 75 75 75 69 89 72 72 75 70 85 72 67 67 64 64 67 64 78 59
WYCOSPHA89ELLA-GRAJAN1COLA 66 86 89 97 97 1130 86 81 72 72 72 75 72 72 72
72 72 72 69 T2 72 72 72 72 72 72 72 72 72 72 69 69 78 81 69 69 67 57 64 64 69
FA 75 58
CANDIDA-ALEICA601 75 75 81 B3 ea 86 1C0 94 69 93 69 72 69 69 69 59 69 69 67
69 69 6S 69 69 69 59 69 59 ES 89 69 59 75 72 67 69 64 64 61 61 67 61 75 56
P02H1A-STIPITIS 75 75 78 83 83 81 94 100 72 72 72 75 72 72 72 72 72 72 69
72 72 72 72 72 72 72 72 72 67 67 72 72 72 69 67 72 64 64 61 61 64 61 91 56
4RAB6)0PSIS-TH4LIANA 72 72 72 75 75 72 69 72 100 100 97 97 97 97 97 97 97
97 94 94 94 94 94 94 94 94 94 92 75 75 78 81 09 72 69 75 67 67 69 69 67 64 69
58
ERASSICA-RAPA-69U61SP-PEK1N8559 72 72 72 75 75 72 69 72 100 190 97 97 97 97
97 97 97 97 94 94 94 94 94 94 94 94 94 92 75 75 78 81 69 72 69 75 67 67 69 69
67 64 69 58
FEVEA-0RASILIERSIS 72 72 72 75 75 72 69 72 97 97 100 97 97 97 97 97 97 97
94 94 94 94 94 94 94 94 94 92 75 75 78 RI 69 72 69 75 67 67 69 69 67 64 69 93
CCP9S-JAP0NICA-VAR.-0ISSECTA 75 75 75 78 78 76 72 75 97 97 97 100 87 97 97
97 97 97 84 94 94 94 94 94 94 94 94 92 75 75 79 81 69 72 69 75 67 67 69 69 67
64 69 58
SCLENOSTEM0N-SCU1ELLARIOI0ES
72 72 72 75 75 72 69 72 97 97 97 97 100
100 100 100 100 100 97 97 97 94 94 94 94 94 94 92 75 75 78 81 69 72 69 75 67
87 69 69 67 64 72 81 C)
AVENA-SATTVA 72 72 72 75 75 72 69 72 97 97 97 97 100 100 1C0 100 100 100 97
97 97 94 94 94 94 94 94 92 75 75 78 131 69 72 89 75 67 87 69 69 67 64 72 61
49)
DA9JC1JS-CAR0TA 72 72 72 75 75 72 69 72 97 97 97 97 100 199 109 1130 100
100 97 97 97 94 94 94 94 94 94 92 75 75 78 81 69 72 69 75 67 67 69 69 67 64 72
61 n)
SCLANUM-1U8E80S194
72 72 72 75 75 72 69 72 97 97 97 97 180
100 100 100 100 100 97 97 97 94 94 94 94 94 94 92 75 75 78 81 69 72 69 75 67
67 69 69 67 64 72 61 co
SOLANUM-LYCOPER9CU94 72 72 72 75 75 72 59 72 97 97 97 97 100 100 163 100
100 100 97 97 97 94 94 94 94 94 94 92 75 75 78 81 69 72 69 75 67 67 69 69 67
64 72 61
03
co
PROICAGO-TRUNCA11LA 72 72 72 75 75 72 69 72 97 97 97 97 100 100 100 100 100
100 97 97 97 94 94 94 94 94 94 92 75 75 78 81 69 72 89 75 67 67 69 69 67 64 72
81
Ln
OLYCNE-14134 69 69 69 72 72 69 67 69 94 94 94 94 97 97 97 97 97 97 100 91
94 92 92 92 92 92 92 ag 72 72 75 78 67 69 67 72 64 64 67 67 64 61 69 56
7.,)
HC4RDEUM-VUL0A61E
75 75 75 75 75 72 69 72 94 94 94 94 97 97
97 97 97 97 94 163 100 92 92 94 94 94 97 63 78 78 81 83 72 75 72 78 64 64 67
67 64 67 75 54 N)
0RYZA-SATIYA 75 75 75 75 75 72 68 72 94 94 94 94 87 97 97 97 97 97 94 100
100 92 92 94 94 94 97 89 78 76 81 83 72 75 72 76 64 64 67 67 64 67 75 64
SOR0MUM-aCOLCR 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 94 92 92
92 100 100 94 94 94 94 22 75 75 78 81 69 72 69 75 67 87 89 69 67 64 89 58 1-
4
ZEA-MAYS 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 94 92 92 92
100 100 94 94 94 94 92 75 75 78 81 69 72 69 75 87 67 69 69 67 64 69 58
(9
.413095005_OLUC4ARIMB 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 84
92 94 94 94 94 100 163 100 97 92 75 75 78 61 69 72 69 75 64 64 67 67 64 64 69
56
OTAU811 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 94 92 94 94
94 94 100 100 100 87 92 75 75 76 81 SS 72 89 75 64 64 67 67 64 64 69 58 CD
MICRO640NASASILLA-CCMP1545 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94
94 94 92 94 94 94 94 109 100 100 97 92 75 75 78 81 69 72 69 75 64 64 67 67 64
64 69 58
TRI11CU4-AES11VU8 75 75 75 75 75 72 69 72 99 94 94 94 94 94 94 94 94 94 92
97 97 94 99 97 97 97 160 92 78 78 at 83 72 75 72 78 64 54 67 67 64 67 72 81
1CO11ANA-BENT'9A96ANA 75 75 75 75 75 72 69 72 92 92 92 92 92 92 92 92 92 92
89 89 89 92 92 92 92 92 92 100 78 78 81 83 69 72 72 75 67 67 67 67 67 64 72 61
14940DOCOCCUS-517 69 69 78 59 69 72 69 67 75 75 75 75 75 75 75 75 75 75 72
78 79 75 75 75 75 75 79 78 100 97 92 92 63 83 63 76 69 69 69 69 75 69 78 57
RHODOCOCCUS-ERY1I81OP0US 69 69 78 69 69 72 69 67 75 75 75 75 75 75 75 75 75
75 72 78 78 75 75 75 75 75 78 78 97 100 92 92 83 83 83 78 69 69 69 69 75 69 75
67
STREPTOMYCESAVERKTUS 72 72 78 72 72 69 69 72 78 78 78 78 78 78 78 78 78 78
75 81 81 78 78 78 78 78 81 el 92 92 100 94 81 81 83 83 69 69 69 69 72 72 78 67
JA7.BACTER-SP.91TOC2649 72 72 78 72 72 69 63 72 81 01 e1 81 91 81 &I 131 81
81 78 63 83 61 81 91 81 81 63 83 92 92 94 100 91 83 e3 83 69 69 69 69 72 89 78
87
KCRD1A-AL0IC10A 69 69 70 75 75 78 75 72 69 69 69 59 69 69 69 69 69 69 67 72
72 69 69 69 69 69 72 69 83 63 91 81 100 86 83 83 75 75 72 72 78 72 75 54
LEE1WENHOEKELLA-BLANDENSIS-ME 72 72 81 78 78 81 72 69 72 72 72 72 72 72 72 72
72 72 89 75 75 72 72 72 72 72 75 72 83 83 61 83 136 100 83 83 72 72 69 es 78
72 72 64
PICROPHILUS-TORRIDUS 68 69 78 89 69 69 67 67 69 69 69 69 59 69 69 69 69 69
67 72 72 69 69 69 69 69 72 72 83 83 63 83 53 83 100 86 69 69 69 69 72 69 75 69
1313NATIVA4AS=ADRANTIACA 8697272 7269 69727575757575757575757572 TB
7875757575757875787883 83 8383 88100 T2716969 757272139
V13190-SP -MED222 58 58 69 67 67 67 64 64 67 67 67 67 67 67 67 67 67 67 64
64 64 67 67 64 64 64 64 67 69 69 69 69 75 72 69 72 100 100 94 94 92 81 64 51
4ARINOM0NAS-SP At79121 58 58 69 67 67 67 64 64 67 67 67 67 67 67 67 67 67
67 54 64 64 67 67 64 64 64 64 67 69 69 69 69 75 72 69 72 100 100 94 94 92 91
64 61
SULM08ACTER,99.-64A9-141 58 58 69 64 54 64 61 61 69 69 69 69 69 69 69 69 69
69 67 67 67 69 69 67 67 67 67 67 69 68 69 69 72 69 69 69 94 94 100 100 66 75
64 61
OCEANICOLA-8ATSENSIS-64TCC2597 58 58 69 64 64 64 61 61 69 69 69 69 69 69 69
69 69 69 67 67 67 69 69 67 67 67 67 67 69 69 69 69 72 69 es 69 94 94 100 100
88 75 64 81
PSEDUMONOASE-FLUORESCENCE 58 56 67 67 67 69 67 64 67 67 67 57 87 67 67 67
67 67 64 64 64 67 67 84 64 64 64 67 75 75 72 72 78 78 72 75 92 92 86 86 100 61
64 81
EDELLOVIBRIOZACTERIOVORUS 61 61 67 64 64 64 61 61 64 64 64 64 64 64 64 64
64 64 51 67 67 64 64 64 64 64 67 64 69 69 72 63 72 72 69 72 81 81 75 75 81 100
64 61
EL9PHARISMA-JAPONICUS 81 81 83 78 78 75 75 81 69 69 69 69 72 72 72 72 72 72
69 75 75 69 69 69 69 69 72 72 78 75 78 76 75 72 75 72 64 64 64 64 64 64 100 72
991,ECH000CCUS-59. 64 64 67 58 S3 56 56 56 58 58 58 58 61 61 61 61 61 61 58
64 64 58 58 58 58 58 61 61 67 67 67 67 54 64 69 69 61 61 61 61 61 61 72 100

CA 02808152 2013-03-05
. .
49
Table 5a: Amino acid positions which are strictly conserved shown for the
crystal
structures
Arabidopsis Pseudomonas Streptomyces Homos Rattus
thaliana fluorescens avermitilis apiens norvegicus
Amino Amino Amino Amino
Amino
Position Acid Position Acid Position Acid Position Acid Position Acid
226 H 162 H 187 H 183 H 183 H
267 S 202 S 230 S 226 S 226 S
282 N 217 N 245 N 241 N 241 N
308 H 241 H 270 H 266 H 266 H
342 Y 271 Y 299 Y 295 Y 295 Y
394 E 323 E 349 E 349 E 349 E
420 G 334 G 360 G 360 G 360 G
423 N 337 N 363 N 363 N 363 N

CA 02808152 2013-03-05
Table 5b: Amino acid position with the amino acids at this positions in the
known
crystal structures and the amino acids a these position within all HPPD
sequences
Arabidopsis Pseudomonas Streptomyces Homo Rattus
Natural
thaliana tluorescens ayennitilis sapiens nowegicus
Variabaility
Amino Amino Amino Amino Amino Amino
Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos, Acid Acid
228 V 164 T 189 V 185 V 185 V VTCAG
248 H 186 R 211 T 207 H 207 H HRQTKEYLAGSN
250 F 188 A 213 M 209 F 209 F FYILVAQEDGTSMRK
251 A 189 R 214 K 210 W 210 W AWILSRKHDEPGNY
252 E 190 Y 215 E 211 S 211 S ESTYFHQNGLMVILIR
253 F 191 F 216 F 212 V 212 V FVIALVVMQHY
265 L 200 L 22E3 L 224 L 224 L LMVIA
268 A 203 K 231 K 227 I 227 I AVLMIKRQY
269 V 204 A 232 V 228 V 228 V VAT
270 L 205 M 233 V 229 V 229 V LVMIA
271 A 206 S 234 A 230 A 230 A ASTVRKELIMHG
280 P 215 P 243 P 239 P 239 P PAVTNI
293 Q 226 Q 255 0 251 Q 251 Q QLAVRSGVFM
294 I 227 I 256 I 252 I 252 I IMVTASP
307 Q 240 Q 269 Q 265 Q 265 Q QHN
335 M 264 M 293 L 289 L 289 L MLIN
368 L 295 L 323 L 323 L 323 L LM
379 Q 310 0 334 Q 334 Q 334 0 QH
381 F 312 F 336 F 336 F 336 F FY
392 F 321 F 347 F 347 F 347 F FS
419 F 333 F 359 F 359 F 359 F FY
421 K 335 E 361 K 361 A 361 A KQAILVNDEGS
422 G 336 G 362 G 362 G 362 G GAPVTM
424 F 338 F 364 F 364 F 364 F FAVIL
425 S 339 K 365 K 365 N 365 N SNKGRAPSIKQR
426 E 340 A 366 A 366 S 366 S EQSAIVFT
427 L 341 L 367 L 367 L 367 L LR
431 I 345 i 371 I 371 F 371 F IFVMLQR

CA 02808152 2013-03-05
,
51
Table 5c: Amino acid position with the amino acids at this positions in the
known
crystal structures and most common amino acids a these position within all
HPPD
sequences
Arabidopsis Pseudomonas Streptomyces Homo Rattus Most
thaliana fluorescens avermitilis sapiens norvegicus common
Amino Amino Amino Amino Amino Amino
Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid Acid
228 V 164 7 189 V 185 V 185 V VT-CA
248 H 186 R 211 T 207 H 207 H HRQTKEYA
250 F 188 A 213 M 209 F 209 F FYILVAEDTM
251 A 189 R 214 K 210 W 210 W AWILRKHDEPY
252 E 190 Y 215 E 211 S 211 S ESTRY
253 F 191 F 216 F 212 V 212 V FVIALVVY
265 L 200 L 228 L 224 L 224 L LMVA
268 A 203 K 231 K 227 I 227 I AVLMIKRQY
269 V 204 A 232 V 228 V 228 V VA
270 L 205 M 233 V 229 V 229 V LVM1
271 A 206 S 234 A 230 A 230 A ASTVREGK
280 P 215 P 243 P 239 P 239 P PAVT
293 Q 226 0 255 Q 251 Q 251 0 0LA
294 I 227 I 256 I 252 I 252 I IMVA
307 0 240 0 269 0 265 Q 265 Q QHN
335 M 264 M 293 L 289 L 289 L MU
368 L 295 L 323 L 323 L 323 L LM
379 0 310 0 334 0 334 Q 334 Q QH
381 F 312 F 336 F 336 F 336 F FY
392 F 321 F 347 F 347 F 347 F F
419 F 333 F 359 F 359 F 359 F FY
421 K 335 E 361 K 361 A 361 A KQAIVLDE
422 G 336 G 362 G 362 G 362 G GA
424 F 338 F 364 F 364 F 364 F FAVL
425 S 339 K 365 K 365 N 365 N SNKRA1Q
426 E 340 A 366 A 366 S 366 S ESAIVF

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Arabidopsis Pseudomonas Streptomyces Homo Raftus Most
thaliana fluorescens avermitilis sapiens norvegicus common
Amino Amino Amino Amino Amino Amino
Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos, Acid Acid
427 L 341 L 367 L 367 L 367 L LR
431 I 345 I 371 I 371 F 371 F IFVMQRL
Not all amino acids occurring at the variable positions have the same
likelihood to be
present in an active protein. In some cases most sequences have the same amino

acids at a particular position while other amino acids are present at that
position in
only few HPPD sequences. An example is position 392. Most sequences have at a
corresponding position a phenylalanine while few i.e. the burkholdeda
sequences
have a serine. Whereas in some rare cases, the rare amino acids may be the
result
of a sequencing error, in most other cases the resulting protein is active.
Table 5c
shows a list with the most common amino acids at the variable positions.
The influence of the amino acids at the variable positions is different. Some
of these
positions are crucial for catalysis and/or the interaction of HPPD with an
inhibitor
while others may have less impact. For instances changes at positions 269 and
280
which are in direct contact with the inhibitor and the substrate will very
likely have a
great impact upon catalysis and inhibitor binding. Also modifications at
positions
involved in the helix movement induced by inhibitor or substrate binding such
as
positions 252, 421 and 422 are likely to have high impact on inhibitor
binding. In
contrast modifications at positions such as 293 are less likely to influence
inhibitor
binding because this position is quite distant from the active site. Table 6a
shows the
position with very high impact and Table 6b with high impact on activity and
inhibitor
binding.
From these observations, the present inventors drew the conclusion that amino
acids
are preferred in the binding sites which naturally occur at the corresponding
positions.
This means, that those modifications which exchange a naturally occurring
amino
acid into another are likely providing a catalytically active HPPD protein
which may
exert a modified or even increased tolerance to HPPD herbicide inhibitors.
Even

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53
more promising are those mutant proteins which have the amino acid most often
found in nature at the variable position chosen from Table 5c.
Table 6a: Amino acid positions with high priority shown for the x-ray
structures
Arabidopsis Pseudomonas Streptomyces Homo Rattus
thaliana tluorescens avermitilis sapiens
norvegicus
Amino Amino Amino Amino Amino
Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid
228 V 164 T 189 V 185 V 185 V
250 F 188 A 213 M 209 F 209 F
251 A 189 R 214 K 210 W 210 W
252 E 190 Y 215 E 211 S 211 S
253 F 191 F 216 F 212 V 212 V
265 L 200 L 228 L 224 L 224 L
268 A 203 K 231 K 227 I 227 I
269 V 204 A 232 V 228 V 228 V
270 L 205 M 233 V 229 V 229 V
271 A 206 S 234 A 230 A 230 A
280 P 215 P 243 P 239 P 239 P
307 Q 240 Q 269 Q 265 Q 265 Q
335 M 264 M 293 L 289 L 289 L
368 L 295 L 323 L 323 L 323 L
379 Q 310 Q 334 Q 334 Q 334 Q
392 F 321 F 347 F 347 F 347 F
421 K 335 E 361 K 361 A 361 A
422 G 336 G 362 G 362 G 362 G
426 E 340 A 366 A 366 S 366 S .
427 L 341 L 367 L 367 L 367 L

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54
Table 6b: Amino acid positions with very high priority shown for the x-ray
structures
Arabidopsis Pseudomonas Streptomyces Homo Rattus
Ma'am fluorescens avermitilis sapiens
norvegicus
Amino Amino Amino Amino Amino
Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid
252 E 190 Y 215 E 211 S 211 S
269 V 204 A 232 V 228 V 228 V
280 P 215 P 243 P 239 P 239 P
335 M 264 M 293 L 289 L 289 L
368 L 295 L 323 L 323 L 323 L
421 K 335 E 361 K 361 A 361 A
422 G 336 G 362 G 362 G 362 G
In another embodiment, in the isolated nucleic acid of the invention as
defined above,
said at least on amino acid is selected from
a) Ala, Asp, Glu, Phe, Thr, Val or Tyr at a position in an HPPD protein,
said
position corresponding to position 250 of the amino acid sequence of SEQ ID
No. 2;
b) Ala, Asp, Glu, Ile, Lys, Leu, Pro or Arg at a position in an HPPD
protein, said
position corresponding to position 251 of the amino acid sequence of SEQ ID
No. 2;
c) Glu, Thr, Arg, Ser or Tyr at a position in an HPPD protein, said
position
corresponding to position 252 of the amino acid sequence of SEQ ID No. 2;
d) Ala, Phe, Val or Trp at a position in an HPPD protein, said position
corresponding to position 253 of the amino acid sequence of SEQ ID No. 2;
e) Leu or Val at a position in an HPPD protein, said position corresponding
to
position 265 of the amino acid sequence of SEQ ID No. 2;
f) Leu, Gin, Arg, Val or Tyr at a position in an HPPD protein, said
position
corresponding to position 268 of the amino acid sequence of SEQ ID NO: 2;
g) Ala or Val at a position in an HPPD protein, said position corresponding to
position 269 of the amino acid sequence of SEQ ID No. 2;

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h) Ala, Pro, Thr or Val at a position in an HPPD protein, said position
corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i) Gin at a position in an HPPD protein, said position corresponding to
position
307 of the amino acid sequence of SEQ ID No. 2;
5 j) Ile or Met at a position in an HPPD protein, said position
corresponding to
position 335 of the amino acid sequence of SEQ ID No. 2;
k) Leu at a position in an HPPD protein, said position corresponding to
position
368 of the amino acid sequence of SEQ ID No. 2;
I) Phe at a position in an HPPD protein, said position corresponding to
position
10 392 of the amino acid sequence of SEQ ID No. 2;
m) Asp, Lys, or Asn at a position in an HPPD protein, said position
corresponding
to position 421 of the amino acid sequence of SEQ ID No. 2;
n) Ala or Gly at a position in an HPPD protein, said position corresponding
to
position 422 of the amino acid sequence of SEQ ID No. 2;
15 o) Ile, Arg or Ser at a position in an HPPD protein, said position
corresponding to
position 425 of the amino acid sequence of SEQ ID No. 2
p) Glu, Phe or Val at a position in an HPPD protein, said position
corresponding to
position 426 of the amino acid sequence of SEQ ID No. 2;
q) Ile, Met, Gin or Val or Phe at a position in an HPPD protein, said
position
20 corresponding to position 431 of the amino acid sequence of SEQ ID No.
2;
r) Ala, Cys, Thr or Val at a position in an HPPD protein, said position
corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
s) Ala, Glu, His, Lys, Gln, Arg, Thr or Tyr at a position in an HPPD
protein, said
position corresponding to position 248 of the amino acid sequence of SEQ ID
25 No. 2;
t) Ile, Leu, Met or Val at a position in an HPPD protein, said position
corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
u) Ala, Glu, Lys, Arg, Ser, Thr or Val at a position in an HPPD protein,
said
position corresponding to position 271 of the amino acid sequence of SEQ ID
30 No. 2;
v) His or Gin at a position in an HPPD protein, said position corresponding
to
position 379 of the amino acid sequence of SEQ ID No. 2;

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w) Leu or Arg at a position in an HPPD protein, said position
corresponding to
position 427 of the amino acid sequence of SEQ ID No. 2.
In an alternative embodiment of the nucleic acid of the invention, said at
least one
.. amino acid is selected from
a) Glu, Thr, Arg, Ser or Tyr at a position in an HPPD protein, said
position
corresponding to position 252 of the amino acid sequence of SEQ ID No. 2;
b) Ala or Val at a position in an HPPD protein, said position corresponding
to
position 269 of the amino acid sequence of SEQ ID No. 2;
c) Ala, Pro, Thr or Val at a position in an HPPD protein, said position
corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
d) Ile or Met at a position in an HPPD protein, said position corresponding
to
position 335 of the amino acid sequence of SEQ ID No. 2;
e) Leu at a position in an HPPD protein, said position corresponding to
position
368 of the amino acid sequence of SEQ ID No. 2;
f) Aspõ Asn or Lys at a position in an HPPD protein, said position
corresponding
to position 421 of the amino acid sequence of SEQ ID No. 2;
g) Ala or Gly at a position in an HPPD protein, said position corresponding
to
position 422 of the amino acid sequence of SEQ ID No. 2;
h) Ala, Cys, Thr or Val at a position in an HPPD protein, said position
corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
i) Ala, Glu, His, Lys, Gin, Arg, Thr or Tyr at a position in an HPPD
protein, said
position corresponding to position 248 of the amino acid sequence of SEQ ID
No. 2;
j) Ile, Leu, Met or Val at a position in an HPPD protein, said position
corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
k) Ala, Glu, Lys, Arg, Ser, Thr or Val at a position in an HPPD protein,
said
position corresponding to position 271 of the amino acid sequence of SEQ ID
No. 2;
I) His or Gin at a position in an HPPD protein, said position corresponding
to
position 379 of the amino acid sequence of SEQ ID No. 2;

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57
m) Leu or Arg at a position in an HPPD protein, said position
corresponding to
position 427 of the amino acid sequence of SEQ ID No. 2.
Various sequences of HPPD proteins or predicted HPPD proteins are known in the
art. These include the HPPD sequences of Streptomyces avermitilis
(Genebank SAV11864), Daucus carota (Genebank DCU 87257), Arabidopsis
thaliana (Genebank AF047834), Mycosphaerella graminicola (Genebank AF038152),
oryza sativa / rice [8A026248], Zea mays / corn [ACN36372], avena sativa
[ABZ23427], Pseudomonas fluorescens [ABF50055], Synechococcus sp.
[YP_473959], Blepharisma japonicum [BAF91881], Rhodococcus RHA1 sp. ro0240
FP_7020051, Rhodococcus RHA1 sp. ro0341 [Y13_703002], Picrophilus torridus
[YP_0241471, Kordia algicida [ZP_02161490], Sorghum bicolor [X13_0024533591,
Triticum aestivum / wheat [AAZ67144], or Hordeum vulgare / barley [048604].
The sequence of the HPPD protein taken as a starting point may be any amino
acid
sequence encoding a catalytically active HPPD protein. In one embodiment of
the
isolated nucleic acid of the invention, said HPPD protein comprises the amino
acid
sequence of SEQ ID No. 4 [Oryza sativa], wherein the resulting amino acid
sequence comprises at least one amino acid selected from
a) Ala, Asp, Glu, Gly, Lys, Gln, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at
position
247 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 250
of SEQ ID No. 2);
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position
248 of the
amino acid sequence of SEQ ID No. 4 (corresponding to position 251 of SEQ ID
No. 2);
Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position
249 of
the amino acid sequence of SEQ ID No. 4 (corresponding to position 252 of SEQ
ID No. 2) ;
d) Ala, His, Gin, Val, Trp, lie, Leu or Met at position 250 of the amino acid
sequence
of SEQ ID No. 4 (corresponding to position 253 of SEQ ID No. 2);

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e) Val or Met at position 262 of the amino acid sequence of SEQ ID No. 4
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 265 of the amino acid
sequence
of SEQ ID No. 4 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 266 of the amino acid sequence of SEQ ID No. 4
(corresponding to position 269 of SEQ ID No. 2);
h) Ala, Ile, Asn, Thr or Val at position 277 of the amino acid sequence of SEQ
ID No.
4 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 290 of the amino
acid
sequence of SEQ ID No. 4 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 291 of the amino acid
sequence of SEQ
ID No. 4 (corresponding to position 294 of SEQ ID No. 2);
k) Ile, Met or Asn at position 332 of the amino acid sequence of SEQ ID No. 4
(corresponding to position 335 of SEQ ID No. 2);
l) Met at position 365 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 368 of SEQ ID No. 2);
m) Tyr at position 378 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 381 of SEQ ID No. 2);
n) Ser at position 389 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 392 of SEQ ID No. 2);
o) Tyr at position 416 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 419 of SEQ ID No. 2);
p) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val
at
position 418 of the amino acid sequence of SEQ ID No. 4 (corresponding to
position 421 of SEQ ID No. 2);
q) Ala, Met, Pro or Thr at position 419 of the amino acid sequence of SEQ ID
No. 4
(corresponding to position 422 of SEQ ID No. 2);
r) Ala, Ile or Val at position 421 of the amino acid sequence of SEQ ID No. 4
(corresponding to position 424 of SEQ ID No. 2);
s) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 422 of the amino acid
sequence
of SEQ ID No. 4 (corresponding to position 425 of SEQ ID No. 2);

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t) Phe, Thr, Val, Ala, Gin or Ser at position 423 of the amino acid
sequence of SEQ
ID No. 4 (corresponding to position 426 of SEQ ID No. 2);
u) Met, Gin, Val, Phe or Leu at position 428 of the amino acid sequence of SEQ
ID
No. 4 (corresponding to position 431 of SEQ ID No. 2);
v) Phe at position 431 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 434 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 225 of the amino acid sequence of SEQ ID
No. 4
(corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 245 of
the amino
acid sequence of SEQ ID No. 4 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Met or Val at position 267 of the amino acid sequence of SEQ ID
No. 4
(corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 268 of the
amino acid
sequence of SEQ ID No. 4 (corresponding to position 271 of SEQ ID No. 2);
aa)His at position 376 of the amino acid sequence of SEQ ID No. 4
(corresponding
to position 379 of SEQ ID No. 2);
bb)Arg at a position in an HPPD protein, said position corresponding to
position 424
of the amino acid sequence of SEQ ID No. 4 (corresponding to position 427 of
SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 6 [Zea mays], wherein
the resulting amino acid sequence comprises at least one amino acid selected
from
a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met
at position
224 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 250
of SEQ ID No. 2);
b) Asp, Glu, lie, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position
225 of the
amino acid sequence of SEQ ID No. 6 (corresponding to position 251 of SEQ
ID No. 2);
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at
position 226 of
the amino acid sequence of SEQ ID No. 6 (corresponding to position 252 of
SEQ ID No. 2);

CA 02808152 2013-03-05
,
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 227 of the amino
acid
sequence of SEQ ID No. 6 (corresponding to position 253 of SEQ ID No. 2);
e) Val or Met at position 239 of the amino acid sequence of SEQ ID No. 6
(corresponding to position 265 of SEQ ID No. 2);
5 f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 242 of the
amino acid
sequence of SEQ ID No. 6 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 243 of the amino acid sequence of SEQ ID No. 6
(corresponding to position 269 of SEQ ID No. 2);
h) Ala, Ile, Asn, Thr or Val at position 254 of the amino acid sequence of
SEQ ID
10 No. 6 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 267 of the amino
acid
sequence of SEQ ID No. 6 (corresponding to position 293 of SEQ ID No. 2);
j) Met at position 268 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 294 of SEQ ID No. 2);
15 k) His or Asn at position 281 of the amino acid sequence of SEQ ID No. 6
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 309 of the amino acid sequence of SEQ ID
No. 6
(corresponding to position 335 of SEQ ID No. 2);
m) Met at position 342 of the amino acid sequence of SEQ ID No. 6
(corresponding
20 to position 368 of SEQ ID No. 2);
n) Tyr at position 355 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 381 of SEQ ID No. 2);
o) Ser at position 366 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 392 of SEQ ID No. 2);
25 p) Tyr at position 393 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr
or Val at
position 395 of the amino acid sequence of SEQ ID No. 6 (corresponding to
position 421 of SEQ ID No. 2);
30 r) Ala, Met, Pro or Thr at position 396 of the amino acid sequence of
SEQ ID No.
6 (corresponding to position 422 of SEQ ID No. 2);

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61
s) Ala, Ile or Val at position 398 of the amino acid sequence of SEQ ID No.
6
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 399 of the amino
acid
sequence of SEQ ID No. 6 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Ala or Ser at position 400 of the amino acid sequence
of
SEQ ID No. 6 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Val, Phe or Leu at position 405 of the amino acid sequence of
SEQ ID
No. 6 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position
corresponding to position 202 of the amino acid sequence of SEQ ID No. 6
(corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 222
of the
amino acid sequence of SEQ ID No. 6 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 244 of the amino acid sequence of SEQ ID
No. 6
(corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 245 of
the amino
acid sequence of SEQ ID No. 6 (corresponding to position 271 of SEQ ID No.
2);
aa) Gin at position 353 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 379 of SEQ ID No. 2);
ab) Arg at position 401 of the amino acid sequence of SEQ ID No. 6
(corresponding
to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 8 [Avena sativa],
wherein
the resulting amino acid sequence comprises at least one amino acid selected
from
a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or
Met at position
241 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 250
of SEQ ID No. 2), preferably Ala;

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62
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position
242 of the
amino acid sequence of SEQ ID No. 8 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at
position 243 of
the amino acid sequence of SEQ ID No. 8 (corresponding to position 252 of
SEQ ID No. 2), preferably Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 244 of the amino
acid
sequence of SEQ ID No. 8 (corresponding to position 253 of SEQ ID No. 2),
preferably Val;
e) Val or Met at position 256 of the amino acid sequence of SEQ ID No. 8
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 259 of the amino
acid
sequence of SEQ ID No. 8 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 260 of the amino acid sequence of SEQ ID No. 8
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 271 of the amino acid sequence of
SEQ ID
No. 8 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 284 of the amino
acid
sequence of SEQ ID No. 8 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 285 of the amino acid
sequence of
SEQ ID No. 8 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 265 of the amino acid sequence of SEQ ID
No. 8
(corresponding to position 307 of SEQ ID No. 2);
I) Ile or Asn or Leu at position 326 of the amino acid sequence of
SEQ ID No. 8
(corresponding to position 335 of SEQ ID No. 2);
m) Met at position 359 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 368 of SEQ ID No. 2);
n) Tyr at position 372 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 381 of SEQ ID No. 2);
o) Ser at position 383 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 392 of SEQ ID No. 2);

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63
p) Tyr at position 410 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or
Val at
position 412 of the amino acid sequence of SEC) ID No. 8 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 413 of the amino acid sequence of SEQ
ID No.
8 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 415 of the amino acid sequence of SEQ ID No.
8
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 416 of the amino
acid
sequence of SEQ ID No. 8 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr, Val, Ala, Gin or Ser at position 417 of the amino acid
sequence of
SEQ ID No. 8 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Val, Phe or Leu at position 422 of the amino acid sequence of
SEQ ID
No. 8 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 219 of the amino acid sequence of SEQ
ID No.
8 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leta, Asn, Gin, Arg, Ser, Thr or Tyr at position 239
of the
amino acid sequence of SEQ ID No. 8 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 261 of the amino acid sequence of SEQ
ID No. 8
(corresponding to position 270 of SEQ ID No, 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Vat at position 262 of
the amino
acid sequence of SEQ ID No. 8 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 370 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 379 of SEQ ID No. 2);
bb) Arg at position 418 of the amino acid sequence of SEQ ID No. 8
(corresponding
to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said
HPPD
protein comprises the amino acid sequence of SEQ ID No. 10 [Pseudomonas

CA 02808152 2013-03-05
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64
fluorescens], wherein the resulting amino acid sequence comprises at least one

amino acid selected from
a) Phe, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met
at position
188 of the amino acid sequence of SEQ ID No. 10 (corresponding to position
250 of SEQ ID No. 2), preferably Ala;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Ser, Gly, His or Tyr at position
189 of the
amino acid sequence of SEQ ID No. 10 (corresponding to position 251 of SEQ
ID No. 2), preferably Ala or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Glu or Thr at
position 190
of the amino acid sequence of SEQ ID No. 10 (corresponding to position 252 of
SEQ ID No. 2), preferably Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 191 of the amino
acid
sequence of SEQ ID No. 10 (corresponding to position 253 of SEQ ID No. 2),
preferably Phe or Val;
e) Val or Met at position 200 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val Tyr, Ala, Ile or Met at position 203 of the amino
acid
sequence of SEQ ID No. 10 (corresponding to position 268 of SEQ ID No. 2);
g) Thr or Val at position 204 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 269 of SEQ ID No. 2), preferably Val;
h) Ala, Ile, Asn, Thr or Val at position 215 of the amino acid sequence of
SEQ ID
No. 10 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 226 of the amino
acid
sequence of SEQ ID NO:SEQ ID No. 10 (corresponding to position 293 of SEQ
ID No. 2);
j) Met, Thr, Ala, Pro, Ser or Val at position 227 of the amino acid
sequence of
SEQ ID No. 10 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 240 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 264 of the amino acid sequence of SEQ ID No.
10
(corresponding to position 335 of SEQ ID No. 2);

CA 02808152 2013-03-05
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m) Met at position 295 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 312 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 381 of SEQ ID No. 2);
5 o) Ser at position 321 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 333 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Lys, Asn, Ala, Cys, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val
at position
10 335 of the amino acid sequence of SEQ ID No. 10 (corresponding to
position
421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 336 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 338 of the amino acid sequence of SEQ ID No.
10
15 (corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Ala, Asn, Gly or Gin at position 339 of the amino acid
sequence of SEQ
ID No. 10 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr or Val at position 340 of the amino acid sequence of SEQ ID No.
10
(corresponding to position 426 of SEQ ID No. 2);
20 v) Met, Phe, Leu, Gin or Val at position 345 of the amino acid sequence
of SEQ ID
No. 10 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Val at position 164 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Ser, Thr or Tyr at position 186
of the
25 amino acid sequence of SEQ ID No. 10 (corresponding to position 248 of
SEQ
ID No. 2);
y) Ala, Ile, Leu or Val at position 205 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at position 206 of
the amino
30 acid sequence of SEQ ID No. 10 (corresponding to position 271 of SEQ ID
No.
2);

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66
aa) His at position 310 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 341 of the amino acid sequence of SEQ ID No. 10
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 14 [Synechococcus
sp.],
wherein the resulting amino acid sequence comprises at least one amino acid
selected from
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Met at
position
169 of the amino acid sequence of SEQ ID No. 14 (corresponding to position
250 of SEQ ID No. 2), preferably Ala or Phe;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg or Ser, Gly or His at
position 170 of
the amino acid sequence of SEQ ID No. 14 (corresponding to position 251 of
SEQ ID No. 2), preferably Ala, Arg or Lys;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Ser, Val, Tyr or Thr at
position 171 of
the amino acid sequence of SEQ ID No. 14 (corresponding to position 252 of
SEQ ID No. 2), preferably Glu, Ser or Tyr;
d) Ala, Phe, His, Gin, Val or Ile, Leu or Met at position 172 of the amino
acid
sequence of SEQ ID No, 14 (corresponding to position 253 of SEQ ID No. 2),
preferably Phe or Val;
e) Val or Met at position 181 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 184 of the amino
acid
sequence of SEQ ID No. 14 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 185 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ile, Asn, Pro, Thr or Val at position 196 of the amino acid sequence of
SEQ ID
No. 14 (corresponding to position 280 of SEQ ID No. 2), preferably Pro;
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 206 of the amino
acid
sequence of SEQ ID No. 14 (corresponding to position 293 of SEQ ID No. 2);

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j) Met , Ala, Pro, Ser, Thr or Val at position 207 of the amino acid
sequence of
SEQ ID No. 14 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 220 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 244 of the amino acid sequence of SEQ ID No.
14
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 286 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 368 of SEQ ID No. 2);
n) Phe or Tyr at position 301 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 312 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 325 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val
at
position 327 of the amino acid sequence of SEQ ID No. 14 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Gly, Met, Pro or Thr at position 328 of the amino acid sequence of
SEQ ID No.
14 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 330 of the amino acid sequence of SEQ ID No. 14
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Lys or Asn at position 331 of the amino
acid
sequence of SEQ ID No. 14 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gln or Ser at position 332 of the amino acid
sequence of
SEQ ID No. 14 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Ile, Met, Gln, Val or Phe at position 337 of the amino acid sequence of
SEQ ID
No. 14 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 145 of the amino acid sequence of SEQ
ID No.
14 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gln, Ser, Thr or Tyr at position 167 of
the
amino acid sequence of SEQ ID No. 14 (corresponding to position 248 of SEQ
ID No. 2);

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y) Ala, Ile, Leu or Met at position 186 of the amino acid sequence of SEQ
ID No.
14 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 187
of the
amino acid sequence of SEQ ID No. 14 (corresponding to position 271 of SEQ
ID No. 2);
aa) His at a position at position 299 of the amino acid sequence of SEQ ID No.
14
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at a position at position 333 of the amino acid sequence of SEQ ID No.
14
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 16 [Blepharisma
japonicum], wherein the resulting amino acid sequence comprises at least one
amino acid selected from
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Ile, Leu or Met at
position
209 of the amino acid sequence of SEQ ID No. 16 (corresponding to position
250 of SEQ ID No. 2), preferably Ala or Phe;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at
position 210 of
the amino acid sequence of SEQ ID No. 16 (corresponding to position 251 of
SEQ ID No. 2), preferably Ala, Arg, Lys;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr at
position 211 of
the amino acid sequence of SEQ ID No. 16 (corresponding to position 252 of
SEQ ID No. 2), preferably Glu or Tyr;
d) Phe, His, Gin, Val, Trp, Ile, Leu or Met at position 212 of the amino
acid
sequence of SEQ ID No. 16 (corresponding to position 253 of SEQ ID No. 2),
preferably Phe or Val;
e) Val or Met at position 224 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 227 of the amino
acid
sequence of SEQ ID No. 16 (corresponding to position 2268 of SEQ ID No. 2);
g) Ala or Thr at position 228 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;

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69
h) Ala, Ile, Asn, Thr or Val at position 239 of the amino acid sequence of
SEQ ID
No. 16 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 251 of the amino
acid
sequence of SEQ ID No. 16 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 252 of the amino acid
sequence of
SEQ ID No. 16 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 265 of the amino acid sequence of SEQ ID No.
16
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 289 of the amino acid sequence of SEQ ID
No. 16
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 323 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 336 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 347 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 292 of SEQ ID No. 2);
p) Tyr at position 359 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or
Val at
position 361 of the amino acid sequence of SEQ ID No. 16 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 362 of the amino acid sequence of SEQ
ID No.
16 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 364 of the amino acid sequence of SEQ ID No.
16
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 365 of the amino
acid
sequence of SEQ ID No. 16 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gln or Ser at position 366 of the amino acid
sequence of
SEQ ID No. 16 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Ile, Met, Gln, Val or Phe at position 371 of the amino acid sequence of SEQ
ID
No. 16 (corresponding to position 431 of SEQ ID No. 2);

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w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position
corresponding to position 185 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at a position in
an HPPD
5 protein, said position corresponding to position 207 of the amino acid
sequence
of SEQ ID No. 16 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position
corresponding to position 229 of the amino acid sequence of SEQ ID No. 16
(corresponding to position 270 of SEQ ID No. 2);
10 z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at a position in
an HPPD
protein, said position corresponding to position 230 of the amino acid
sequence
of SEQ ID No. 16 (corresponding to position 271 of SEQ ID No. 2);
aa) His at a position in an HPPD protein, said position corresponding to
position
334 of the amino acid sequence of SEQ ID No, 16 (corresponding to position
15 379 of SEQ ID No. 2);
bb) Arg at a position in an HPPD protein, said position corresponding to
position
367 of the amino acid sequence of SEQ ID No, 16 (corresponding to position
427 of SEQ ID No. 2).
20 In another embodiment, of the isolated nucleic acid of the invention,
said HPPD
protein comprises the amino acid sequence of SEQ ID No. 18 [Rhodococcus RHA1
sp. ro0240], wherein the resulting amino acid sequence comprises at least one
amino acid selected from
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Leu
at position
25 231 of the amino acid sequence of SEQ ID No, 18 (corresponding to
position
250 of SEQ ID No. 2), preferably Phe or Ala;
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position
232 of the
amino acid sequence of SEQ ID No. 18 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
30 c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at
position 233 of
the amino acid sequence of SEQ ID No. 18 (corresponding to position 252 of
SEQ ID No. 2), preferably Ser or Tyr;

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71
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 234 of the amino
acid
sequence of SEQ ID No. 18 (corresponding to position 253 of SEQ ID No. 2),
preferably Val;
e) Val or Met at position 246 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 249 of the amino
acid
sequence of SEQ ID No. 18 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 250 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 261 of the amino acid sequence of SEQ
ID
No. 18 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 273 of the amino
acid
sequence of SEQ ID No. 18 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 274 of the amino acid
sequence of
SEQ ID No. 18 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 287 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 311 of the amino acid sequence of SEQ ID
No. 18
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 343 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 356 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 381 of SEQ ID No. 2);
a) Ser at position 367 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 379 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or Val
at position
381 of the amino acid sequence of SEQ ID No. 18 (corresponding to position
421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 382 of the amino acid sequence of SEQ
ID No.
18 (corresponding to position 422 of SEQ ID No. 2);

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s) Ala, Ile or Val at position 384 of the amino acid sequence of SEQ ID No,
18
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 385 of the amino
acid
sequence of SEQ ID No. 18 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 386 of the amino acid sequence
of
SEQ ID No. 18 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 391 of the amino acid
sequence of
SEQ ID No. 18 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position
corresponding to position 207 of the amino acid sequence of SEQ ID No. 1
(corresponding to position 228 of SEQ ID No. 2)8;
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser or Tyr at a position in
an HPPD
protein, said position corresponding to position 229 of the amino acid
sequence
of SEQ ID No. 18 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position
corresponding to position 251 of the amino acid sequence of SEQ ID No. 18
(corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at a position in
an HPPD protein,
said position corresponding to position 252 of the amino acid sequence of SEQ
ID No. 18 (corresponding to position 271 of SEQ ID No. 2);
aa) His at a position in an HPPD protein, said position corresponding to
position
354 of the amino acid sequence of SEQ ID No. 18 (corresponding to position
379 of SEQ ID No. 2);
bb) Arg at a position in an HPPD protein, said position corresponding to
position
387 of the amino acid sequence of SEQ ID No. 18 (corresponding to position
427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said
HPPD
protein comprises the amino acid sequence of SEQ ID No. 30 [Rhodococcus RHA1
sp. 0341], wherein the resulting amino acid sequence comprises at least one
amino
acid selected from

CA 02808152 2013-03-05
73
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Leu at
position
232 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 250
of SEQ ID No. 2), preferably Phe or Ala;
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position
233 of the
amino acid sequence of SEQ ID No. 30 (corresponding to position 251 of SEQ ID
No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at
position 234 of
the amino acid sequence of SEQ ID No. 30 (corresponding to position 252 of
SEC) ID No. 2), preferably Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 235 of the amino acid
sequence
of SEQ ID No. 30 (corresponding to position 253 of SEQ ID No. 2), preferably
Val;
e) Val or Met at position 247 of the amino acid sequence of SEQ ID No. 30
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 250 of the amino acid
sequence
of SEQ ID No. 30 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 251 of the amino acid sequence of SEQ ID No. 30
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 262 of the amino acid sequence of SEQ
ID No.
30 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 274 of the amino acid
sequence of SEQ ID No. 30 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 275 of the amino acid
sequence of SEQ
ID No. 30 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 288 of the amino acid sequence of SEQ ID No. 30
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 312 of the amino acid sequence of SEQ ID
No. 30
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 344 of the amino acid sequence of SEQ ID No. 30
(corresponding
to position 368 of SEQ ID No. 2);
n) Tyr at position 357 of the amino acid sequence of SEQ ID No. 30
(corresponding
to position 381 of SEQ ID No. 2);

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0) Ser at position 368 of the amino acid sequence of SEQ ID No. 30
(corresponding
to position 392 of SEQ ID No. 2);
p) Tyr at position 380 of the amino acid sequence of SEQ ID No. 30
(corresponding
to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or Val
at
position 382 of the amino acid sequence of SEQ ID No. 30 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 383 of the amino acid sequence of SEQ ID
No. 30
(corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 385 of the amino acid sequence of SEQ ID No. 30
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 386 of the amino acid
sequence
of SEQ ID No. 30 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 387 of the amino acid sequence
of SEQ
ID No. 30 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 392 of the amino acid sequence
of
SEQ ID No. 30 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position
corresponding
to position 208 of the amino acid sequence of SEQ ID No. 30 (corresponding to
position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser or Tyr at a position in an
HPPD
protein, said position corresponding to position 230 of the amino acid
sequence
of SEQ ID No. 30 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position
corresponding
to position 252 of the amino acid sequence of SEQ ID No. 30 (corresponding to
position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at a position in an HPPD
protein, said
position corresponding to position 253 of the amino acid sequence of SEQ ID
No.
(corresponding to position 271 of SEQ ID No. 2);
30 aa)His at a position in an HPPD protein, said position corresponding to
position 355
of the amino acid sequence of SEQ ID No. 30 (corresponding to position 379 of
SEQ ID No. 2);

CA 02808152 2013-03-05
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,
bb)Arg at a position in an HPPD protein, said position corresponding to
position 388
of the amino acid sequence of SEQ ID No. 30 (corresponding to position 427 of
SEQ ID No. 2).
5 In another embodiment, of the isolated nucleic acid of the invention,
said HPPD
protein comprises the amino acid sequence of SEQ ID No. 20 [Picrophilus
torridus],
wherein the resulting amino acid sequence comprises at least one amino acid
selected from
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Met
at position
10 201 of the amino acid sequence of SEQ ID No. 20 (corresponding to
position
250 of SEQ ID No. 2), preferably Phe or Ala;
b) Ala, Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at
position 202 of
the amino acid sequence of SEQ ID No. 20 (corresponding to position 251 of
SEQ ID No. 2), preferably Ala, Arg or Lys;
15 c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val or Tyr
at position 203
of the amino acid sequence of SEQ ID No. 20 (corresponding to position 252 of
SEQ ID No. 2), preferably Glu, Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 204 of the amino
acid
sequence of SEQ ID No. 20 (corresponding to position 253 of SEQ ID No. 2),
20 preferably Val;
e) Val or Met at position 216 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 219 of the amino
acid
sequence of SEQ ID No. 20 (corresponding to position 268 of SEQ ID No. 2);
25 g) Ala or Thr at position 220 of the amino acid sequence of SEQ ID
No. 20
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 230 of the amino acid sequence of
SEQ ID
No. 20 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 242 of the amino
acid
30 sequence of SEQ ID No. 20 (corresponding to position 293 of SEQ
ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 243 of the amino acid
sequence of
SEQ ID No. 20 (corresponding to position 294 of SEQ ID No. 2);

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76
k) His or Asn at position 256 of the amino acid sequence of SEQ ID
No. 20
(corresponding to position 307 of SEQ ID No. 2);
I) lie, Met or Asn at position 280 of the amino acid sequence of SEQ
ID No. 20
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 310 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 368 of SEQ ID No. 2);
n) Phe or Tyr at position 323 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 334 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 346 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or
Val at
position 348 of the amino acid sequence of SEQ ID No. 20 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 349 of the amino acid sequence of SEQ
ID No.
(corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 351 of the amino acid sequence of SEQ ID No.
20
(corresponding to position 424 of SEQ ID No. 2);
20 t) Ile, Pro, Arg, Ser, Ala, Gly, Lys or Gin at position 352 of the
amino acid
sequence of SEQ ID No. 20 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Ala or Gin at position 353 of the amino acid
sequence of
SEQ ID No. 20 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 358 of the amino acid
sequence of
SEQ ID No. 20 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 177 of the amino acid sequence of SEQ
ID No.
20 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position
199 of the
amino acid sequence of SEQ ID No. 20 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Leu or Met at a position at position 221 of the amino acid
sequence of
SEQ ID No. 20 (corresponding to position 270 of SEQ ID No. 2);

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z) Ala, Glu, His, Ile, Leu, Met, Arg, Ser, Thr or Val at position
222 of the amino
acid sequence of SEQ ID No. 20 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 321 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 354 of the amino acid sequence of SEQ ID No. 20
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said
HPPD
protein comprises the amino acid sequence of SEQ ID No. 22 [Kordia algicidal
wherein the resulting amino acid sequence comprises at least one amino acid
selected from
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Leu or Met
at position
219 of the amino acid sequence of SEQ ID No. 22 (corresponding to position
250 of SEQ ID No. 2), preferably Phe or Ala;
b) Ala, Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at
position 220 of
the amino acid sequence of SEQ ID No. 22 (corresponding to position 251 of
SEQ ID No. 2), preferably Ala, Arg or Lys;
C) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr
at position 221 of
the amino acid sequence of SEQ ID No, 22 (corresponding to position 252 of
SEQ ID No. 2), preferably Glu or Tyr;
d) Ala, His, Gin, Val Trp, Ile, Leu or Met at position 222 of the
amino acid
sequence of SEQ ID No. 22 (corresponding to position 253 of SEQ ID No. 2),
preferably Val;
e) Val or Met at position 234 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 237 of the amino
acid
sequence of SEQ ID No. 22 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 238 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 249 of the amino acid sequence of
SEQ ID
No. 22 (corresponding to position 280 of SEQ ID No. 2);

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i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 261 of the amino
acid
sequence of SEQ ID No. 22 (corresponding to position 293 of SEQ ID Na. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 262 of the amino acid
sequence of
SEQ ID No. 22 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 275 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 299 of the amino acid sequence of SEQ
ID No. 22
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 329 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 342 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 353 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 365 of the amino acid sequence of SEQ ID No. 22
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin,
Ser or Thr at
position 367 of the amino acid sequence of SEQ ID No. 22 (corresponding to
position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 368 of the amino acid sequence of SEQ ID
No.
22 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 370 of the amino acid sequence of SEQ ID No.
22
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gln at position 371 of the amino
acid
sequence of SEQ ID No. 22 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 372 of the amino acid
sequence of
SEQ ID No. 22 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 377 of the amino acid
sequence of
SEQ ID No. 22 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 195 of the amino acid sequence of SEQ ID
No.
22 (corresponding to position 228 of SEQ ID No. 2);

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x) Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 217
of the
amino acid sequence of SEQ ID No. 22 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Leu or Val at position 239 of the amino acid sequence of SEQ
ID No.
22 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at position 240 of
the amino
acid sequence of SEQ ID No. 22 (corresponding to position 271 of SEQ ID No.
2);
aa) His at a position at position 340 of the amino acid sequence of SEQ ID No.
22
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at a position at position 373 of the amino acid sequence of SEQ ID No.
22
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said
HPPD
protein comprises the amino acid sequence of SEQ ID No. 24 [Sorghum bicolor],
wherein the resulting amino acid sequence comprises at least one amino acid
selected from
a) Ala, Asp, Glu, Gly, Lys,G1n, Arg, Ser, Thr, Val Tyr, Ile, Leu or Met at
position
241 of the amino acid sequence of SEQ ID No. 24 (corresponding to position
250 of SEQ ID No. 2), preferably Ala;
b) Asp, Glu, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Trp at position 242
of the
amino acid sequence of SEQ ID No. 24 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
C) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr at
position 243 of the
amino acid sequence of SEQ ID No. 24 (corresponding to position 252 of SEQ
ID No. 2), preferably Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 244 of the amino
acid
sequence of SEQ ID No. 24 (corresponding to position 253 of SEQ ID No. 2),
preferably Val;
e) Val or Met at position 256 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 265 of SEQ ID No. 2);

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f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 259 of the amino
acid
sequence of SEQ ID No. 24 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 260 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
5 h) Ala, Ile, Asn, Thr or Val at position 271 of the amino acid sequence
of SEQ ID
No. 24 (corresponding to position 280 of SEQ ID No. 2);
i) Leu or Val, Ala, Phe, Gly, Met, Arg or Ser at position 284 of the amino
acid
sequence of SEQ ID No. 24 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 285 of the amino acid
sequence of
10 SEQ ID No. 24 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 298 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 326 of the amino acid sequence of SEQ
ID No. 24
(corresponding to position 335 of SEQ ID No, 2);
15 m) Met at position 359 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 372 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 383 of the amino acid sequence of SEQ ID No. 24
20 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 410 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or
Val at
position 412 of the amino acid sequence of SEQ ID No. 24 (corresponding to
25 position 421 of SEQ ID No. 2);
r) Ala, Met, Pro or Thr at position 413 of the amino acid sequence of SEQ
ID No.
24 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 415 of the amino acid sequence of SEQ ID No.
24
(corresponding to position 424 of SEQ ID No. 2);
30 t) Ile, Pro, Arg, Als, Gly, Lys, Asn or Gin at position 416 of the
amino acid
sequence of SEQ ID No. 24 (corresponding to position 425 of SEQ ID No. 2);

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U) Phe, Thr, Val, Ala, Gin or Ser at position 417 of the amino acid
sequence of
SEQ ID No. 24 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Phe, Val, Phe or Leu at position 422 of the amino acid
sequence of
SEQ ID No. 24 (corresponding to position 431 of SEQ ID No, 2);
w) Ala, Cys, Gly or Thr at position 219 of the amino acid sequence of SEQ ID
No.
24 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position
239 of the
amino acid sequence of SEQ ID No. 24 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 261 of the amino acid sequence of SEQ ID
No.
24 (corresponding to position 270 of SEQ ID No. 2);
z) Giu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 262 of
the amino
acid sequence of SEQ ID No. 24 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 370 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 418 of the amino acid sequence of SEQ ID No. 24
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said I-
IPPD
protein comprises the amino acid sequence of SEQ ID No. 26 [Triticum aestivum
/
wheat], wherein the resulting amino acid sequence comprises at least one amino

acid selected from
a) Ala, Asp, Glu, Gly, Lys,G1n, Arg, Ser, Thr, Val, Tyr, lie, Leu or Met at
position
237 of the amino acid sequence of SEQ ID No. 26 (corresponding to position
250 of SEQ ID No. 2), preferably Ala;
b) Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at position
238 of the
amino acid sequence of SEQ ID No. 26 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val or Tyr or Thr at position
239 of
the amino acid sequence of SEQ ID No. 26 (corresponding to position 252 of
SEQ ID No. 2), preferably Tyr;

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d) Ala, His, Gin, Val Trp, Ile, Leu or Met at position 240 of the amino
acid
sequence of SEQ ID No. 26 (corresponding to position 253 of SEQ ID No. 2),
preferably Val;
e) Val or Met at position 252 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 255 of the amino
acid
sequence of SEQ ID No. 26 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 256 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 267 of the amino acid sequence of SEQ
ID
No. 26 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 280 of the amino
acid
sequence of SEQ ID No. 26 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 281 of the amino acid
sequence of
SEQ ID No. 26 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 294 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 322 of the amino acid sequence of SEQ ID
No. 26
(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 355 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 368 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 379 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 406 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or
Val at
position 408 of the amino acid sequence of SEQ ID No. 26 (corresponding to
position 421 of SEQ ID No. 2);
r) Ala, Met, Pro or Thr at position 409 of the amino acid sequence of SEQ
ID No.
26 (corresponding to position 422 of SEQ ID No. 2);

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s) Ala, Ile or Val at position 411 of the amino acid sequence of SEQ ID No.
26
(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 412 of the amino
acid
sequence of SEQ ID No. 26 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr or Val, Ala, Gin or Ser at position 413 of the amino acid sequence
of
SEQ ID No. 26 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Phe, Leu or Val at position 418 of the amino acid sequence of
SEQ ID
No. 26 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position
corresponding to position 215 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 235
of the
amino acid sequence of SEQ ID No. 26 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 257 of the amino acid sequence of SEQ ID
No.
26 (corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position
258 of the amino
acid sequence of SEQ ID No. 26 (corresponding to position 271 of SEQ ID No.
2);
aa) His at a position at position 366 of the amino acid sequence of SEQ ID No.
26
(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 414 of the amino acid sequence of SEQ ID No. 26
(corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said
HPPD
protein comprises the amino acid sequence of SEQ ID No. 2 [Arabidopsis
thaliana],
wherein the resulting amino acid sequence comprises at least one amino acid
selected from
(a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at
position
250 of the amino acid sequence of SEQ ID No. 2;
(b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Trp at
position 251 of the
amino acid sequence of SEQ ID No. 2;

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(c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at
position 252 of
the amino acid sequence of SEQ ID No. 2;
(d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 253 of the amino
acid
sequence of SEQ ID No. 2;
(e) Val or Met at a position at position 265 of the amino acid sequence of SEQ
ID
No. 2;
(f) Leu, Gin, Arg, Val, Tyr, Ile, Lys or Met at position 268 of the amino
acid
sequence of SEQ ID No. 2;
(g) Ala or Thr at position 269 of the amino acid sequence of SEQ ID No. 2;
(h) Ala, Ile, Asn, Thr or Val at position 280 of the amino acid sequence of
SEQ ID
No. 2;
(i) Gin, Val, Ala, Phe, Gly, Met, Arg or Ser at a in an HPPD protein, said
position
corresponding to position 293 of the amino acid sequence of SEQ ID No. 2;
(j) Met, Ala, Pro, Ser, Thr or Val at position 294 of the amino acid
sequence of
SEQ ID No. 2;
(k) His or Asn at position 307 of the amino acid sequence of SEQ ID No. 2;
(I) Ile, Asn or Leu at position 335 of the amino acid sequence of SEQ ID
No. 2;
(m) Met at position 368 of the amino acid sequence of SEQ ID No. 2;
(n) Tyr at position 381 of the amino acid sequence of SEQ ID No. 2;
(o) Ser at position 392 of the amino acid sequence of SEQ ID No. 2;
(p) Tyr at position 419 of the amino acid sequence of SEQ ID No. 2;
(q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or
Val at
position 421 of the amino acid sequence of SEQ ID No. 2;
(r) Ala, Met, Pro or Thr at position 422 of the amino acid sequence of SEQ
ID No.
2;
(s) Ala, Ile or Val at position 424 of the amino acid sequence of SEQ ID
No. 2;
(t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 425 of the amino
acid
sequence of SEQ ID No. 2;
(u) Phe, Thr, Val, Ala, Gin or Ser at position 426 of the amino acid
sequence of
SEQ ID No. 2;
(v) Met, Gin, Val, Phe or Leu position 431 of the amino acid sequence of
SEQ ID
No. 2;

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(w) Ala, Cys, Gly or Thr at position 228 of the amino acid sequence of SEQ ID
No.
2;
(x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 248
of the
amino acid sequence of SEQ ID No. 2;
5 (y) Ala, Ile, Met or Val at position 270 of the amino acid sequence of
SEQ ID No. 2;
(z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position
271 of the amino
acid sequence of SEQ ID No. 2;
(aa) His at position 379 of the amino acid sequence of SEQ ID No. 2; or
(bb) Arg at position 427 of the amino acid sequence of SEQ ID No. 2.
In a further embodiment of the nucleic acid of the invention, in said mutated
HPPD
protein at least one amino acid has been replaced so that the resulting amino
acid
sequence comprises (a) Glu, Ser or Tyr at position 252; (b) Val or Ala at
position 269;
(c) Pro, Val or Ala at position 280; (d) Asp, Lys or Asn at position 421; and
(e) Gly or
Ala at postion 42Z These positions correspond to amino acid sequence of SEQ ID
No 2.
In a further embodiment of the nucleic acid of the invention, in said mutated
HPPD
protein at least two amino acids have been replaced.
An isolated nucleic acid comprising a nucleotide sequence encoding a mutated
HPPD protein,
wherein said mutated HPPD protein has HPPD activity,
wherein in said mutated HPPD protein at least one amino acid at position 228,
248,
270, 271, 379 and/or 427 has been replaced by another amino acid.
In an alternative embodiment of the nucleic acid of the invention having at
least one
amino acid at position 228, 248, 270, 271, 379 and/or 427 deleted or replaced
by
another amino acid as defined above, said mutated HPPD protein comprises
a) a His at a position in an HPPD protein, said position corresponding to
position
226 of the amino acid sequence of SEQ ID No. 2;

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b) a Ser at a position in an HPPD protein, said position corresponding to
position
267 comprising the amino acid sequence of SEQ ID No. 2 or at a position
corresponding thereto in a different HPPD enzyme;
c) an Asn at a position in an HPPD protein, said position corresponding to
position
282 of the amino acid sequence of SEQ ID No. 2;
d) a His at a position in an HPPD protein, said position corresponding to
position
308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr at a position in an HPPD protein, said position corresponding to
position
342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu at a position in an HPPD protein, said position corresponding to
position
394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly at a position in an HPPD protein, said position corresponding to
position
420 of the amino acid sequence of SEQ ID No. 2; and
h) an Asn at a position in an HPPD protein, said position corresponding to
position
423 of the amino acid sequence of SEQ ID No. 2_
In another embodiment of the isolated nucleic acid as defined above, in said
mutated
HPPD protein at least one amino acid has been replaced so that the resulting
amino
acid sequence has at least one selected from
a. Ala, Cys, Gly, Thr or Val at a position in an HPPD protein, said position
corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
b. Ala, Glu, Gly, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr or Tyr at a
position in an
HPPD protein, said position corresponding to position 248 of the amino acid
sequence of SEQ ID No. 2;
c. Ala, Ile, Leu, Met or Val at a position in an HPPD protein, said
position
corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
d. Ala, Glu, Hits, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at a
position in an HPPD
protein, said position corresponding to position 271 of the amino acid
sequence
of SEQ ID No. 2;
e. His or Gin at a position in an HPPD protein, said position corresponding to
position 379 of the amino acid sequence of SEQ ID No. 2; and

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f. Leu or Arg at a position in an HPPD protein, said position
corresponding to
position 427 of the amino acid sequence of SEQ ID No. 2.
In another embodiment of the isolated nucleic acid as defined above, in said
mutated
HPPD protein at least one amino acid has been replaced so that the resulting
amino
acid sequence has at least one selected from
a. Val or Thr at a position in an HPPD protein, said position corresponding
to
position 228 of the amino acid sequence of SEQ ID No. 2;
b. Leu, Met or Val at a position in an HPPD protein, said position
corresponding to
position 270 of the amino acid sequence of SEQ ID No. 2;
c. Ala or Ser at a position in an HPPD protein, said position corresponding
to
position 271 of the amino acid sequence of SEQ ID No. 2,
d. Gin at a position in an HPPD protein, said position corresponding to
position
379 of the amino acid sequence of SEQ ID No. 2; and
e. Leu at a position in an HPPD protein, said position corresponding to
position
427 of the amino acid sequence of SEQ ID No. 2.
In another embodiment of the nucleic acid as defined above, said mutated HPPD
protein is capable of increasing the tolerance of a plant to at least one
herbicide
acting on HPPD (also called HPPD inhibitor herbicide).
In another embodiment, the present invention relates to a protein encoded by
the
isolated nucleic acid of the invention.
In a further embodiment, the present invention relates to a chimeric gene
comprising
a coding sequence comprising the nucleic acid of the invention operably linked
to a
plant-expressible promoter and optionally a transcription termination and
polyadenylation region.
As a regulatory sequence which functions as a promoter in plant cells and
plants,
use may be made of any promoter sequence of a gene which is naturally
expressed
in plants, in particular a promoter which is expressed especially in the
leaves of

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plants, such as for example "constitutive" promoters of bacterial, viral or
plant origin,
or "light-dependent" promoters, such as that of a plant ribulose-
biscarboxylase/oxygenase (RuBisCO) small subunit gene, or any suitable known
promoter-expressible which may be used. Among the promoters of plant origin,
mention will be made of the histone promoters as described in EP 0 507 698 Al,
the
rice actin promoter (US 5,641,876), or a plant ubiquitin promoter (US
5,510,474).
Among the promoters of a plant virus gene, mention will be made of that of the

cauliflower mosaic virus (CaMV 19S or 35S, Sanders et al. (1987), Nucleic
Acids
Res. 15(4)1543-58.), the circovirus (AU 689 311) or the Cassava vein mosaic
virus
(CsVMV, US 7,053,205).
In one embodiment of this invention, a promoter sequence specific for
particular
regions or tissues of plants can be used to express the HPPD proteins of the
invention, such as promoters specific for seeds (Datla, R. et al., 1997,
Biotechnology
Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378 Al), the
phaseolin
promoter, the glutenin promoter, the helianthinin promoter (WO 92/17580), the
albumin promoter (WO 98/45460), the oleosin promoter (WO 98/45461), the SAT1
promoter or the SAT3 promoter (PCT/US98/06978).
Use may also be made of an inducible promoter advantageously chosen from the
phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase,
glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase (nos)
and
vspB promoters (US 5 670 349, Table 3), the HMG2 promoter (US 5 670 349), the
apple beta-galactosidase (ABG1) promoter and the apple aminocyclopropane
carboxylate synthase (ACC synthase) promoter (WO 98/45445).
According to the invention, use may also be made, in combination with the
promoter,
of other regulatory sequences, which are located between the promoter and the
coding sequence, such as transcription activators ("enhancers"), for instance
the
translation activator of the tobacco mosaic virus (TMV) described in
Application WO
87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed
1990,

CA 02808152 2013-03-05
. .
,
89
J. Virol. 64: 1590-1597, for example, or introns such as the adh1 intron of
maize or
intron 1 of rice actin.
As a regulatory terminator or polyadenylation sequence, use may be made of any
corresponding sequence of bacterial origin, such as for example the nos
terminator
of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV
35S
terminator, or of plant origin, such as for example a histone terminator as
described
in published Patent Application EP 0 633 317 Al.
A method of obtaining a mutated HPPD protein capable of modulating the
tolerance
of a plant to at least one herbicide acting on HPPD, wherein said mutated HPPD

protein has HPPD activity, the method comprising
i. providing an HPPD protein, said HPPD optionally comprising an amino acid
sequence, wherein
a) a His is present at a position in an HPPD protein, said position
corresponding to position 226 of the amino acid sequence of SEQ ID No. 2;
b) a Ser is present at a position in an HPPD protein, said position
corresponding to position 267 of the amino acid sequence of SEQ ID No. 2;
c) an Asn is present at a position in an HPPD protein, said position
corresponding to position 282 of the amino acid sequence of SEQ ID No. 2;
d) a His is present at a position in an HPPD protein, said position
corresponding to position 308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr is present at a position in an HPPD protein, said position
corresponding to position 342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu is present at a position in an HPPD protein, said position
corresponding to position 394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly is present at a position in an HPPD protein, said
position
corresponding to position 420 of the amino acid sequence of SEQ ID No. 2;
and
h) an Asn is present at a position in an HPPD protein, said position
corresponding to position 423 of the amino acid sequence of SEQ ID No. 2

CA 02808152 2013-03-05
. .
,
ii) replacing at least one amino acid in said HPPD enzyme so that the
resulting
amino acid sequence has at least one selected from
a. Ala, Asp, Glu, Phe, Gly, Lys,G1n, Arg, Ser, Thr, Val, Ile, Leu, Met or Tyr
at a
position in an HPPD protein, said position corresponding to position 250 of
5 the amino acid sequence of SEQ ID No. 2;
b. Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Gly, His, Tyr or Ser at a
position
in an HPPD protein, said position corresponding to position 251 of the amino
acid sequence of SEQ ID No. 2;
c, Giu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Thr or Tyr at a
10 position in an HPPD protein, said position corresponding to position
252 of
the amino acid sequence of SEQ ID No. 2;
d, Ala, Phe, His, Gin, Val, Ile, Leu, Met or Trp at a position in an HPPD
protein,
said position corresponding to position 253 of the amino acid sequence of
SEQ ID No. 2;
15 e. Leu, Met or Val at a position in an HPPD protein, said position
corresponding to position 265 of the amino acid sequence of SEQ ID No. 2;
f. Leu, Gin, Arg, Val, Ala, lie, Lys, Met or Tyr at a position in
an HPPD protein,
said position corresponding to position 268 of the amino acid sequence of
SEQ ID No. 2;
20 g. Ala, Thr or Val at a position in an HPPD protein, said position
corresponding
to position 269 of the amino acid sequence of SEQ ID No. 2;
h. Ala, Ile, Asn, Pro, Thr or Val at a position in an HPPD protein, said
position
corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i. Leu, Gin, Ala, Phe, Gly, Met, Arg, Ser or Val at a position in an HPPD
25 protein, said position corresponding to position 293 of the amino
acid
sequence of SEQ ID No. 2;
j. Ile, ala, Pro, Ser, Thr, Val or Met at a position in an HPPD protein,
said
position corresponding to position 294 of the amino acid sequence of SEQ
ID No. 2;
30 k. Gin, His or Asn at a position in an HPPD protein, said position
corresponding to position 307 of the amino acid sequence of SEQ ID No. 2;

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91
I. Ile, Met, Leu or Asn at a position in an HPPD protein, said
position
corresponding to position 335 of the amino acid sequence of SEQ ID No. 2;
m. Leu or Met at a position in an HPPD protein, said position corresponding to
position 368 of the amino acid sequence of SEQ ID No. 2;
n. Phe or Tyr at a position in an HPPD protein, said position corresponding to
position 381 of the amino acid sequence of SEQ ID No. 2;
o. Phe or Ser at a position in an HPPD protein, said position corresponding to

position 392 of the amino acid sequence of SEQ ID No. 2;
p. Phe or Tyr at a position in an HPPD protein, said position corresponding to
position 419 of the amino acid sequence of SEQ ID No. 2;
q. Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr
or
Val at a position in an HPPD protein, said position corresponding to position
421 of the amino acid sequence of SEQ ID No. 2;
r. Ala, Gly, Met, Pro or Thr at a position in an HPPD protein, said position
corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;
s. Ala, Phe, Ile or Val at a position in an HPPD protein, said position
corresponding to position 424 of the amino acid sequence of SEQ ID No. 2
or;
t. Ile, Pro, Arg, Ala, Gly, Lys, Asn, Gin or Ser at a position in an HPPD
protein,
said position corresponding to position 425 of the amino acid sequence of
SEQ ID No. 2;
u. Glu, Phe, Thr, Ala, Gin, Ser or Val at a position in an HPPD protein, said
position corresponding to position 426 of the amino acid sequence of SEQ
ID No. 2;
v. Ile, Met, Gin, Phe, Leu or Val at a position in an HPPD protein, said
position
corresponding to position 431 of the amino acid sequence of SEQ ID No. 2;
w. an amino acid deletion or replacement at least one position in an HPPD
protein, said position corresponding to at least one of positions 228, 248,
270, 271, 379 and 427 of the amino acid sequence of SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one
herbicide acting on HPPD;

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92
wherein an inhibition of the resulting protein of less or more than that
observed
with a reference HPPD protein is indicative that the resulting protein is
capable
of modulating the tolerance of a plant to said herbicide.
It is to be understood that also the (more specific) amino acids and positions
listed
above for other embodiments, such as the nucleic acid of the invention, may be

applied to the method of obtaining a mutated HPPD protein as described above.
In an alternative embodiment of the method of obtaining a mutated HPPD protein
as
described above, said mutated HPPD protein is capable of increasing the
tolerance
of a plant to at least one herbicide acting on HPPD.
Within the above method of obtaining in mutated HPPD protein, different
herbicides
acting on HPPD may be chosen. Accordingly, in another embodiment of the method
of obtaining a mutated HPPD protein as described above, wherein said mutated
HPPD protein is capable of increasing the tolerance of a plant to at least one

herbicide acting on HPPD, the herbicide acting on HPPD is selected from
triketones,
or pyrazolinates, preferably tembotrione, mesotrione, topramezone or
sulcotrione,
bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryitrione,
particularly
tembotrione and such plants containing the HPPD of the invention have an
agronomically acceptable tolerance to an HPPD inhibitor herbicide particularly
to
triketones, or pyrazolinates, preferably tembotrione, mesotrione, topramezone
or
sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and
tefuryltrione,
particularly tembotrione.
In another embodiment, the present invention relates to a method of producing
a
transgenic plant comprising introducing into a said plant genome the nucleic
acid of
the present invention operably linked to a plant expressible promoter, the
chimeric
gene of the invention or a nucleic acid encoding the HPPD enzyme identified by
the
method of of claim 27 or 28.

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93
In an alternative embodiment of the method of producing a transgenic plant as
described above, the nucleic acid of the invention, wherein said mutated HPPD
protein is capable of increasing the tolerance of a plant to at least one
herbicide
acting on HPPD, or a nucleic acid identified by the method of obtaining a
mutated
HPPD protein, wherein said mutated HPPD protein is capable of increasing the
tolerance of a plant to at least one herbicie acting on HPPD, both operably
linked to
a plant expressible promoter, or the chimeric gene of the invention comprising
a
nucleic acid, wherein said mutated HPPD protein is capable of increasing the
tolerance of a plant to at least one herbicie acting on HPPD, is introduced
into said
plant.
In another embodiment, the present invention relates to a plant cell
comprising the
isolated nucleic acid of the invention or the chimeric gene of the invention
in its
genetic information.
The present invention also relates to a plant, a part of a plant or plant
tissue
consisting essentially of the plant cells of the invention.
Furthermore, the present invention relates to a plant obtainable from the
method of
obtaining a mutated HPPD protein capable of modulating or increasing the
tolerance
of a plant to at least one herbicide acting on HPPD in all alternative aspects

described above.
The plant of the present invention can be any plant. Non-limiting examples of
plants
of the invention include wheat, cotton, canola, rice, corn, soy bean,
sorghum,canola,
sunflower, tobacco, sugarbeet, cotton, maize, wheat, barley, rice, sorghum,
tomato,
mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce,

cabbage, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus,
alfalfa,
rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing
plants.
.. The present invention also relates to a seed of the plant of the invention.

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94
Furthermore, the invention relates to progeny of the plant of the invention or
the seed
of the invention.
In a further embodiment, the present invention relates to a method of
modulating a
plant's tolerance to at least one herbicide acting on HPPD comprising
introducing the
isolated nucleic acid of the invention operably linked to a plant expressible
promoter
or the chimeric gene of the invention into a plant's genome
In an alternative embodiment, the present invention relates to a method of
increasing
a plant's tolerance to at least one herbicide acting on HPPD or of obtaining a
plant
tolerant to an HPPD inhibitor herbicide comprising introducing the isolated
nucleic
acid of the invention, wherein said nucleic acid encodes a mutated HPPD
protein
which is capable of increasing the tolerance of a plant to at least one
herbicide acting
on HPPD, operably linked to a plant expressible promoter or the chimeric gene
of the
invention comprising a nucleic acid of the invention, wherein said nucleic
acid
encodes a mutated HPPD protein which is capable of increasing the tolerance of
a
plant to at least one herbicide acting on HPPD, into a plant's genome.
Furthermore, the present invention relates to a method for controlling weeds
comprising spraying at least one herbicide acting on HPPD on or around a crop
plant,
wherein said crop plant comprises the nucleic acid of the present invention,
wherein
said mutated HPPD protein is capable of increasing the tolerance of a plant to
at
least one herbicide acting on HPPD, operably linked to a plant expressible
promoter
or the chimeric gene of the invention comprising the nucleic acid of the
invention,
wherein said mutated HPPD protein is capable of increasing the tolerance of a
plant
to at least one herbicide acting on HPPD. In alternative embodiment of the
method of
controlling weeds, the tolerance of said plant to at least one herbicie acting
on HPPD
is increased.
In addition, the present invention relates to the use of a chimeric gene of
the
invention or the nucleic acid of the invention operably linked to a plant
expressible

CA 02808152 2013-03-05
promoter for modulating the tolerance of a plant to at least one herbicide
acting on
HPPD.
In an alternative embodiment, the present invention relates to the use of a
chimeric
5 gene of the invention or the nucleic acid of the invention operably
linked to a plant
expressible promoter for increasing the tolerance of a plant to at least one
herbicide
acting on HPPD. In this embodiment of the invention, the chimeric gene used
comprises the nucleic acid of the invention, wherein the mutated HPPD protein
encoded thereby is capable of increasing the tolerance of a plant to at least
one
10 herbicide acting on HPPD. Alternatively, if a nucleic acid operably
linked to a plant
expressible promoter is used, said nucleic is chose that the mutated HPPD
protein
encoded thereby is capable of increasing the tolerance of a plant to at least
one
herbicide acting on HPPD.
15 The present invention also relates to the plant cell of the invention
and the plant of
the invention which may comprise a further useful trait as described further
below.
While a number of herbicide-tolerant crop plants are presently commercially
available, one issue that has arisen for many commercial herbicides and
20 herbicide/crop combinations is that individual herbicides typically have
incomplete
spectrum of activity against common weed species. For most individual
herbicides
which have been in use for some time, populations of herbicide resistant weed
species and biotypes have become more prevalent (see, e.g., Tranel and Wright
(2002) Weed Science 50: 700-712; Owen and Zelaya (2005) Pest Manag. Sci. 61:
25 301-311). Transgenic plants which are resistant to more than one
herbicide have
been described (see, e.g., W02005/012515). However, improvements in every
aspect of crop production, weed control options, extension of residual weed
control,
and improvement in crop yield are continuously in demand.
30 The HPPD protein or gene of the invention is advantageously combined in
plants
with other genes which encode proteins or RNAs that confer useful agronomic
properties to such plants. Among the genes which encode proteins or RNAs that

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96
confer useful agronomic properties on the transformed plants, mention can be
made
of the DNA sequences encoding proteins which confer tolerance to one or more
herbicides that, according to their chemical structure, differ from HPPD
inhibitor
herbicides, and others which confer tolerance to certain insects, those which
confer
tolerance to certain diseases, DNAs that encodes RNAs that provide nematode or
insect control, etc...
Such genes are in particular described in published PCT Patent Applications WO
91/02071 and W095/06128.
Among the DNA sequences encoding proteins which confer tolerance to certain
herbicides on the transformed plant cells and plants, mention can be made of a
bar
or PAT gene or the Streptomyces coelicolor gene described in W02009/152359
which confers tolerance to glufosinate herbicides, a gene encoding a suitable
EPSPS which confers tolerance to herbicides having EPSPS as a target, such as
glyphosate and its salts (US 4,535,060, US 4,769,061, US 5,094,945, US
4,940,835,
US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US
5,627,061, US 5,633,435), or a gene encoding glyphosate oxydoreductase (US
5,463,175).
Among the DNA sequences encoding a suitable EPSPS which confer tolerance to
the herbicides which have EPSPS as a target, mention will more particularly be
made of the gene which encodes a plant EPSPS, in particular maize EPSPS,
particularly a maize EPSPS which comprises two mutations, particularly a
mutation
at amino acid position 102 and a mutation at amino acid position 106
(WO 2004/074443), and which is described in Patent Application US 6566587,
hereinafter named double mutant maize EPSPS or 2mEPSPS, or the gene which
encodes an EPSPS isolated from Agrobacterium and which is described by
sequence ID No. 2 and sequence ID No. 3 of US Patent 5,633,435, also named
CP4.
Among the DNA sequences encoding a suitable EPSPS which confer tolerance to
the herbicides which have EPSPS as a target, mention will more particularly be

made of the gene which encodes an EPSPS GRG23 from Arthrobacter globiformis,
but also the mutants GRG23 ACEI, GRG23 ACE2, or GRG23 ACE3, particularly the
mutants or variants of GRG23 as described in W02008/100353, such as
GRG23(ace3)R173K of SEQ ID No. 29 in W02008/100353.

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97
In the case of the DNA sequences encoding EPSPS, and more particularly
encoding
the above genes, the sequence encoding these enzymes is advantageously
preceded by a sequence encoding a transit peptide, in particular the
"optimized
transit peptide" described in US Patent 5,510,471 or 5,633,448.
In WO 2007/024782, plants being tolerant to glyphosate and at least one ALS
(acetolactate synthase) inhibitor are disclosed. More specifically plants
containing
genes encoding a GAT (Glyphosate-N-Acetyltransferase) polypeptide and a
polypeptide conferring resistance to ALS inhibitors are disclosed.
In US 6855533, transgenic tobacco plants containing mutated Arabidopsis
ALS/AHAS genes were disclosed.
In US 6,153,401, plants containing genes encoding 2,4-D-monooxygenases
conferring tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) by
metabolisation are
disclosed.
In US 2008/0119361 and US 2008/0120739, plants containing genes encoding
Dicamba monooxygenases conferring tolerance to dicamba (3,6-dichloro-2-
methoxybenzoic acid) by metabolisation are disclosed.
All the above mentioned herbicide tolerance traits can be combined with those
performing HPPD tolerance which are subject matter of this invention.
Among the DNA sequences encoding proteins concerning properties of tolerance
to
insects, mention will more particularly be made of the Bt proteins widely
described in
the literature and well known to those skilled in the art. Mention will also
be made of
proteins extracted from bacteria such as Photorhabdus (WO 97/17432 & WO
98/08932).
Among such DNA sequences encoding proteins of interest which confer novel
properties of tolerance to insects, mention will more particularly be made of
the Bt
Cry or VIP proteins widely described in the literature and well known to those
skilled
in the art. These include the Cry1F protein or hybrids derived from a Cryl F
protein
(e.g., the hybrid Cry1A-Cry1F proteins described in US 6,326,169; US
6,281,016; US

CA 02808152 2013-03-05
98
6,218,188, or toxic fragments thereof), the Cry1A-type proteins or toxic
fragments
thereof, preferably the Cry1Ac protein or hybrids derived from the Cry1Ac
protein
(e.g., the hybrid Cryl Ab-Cry1Ac protein described in US 5,880,275) or the
Cry1Ab or
Bt2 protein or insecticidal fragments thereof as described in EP451878, the
Cry2Ae,
Cry2Af or Cry2Ag proteins as described in W002/057664 or toxic fragments
thereof,
the Cry1A.105 protein described in WO 2007/140256 (SEQ ID No. 7) or a toxic
fragment thereof, the VIP3Aa19 protein of NCBI accession ABG20428, the
VIP3Aa20 protein of NCB! accession ABG20429 (SEQ ID No. 2 in WO
2007/142840), the VIP3A proteins produced in the C0T202 or C0T203 cotton
events (WO 2005/054479 and WO 2005/054480, respectively), the Cry proteins as
described in W001/47952, the VIP3Aa protein or a toxic fragment thereof as
described in Estruch et al. (1996), Proc Natl Acad Sci U S A. 28;93(11):5389-
94 and
US 6,291,156, the insecticidal proteins from Xenonhabdus (as described in
W098/50427), Serratia (particularly from S. entomophila) or Photorhabdus
species
strains, such as Tc-proteins from Photorhabdus as described in W098/08932
(e.g.,
Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24; Ffrench-
Constant
and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33). Also any variants or
mutants of
any one of these proteins differing in some (1-10, preferably 1-5) amino acids
from
any of the above sequences, particularly the sequence of their toxic fragment,
or
.. which are fused to a transit peptide, such as a plastid transit peptide, or
another
protein or peptide, is included herein.

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99
The FIGURES show:
FIGURE1: Superpositon of the x-ray structure of HPPD from
Arabidopsis
thaliana (dark grey) and (a) Pseudomonas fluorescens (light
grey), (b) Streptomyces avermitilis, (c) Homo sapiens, (d) Rattus
norvegicus. The structures are shown as ribbon plot.
F1GURE2: Amino acids forming the binding site of (a) Arabidopsis
thaliana,
(b) Pseudomonas fluorescens (c) Streptomyces avermitilis (d)
Homo sapiens, and (e) Rattus norvegicus (represented by bold
lines). The core of the HPPD protein is shown as Caoha trace
and the iron is marked.
F1GURE3: Brown test color in Escherichia coil
SEQUENCES LISTING
SEQ ID No. 1: Nucleic acid sequence encoding Arabidopsis thaliana HPPD
SEQ ID No. 2: Protein encoded by SEQ ID No. 1
SEQ ID No. 3: Nucleic acid sequence encoding Oryza sativa HPPD
SEQ ID No. 4: Protein encoded by SEQ ID No. 3
SEQ ID No. 5: Nucleic acid sequence encoding Zea mays HPPD
SEQ ID No. 6: Protein encoded by SEQ ID No. 5
SEQ ID No. 7: Nucleic acid sequence encoding Avena sativa HPPD
SEQ ID No. 8: Protein encoded by SEQ ID No. 7
SEQ ID No. 9: Nucleic acid sequence encoding Pseudomonas fluorescens
HPPD
SEQ ID No. 10: Protein encoded by SEQ ID No. 9
SEQ ID No. 13: Nucleic acid sequence encoding Synechococcus sp. HPPD
SEQ ID No. 14: Protein encoded by SEQ ID No. 13
SEQ ID No. 15: Nucleic acid sequence encoding Blepharsima japonicum HPPD
SEQ ID No. 16: Protein encoded by SEQ ID No 15

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. .
100
SEQ ID No. 17: Nucleic acid sequence encoding Rhodococcus RHA1
isolate
ro0341 HPPD
SEQ ID No. 18: Protein encoded by SEQ ID No. 17
SEQ ID No. 19: Nucleic acid sequence encoding Picrophilus torridus
HPPD
SEQ ID No. 20: Protein encoded by SEQ ID No. 19
SEQ ID No. 21: Nucleic acid sequence encoding Kordia algicida HPPD
SEQ ID No. 22: Protein encoded by SEQ ID No. 21
SEQ ID No. 23: Nucleic acid sequence encoding Sorghum bicolor HPPD
SEQ ID No. 24: Protein encoded by SEQ ID No. 23
SEQ ID No. 25: Nucleic acid sequence encoding Triticum aestivum
HPPD
SEQ ID No. 26: Protein encoded by SEQ ID No. 25
SEQ ID No. 27: Nucleic acid sequence encoding Hordeum vulgare HPPD
SEQ ID No. 28: Protein encoded by SEQ ID No. 27
SEQ ID No. 29: Nucleic acid sequence encoding Rhodococcus RHA1 isolate
ro0240 HPPD
SEQ ID No. 30: Protein encoded by SEQ ID No. 29
SEQ ID No. 31: Nucleic acid sequence encoding Arabidopsis thaliana
wild type
HPPD polypeptide, plus containing at the 5' end a nucleic acid
encoding an alanine and 6 histidine amino acids
SEQ ID No. 32: Protein encoded by SEQ ID No. 31
SEQ ID No. 33: Nucleic acid sequence encoding mutant HPPD
polypeptide, plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 34: Protein encoded by SEQ ID No. 33,
SEQ ID No. 35: Nucleic acid sequence encoding mutant HPPD
polypeptide, plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 36: Protein encoded by SEQ ID No. 35,
SEQ ID No. 37: Nucleic acid sequence encoding mutant HPPD polypeptide, plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids

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101
SEQ ID No. 38: Protein encoded by SEQ ID No. 37,
SEQ ID No. 39: Nucleic acid sequence encoding mutant HPPD polypeptide,
plus
containing at the 5' end a nucleic acid encoding art alanine and
6 histidine amino acids
SEQ ID No. 40: Protein encoded by SEQ ID No. 39,
SEQ ID No. 41: Nucleic acid sequence encoding mutant HPPD polypeptide,
plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 42: Protein encoded by SEQ ID No. 41,
SEQ ID No. 43: Nucleic acid sequence encoding mutant HPPD polypeptide, plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 44: Protein encoded by SEQ ID No. 43,
SEQ ID No. 45: Nucleic acid sequence encoding mutant HPPD polypeptide,
plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 46: Protein encoded by SEQ ID No. 45
SEQ ID No. 47: Nucleic acid sequence encoding mutant HPPD polypeptide,
plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 48: Protein encoded by SEQ ID No. 47
SEQ ID No. 49: Nucleic acid sequence encoding mutant HPPD polypeptide,
plus
containing at the 5' end a nucleic acid encoding an alanine and
6 histidine amino acids
SEQ ID No. 50: Protein encoded by SEQ ID No. 49,

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=
102
The Examples illustrate the invention
Example1:
A mutation of strictly conserved amino acids leads to an inactive protein.
The point mutants S267A (SEQ ID No. 34) in A.thaliana and N282A (SEQ ID No.
36)
in A.thaliana are inactive. The amino acid position given are referring to the
position
of SEQ ID No. 2.
Mutant HPPD polypeptides of the presenting invention have amino acids changes
at
one or more positions relative to the starting wild type sequence from which
they are
derived.
The DNA sequence SEQ ID No. 2 encoding the wild HPPD protein from Arabidopsis
thaliana (1335 bp; Genebank AF047834; WO 96/38567) was cloned into the vector
pSE420(RI)NX (modified from the the cloning and expression vector pSE420(RI)NX

(5261 bp) is based on the plasmid pSE420 by Invitrogen (Karlsruhe, Germany)).
At
the 5' end, directly downstream to the ATG was inserted a nucleic acid
sequence
coding for an alanine amino acid and a nucleic acid sequence encoding a N-
terminal
HIS6-Tag (6x HIS, encoded by: cat cat cat cac cat cat). The resulting sequence
is
presented as SEQ ID No. 31. Upstream to the ATG, two additional cysteine base
pairs were added in order to obtain a sequence corresponding to the
recognition site
of the restriction enzyme Ncol and downstream to the stop codon the sequences
corresponding to the recognition site of the restriction enzyme Xbal were
added. The
resulting plasmid was then used to transform E. Coll cells B1_21 (DE3) with
50pg/m1
kanamycin or 100pg/mIcarbenicillin selection as described in European patent
applications "EP09015984.9"; EP09015985.6"; EP 09015986.4" EP 09015987.2";
and EP09015988.0" all filed on 23. December 2009.

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103
Quick test of the activity of HPPD protein: Production of brown color
Colorimetric Screening Test for Active HPPD Enzymes:
A YT-broth-type culture medium with 1% agarose, 5mM L-Tyrosine and 42mM
Succinate, which contains the selection agent for the vector pSE420 is poured
into
deep well plates. E.coli culture in the exponentional growth phase which
contains the
vector pSE420-HPPDx (any gene coding for a putative HPPD enzyme/protein) is
applied to each well. After 16 hours at 37 C, the wells which do not contain
the
culture medium, those which have been seeded with an E. coil culture
containing the
empty vector pSE420 are transparent, or those which have been seeded with an
E.
coli culture containing a vector pSE420 containing a gene coding for an
inactive
HPPD are transparent, while the wells seeded with an E. coil culture
containing the
vector pSE420-HPPD coding for active HPPD are brown. It has been previously
demonstrated that this test refelects the HPPD activity, whatever the orgin of
this
activity, and allows the identification of HPPD activities (US 6,768,044).
As it can be seen on Figure 3, the culture from bacteria containing the gene
coding
for the mutant HPPD did not develop a brown color while the one containing the

gene coding of the wild type HPPD developed a strong brown color reflecting
the
activity of the HPPD enzymes. It can be concluded that the two HPPD mutants
are
not able to convert HPP into homogentisate. The two HPPD mutants are inactive.
One can conclude that the amino acid positions 267 and 282 (referring to
position in
SEQ ID No. 2) are essential for HPPD activity.
The expression of the HPPD protein was then done as following.
Overnight cultures grown at 37 C were used to inoculate LB media at a ratio
1:100.
Cells were allowed to grow until OD reached 0.5, then expression was initiated
from
the trp-lac (trc) promoter by induction with 1 mM IPTG which binds to the lac
.. repressor and causes its dissociation from the lac operon. Expression was
carried
out over 15h at 28 C.

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104
To prepare the pre-starter culture, 2 mL of TB medium (100 pg*mL-1
carbenicillin)
were inoculated with 50 pL of an E coil K-12 BL21 glycerol stock. The pre-
starter
culture was incubated at 37 C with shaking at 140 rpm for 15 h. 200p1 of the
pre-
starter culture was used to initiate the starter culture (5mL TB supplement
with
100 pg*L-1), which was incubated 3 h at 37 C.
To prepare the main culture, 400 mL of TB medium (100 pg*ml.:1 carbenicillin)
were
inoculated with 4 mL of the starter culture. This starter culture was
incubated at
37 C with shaking at 140 rpm until 0D600 0.5 was reached. Then recombinant
protein expression was induced with 400 pl of 1M IPTG solution. The cells were

allowed to grow for an additional hour under these conditions, then the
temperature
was lowered to 28 C and the culture was shaken at 140 rpm for 15 h. Cells were

harvested by centrifugation at 6000 x g for 15 min at 4 C. Then cell pellets
were
stored at -80 C.
Isolation and purification of His6-AtHPPD in native form
Lysis of cells
Cells were lysed using Lysozyme, an enzyme that cleaves the 1,4-0-linkages
between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in
peptidoglycan which forms the bacterial cell wall. Cell membranes were then
disrupted by the internal pressure of the bacterial cell. In addition, the
lysis buffer
contained Benzonase Nuclease, an endonuclease that hydrolyzes all forms of
DNA
and RNA without damaging proteins and thereby largely reduces viscosity of the
cell
lysate. Lysis under native conditions was carried out on ice.
For purification of His6-tagged proteins the QIAexprese Ni-NTA Fast Start Kit
was
used following the user manual instruction.
Purification of His6-tagged proteins by immobilized metal ion affinity
chromatography
(IMAC)

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105
The cleared cell lysate (10 mL) obtained after centrifugation of the lysis
reaction was
loaded onto a Ni-NTA Fast Start Column from the QlAexpress Ni-NTA Fast Start
Kit
(Qiagen, Hi!den, Germany) and purification was carried out according to the
instruction manual. The Hiss-tagged protein was eluted with 2.5 mL of elution
buffer.
Desalting of HPPD solutions by gel filtration
I-IPPD solutions eluted from a Ni-NTA Fast Start Column with 2.5 mL of elution
buffer
were applied to a Sephadex G-25 PD-10 column (GE Healthcare, Freiburg,
Germany) following the user manual instruction. After the whole sample had
entered
the gel bed, elution was performed with 3.5 mL of storage buffer.
The HPPD solutions eluted from the desalting column were frozen at -80 C in 1
mL
aliquots.
Determination of HPPD protein concentration using the Bradford protein assay
Protein concentration was determined using the standard Bradford assay
(Bradford,
(1976), Anal Biochem 72: 248-254).
Determination of purity of HPPD solutions using SDS-PAGE
The integrity of the eluted protein was checked by SDS-PAGE protein gel
electrophoresis using the gel NuPAGE Novex 4-12 % Bis-Tris Gels (Invitrogen,
Karlsruhe, Germany), approximately 10 pg of protein were loaded. 10 pL of
Laemmli
Sample Buffer was added to 1-10 pL of protein solution and the mixture was
incubated at 90 C for 10 min. After short centrifugation step, the whole
mixture was
loaded into a slot of an SDS gel previously fixed in a XCell SureLockTM Novex
Mini-
Cell gel chamber filled with NuPAGE MOPS SDS Running Buffer (diluted from the

20 x-solution with ddH20). A voltage of 150 was then applied to the gel
chamber for
1 h. For staining of protein bands, the gel was immersed in Coomassie
Brilliant Blue
R-250 Staining Solution. For destaining of the polyacrylamide gel, it was
immersed in
Coomassie Brilliant Blue R-250 Destaining Solution until protein bands appear
blue
on a white gel.

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106
The HPPD activity was checked by the standard spectrophotmetric assay (method
extensively described in WO 2009/144079)
In this content, p150-value means the log value of the concentration of
inhibitor
__ necessary to inhibit 50% of the enzyme activity in molar concentration.
p150-values for HPPD inhibitors were determined from dose-response plots of
HPPD
activity versus inhibitor concentration using the assay extensively described
in
WO 2009/144079 at 2 mM fixed HPP concentration and 3 minutes fixed incubation
time using the ID Business Solutions Ltd. XLfit software suite.
Table 7: Determination of pI50 HPPD enzymes (Arabidopsis thaliana wild type
HPPD "SEQ ID No. 32", the mutants S267A (SEQ ID No. 34) and N282A
__ (SEQ ID No. 36)" and the tolerance to the several listed below HPPD
inhibitors
tembotrione, diketonitrile, mesotrione, bicyclopyrone, pyrasulfotole,
sulcotrione,
pyrazolate, tefuryltrione, and benzofenap. The symbol ">" means that the value
was
far higher than the one indicated but could not be precisely calculated within
in the
range of concentration of inhibitor tested (2.5x10-6, 5.0x10-6, 1.0x10-5,
2.5x10-5,
__ 6.3x10-5, and 2.5x10-4M).
Bicyclopyrone Benzofenap Diketonitrile Mesotrione
VVT
5.2 >5.6 >5.6 >5.6
(SEQ ID No. 32)
S267A
nd - la (P) nd - la (P) nd la (P) nd - la (P)
(SEQ ID No. 34)
N282A
nd - la (P) nd - la (P) nd - la (P) nd la (P)
(SEQ ID No. 36)

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107
Pyrasulfotole Pyrazolate Sulcotrione Tefuryltrione Tembotrione
WT
5.4 5.4 >5.6 >5.6 >5.6
(SEQ ID No. 32)
S267A
nd - la (P) nd - la (P) nd - la (P) nd - la (P)
nd - la (P)
(SEQ ID No. 34)
N282A
nd la (P) nd - la (P) nd - la (P) nd - la (P)
nd - la (P)
(SEQ ID No. 36)
As previously demonstrated, the 2 protein mutants are not able to convert HPP
into
Homogentisate, confirming that the 2 mutant proteins are inactive. It confirms
the
hypothesis that the position 267 and 282 (referring to position in SEQ ID No.
2) are
absolutely essential for obtaining an active HPPD
Example 2: Single point mutants displayed increased tolerance to HPPD
inhibitor
herbicides:
Kinetic characterization and evaluation of tolerance to HPPD inhibitors of
HPPD
enzyme "SEQ ID No. 32".
.. The HPPD activity was checked by the standard spectrophotmetric assay
(method
extensively described in WO 2009/144079)
=
Determination of HPPD in vitro kinetic properties
Km, Vmax, and kcat values for different HPPD enzyme preparations and K1,
K1=k0n,
and Ki=Koff for different HPPD inhibitors were determined using a HPLC assay
for
measurements of HPPD activity. The assay mixtures contained in a volume of 1
ml
150 mM Tris-HCI buffer at pH 7.8, 10 mM sodium ascorbate, 650 units of bovine
catalase (Sigma C30 (Sigma-Aldrich, Munich,Germany), 34 mg protein/ml, 23,000
.. units/mg), and appropriate amounts of HPP, purified HPPD enzyme and HPPD
inhibitors. For Km, Vmax, and kcat value determination HPP concentrations in
the

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assay mixture were varied between 10 and 400 pM. For K., Ki=Kon, and KA=Koff
value determination 2 mM HPP was used. All assays were started by the addition
of
HPPD enzyme to the assay mixture and stopped at a series of times between 0
and
240 s by addition of 200 pl of the reaction mixture to reaction assay tubes
containing
__ 20 p110% perchloric acid. Precipitated protein was pelleted by a 5 minute
centrifugation at 10,000 g. 100 pl of the supernatant were loaded onto a 250 x
4mm
Knauer (Berlin, Germany) Eurospher 100-5 C18-column equilibrated with 10%
methanol, 0.1% trifluoroacetic acid (buffer A). The column was eluted, also at
1.5
ml/min, using a 4 minute wash with buffer A, followed by a 3 min wash with 95%
__ methanol and by a further 2 minute wash with buffer A. The elution of HGA
(homogentisic acid) and HPP (hydroxyphenylpyruvate) was monitored at 292 nm.
HGA elutes at around 5 minutes and HPP elutes later. A standard set of
concentrations of HGA were used to provide a standard curve in order to
calibrate
the 292 nm absorbance of the HGA peak versus HGA concentration.
__ For Km and Võax value determinations the initial rates of the HPPD reaction
at
different substrate concentrations were determined from plots of HGA formed
versus
time and fitted to the Michaelis-Menten equation for unireactant enzymes using
the
ID Business Solutions Ltd. (www.idbs.com) XLfit software suite. For the
determination of Ki, Ki=Kon, and K.1=K0ff values the time-courses of the HPPD
__ reaction at different inhibitor concentrations were fitted to the equations
for
Mechanism A, competitive inhibition, for tight-binding inhibitors (Cha, S.
(1975) Tight-
binding inhibitors ¨ I. Kinetic behaviour. Biochemical Pharmacology 24, 2177-
2185)
using the ID Business Solutions Ltd. XLfit software suite
__ Table 8
Protein SEQ ID No. 32 consists of protein SEQ ID No. 2 with an insertion of
one
amino acid Ala and six amino acids His directly behind the first methionine.
Kinetic characterization of HPPD enzymes (Arabidopsis thaliana "SEQ ID No. 32"
__ and the respective tolerance to the HPPD inhibitors tembotrione and
diketonitrile.
In below given table1, "Km" (Michaelis-Menten constant) means the kinetic
parameter
that is used to characterise an enzyme, and ills defined as the concentration
of

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109
substrate that permits half maximal rate of the reaction. Km is further
defined as the
substrate concentration at which the reaction rate reaches half of its maximum
value
(Vmax/2) where Vmax has the meaning of being the maximum velocity of the
reaction.
'<0n-A(1 equals the association rate constant of the enzyme-substrate binding
and
Kaff=KA equals the rate constant of the enzyme-inhibitor complex dissociation.
Ki
defines the inhibition constant.
HPP Tembotrione Diketonitrile
Km Vmax k1 k1 Kik1 k1K1
(PM) (IiM) (M-1 s-1) (s-1) (pM) (M-1 s-1) (s-1)
(1A11)
SEQ ID 6.3 1.2 2.3E+05 3.5E-03 0.015 6.1E+05 1.1E-02 0.018
No. 32
The kinetic parameters Km and Vmax of the plant HPPD "SEQ ID No. 32"
summarized
in Table 8 are well in the range of the specific activities measured from
purified plant
HPPD as for example for the carrot HPPD (Garcia et al. 2000, Biochemistry, 39,

7501-7507). The Arabidopsis thaliana HPPD is also sensitive in the smimilar
range
as the carrot HPPD to diketonitrile. It is the first time, that the kinetic
parameter of
Arabidopsis thaliana HPPD in presence of tembotrione are measured and being in
a
comparable range as the one measured in presence of the HPPD inhibitor
diketonitrile.
Determination of HPPD activity in presence of several HPPD inhibitors
In this content, plarvalue means the log value of the concentration of
inhibitor
necessary to inhibit 50% of the enzyme activity in molar concentration.
p150-values for HPPD inhibitors were determined from dose-response plots of
HPPD
activity versus inhibitor concentration using the assay extensively described
in
WO 2009/144079 at 2 mM fixed HPP concentration and 3 minutes fixed incubation
time using the ID Business Solutions Ltd. XLfit software suite.

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110
Table 9: Determination of pI50 HPPD enzymes (Arabidopsis (haliana "SEQ ID
No. 32" and some Arabidopsis HPPD single point mutants "SEQ ID No. 35,
SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44, SEQ ID No, 46, SEQ ID No. 48, and
SEQ ID No. 50") and their respective tolerance to the several listed below
HPPD
inhibitors tembotrione, diketonitrile, mesotrione, bicyclopyrone,
pyrasulfotole,
sulcotrione, pyrazolate, tefuryltrione, and benzofenap. The symbol ">" means
that
the value was far higher than the one indicated but could not be precisely
calculated
within in the range of concentration of inhibitor tested (2.5x10-6, 5.0x10-6,
1.0x10'5,
2.5x10-5, 6.3x10-5, and 2.5x10-4M).
Bicyclopyrone Benzofenap Diketonitrile Mesotrione Pyrasulfotole
SEQ ID No. 32 5.2 >5.6 >5.6 >5.6 5.4
E252S 4.3 5.2 4.9 5.3 4.3
(SEQ ID No. 36)
V269A 4.3 5.2 5.2 5.2 4.4
(SEQ ID No. 40)
P280A 4.3 5.2 5.2 5.3 4.4
SEQ ID No. 42
L368M 4.5 >5.6 5.6 5.4 5.0
SEQ ID No. 44
K4211 4.2 5.1 5.0 5.2 4.3
SEQ ID No. 46
K421Q 4.7 >5.6 >5.6 >5.6 4.8
SEQ ID No. 48
K421V 4.2 5.2 5.1 5.3 4.4
SEQ ID No. 50

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111
Pyrazolat Sulcotrione Tefuryltrione Ternbotrione
SEQ ID No. 32 5.4 >5.6 >5.6 >5.6
E2525 4.7 5.1 5.2 5.5
(SEQ ID No. 35)
V269A 4.4 5.0 5.4 >5.6
(SEQ ID No. 40)
P280A 4.7 5.0 ' 5.5 >5.6
SEQ ID No. 42
L368M 4.9 5.2 >5.6 >5,6
SEQ ID No. 44
- K421I 4.5 5.0 5.2 5.6
SEQ ID No. 46
K421Q 5.0 >5.6 >5.6 >5.6
SEQ ID No. 48
K421V 4.5 5.1 5.4 >5.6
SEQ ID No, 50
Measurements show that the pI50 in case of the point mutants is reduced
compared
to the wild-type protein_
In Table 9, it can be clearly seen that the HPPD mutants are all affected in
their
tolerance to at least one HPPD inhibitors and some to several HPPD inhibitors.
All
the tested mutants are more active (do show an increased tolerance concerning
the
various applyied HPPD inhibitors) in the presence of at least one of the
selected
HPPD inhibitors, therefore the mutant enzyme-inhibitor complex should be less
stable the wild-type enzyme-inhibitor complex. It can be concluded that the
positions
252, 269, 280, 368 and 421 (reference taken in SEQ ID No. 2) are all important
in
the modulation of tolerance of HPPD enzyme to HPPD inhibitors.

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111a
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this
description contains a sequence listing in electronic form in ASCII
text format (file: 30725-1494 Seq 13-MAR-13 vl.txt).
A copy of the sequence listing in electronic form is available from
the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are
reproduced in the following table.
SEQUENCE TABLE.
<110> Bayer CropScience AG
<120> HPPD variants and methods of use
<130> 30725-1494
<140> To be assigned
<141> 2013-03-05
<160> 50
<170> PatentIn version 3.3
<210> 1
<211> 1335
<212> DNA
<213> Arabidopsis thaliana
<400> 1
atgggccacc aaaacgccgc cgtttcagag aatcaaaacc atgatgacgg cgctgcgtcg 60
tcgccgggat tcaagctcgt cggattttcc aagttcgtaa gaaagaatcc aaagtatgat 120
aaattcaagg ttaagcgctt ccatcacatc gagttctggt gcggcgacgc aaccaacgtc 180
gctcgtcgct tctcctgggg tctggggatg agattctccg ccaaatccga tctttccacc 240
ggaaacatgg ttcacgcctc ttacctactc acctccggtg acctccgatt ccttttcact 300
gctccttact ctccgtctct ctccgccgga gagattaaac cgacaaccac agcttctatc 360
ccaaqtttcq atcacggctc ttgtcgttcc ttcttctctt cacatggtct cggtgttaga 420
gccgttgcga ttgaagtaga agacgcagag tcagctttct ccatcagtgt agctaatggc 480
gctattcctt cgtcgcctcc tatcgtcctc aatgaagcag ttacgatcgc tgaggttaaa 540
ctatacggcq atgttgttct ccgatatgtt agttacaaag cagaagatac cgaaaaatcc 600
gaattcttgc cagggttcga gcgtgtagag gatqcgtcgt cgttcccatt ggattatggt 660
atccggcggc ttgaccacgc cgtgggaaac gttcctgagc ttggtccggc tttaacttat 720
gtagcggggt tcactggttt tcaccaattc gdagagttca cagcagacga cgttggaacc 780
gccgagagcg gtttaaattc agcggtcctg gctagcaatg atgaaatggt tcttctaccg 840
attaacgagc cagtgcacgg aacaaagagg aagagtcaga ttcagacgta tttggaacat 900
aacgaaggcg cagggctaca acatctggct ctgatgagtg aagacatatt caggaccctg 960
agagagatga ggaagaggag cagtattgga ggattcgact tcatgccttc tcctccgcct 1020
acttactacc agaatctcaa gaaacgggtc ggcgacgtgc tcagcgatga tcagatcaag 1080
gagtgtgagg aattagggat tcttgtagac agagatgatc aagggacgtt gcttcaaatc 1140

CA 02808152 2013-03-27
111b
ttcacaaaae cactaggtga caggccgacg atatttatag agataatcca gagagtagga 1200
tgcatgatga aagatgagga agggaaggct taccagagtg gaggatgtgg tggttttggc 1260
aaaggcaatt tctctgagct cttcaagtec attgaagaat acgaaaagac tcttgaagcc 1320
aaacagttag tggga 1335
<210> 2
<211> 445
<212> PRT
<213> Arabidopsis thalisns
<400> 2
Met Gly His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp
1 5 10 15
Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe
20 25 30
Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His
35 40 45
His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe
50 55 60
Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Set Thr
65 70 75 80
Gly Asn Met Val His Ala Ser Tyr Leu Lou Thr Ser Gly Asp Leu Arg
85 90 95
Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile
100 105 110
Lys Pro Thr Thr Thr Ala Scr Ile Pro Ser Phe Asp His Gly Ser Cys
115 120 125
Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile
130 135 140
Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly
145 150 155 160
Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile
165 170 175
Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr
160 185 190
Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg
195 200 205
Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Lou
210 215 220
Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr
225 230 235 240
Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp
245 250 255
Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser
260 265 270
Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr
275 280 285
Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala
290 295 300
Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu
305 310 315 320
Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro
325 330 335
Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp
340 345 350

CA 02808152 2013-03-27
111c
Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu
355 360 365
Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro
370 375 380
Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly
385 390 395 400
Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys
405 410 415
Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu
420 425 430
Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly
435 440 445
<210> 3
<211> 1341
<212> DNA
<213> Oryza sativa
<400> 3
atgcctccca ctcccacccc caccgccacc accggcgccg tctcggccgc tgcggcggcg 60
ggggagaacg cggggttccg cctcgtcggg caccgccgct tcgtccgcgc caacccgcgg 120
agcgaccggt tccaggcgct cgcgttccac cacgtogagc tctqgtgcgc cgacgccgcg 180
tccgccgcgg gccggttcgc cttcgccctg ggcgcgccgc tcgccgccag gtccgacctc 240
tccacgggga actccgcgca cgcctccctc ctcctccgct ccgcctccgt cgcgttcctc 300
ttcaccgccc cctacggcgg cgaccacggc gtcggcgcgg acgcggccac caccgcctcc 360
atcccttcct tctccccagg cgccgcgcgg aggttcgccg cggaccacgg cctcgcggtg 420
cacgccgtgg cgctgcgcgt cgccgacgcg gccgacgcct tccgcgccag cgtcgcggcc 480
ggtgcgcgcc cggcgttcca gcccgccgac ctcggcqgtg gcttcggcct cgcggaggtg 540
gagctctacg gcgacgtcgt gctccgattc gtcagccacc cggacggcgc cgacgcgccc 600
ttcctcccgg gtttcgaggg cgtcagcaac ccgggcgccg tggactacgg cctccgccgg 660
ttcgaccacg tcgtcggcaa cgtgccggag ctcgctccgg tagccgcgta catctccggg 720
ttcaccgggt tccacgagtt cgccgagttc accgccgagg acgtgggcac cgccgagagc 780
ggcctcaact cggtggtgct cgccaacaac geggagaccg tgctgctgcc gctcaacgag 840
ccggtgcacg gcaccaagcg gcggagccag atacagacgt acctggacca ccacggcggc 900
ccgggggtgc agcacatcgc gctggccagc gacgacgtgc tcgggacgct gagggagatg 960
cgggcgcgct ccgccatggg cggcttcgag ttcttggcgc cgccgccgcc caactactac 1020
gacggcgtgc ggcggcgcgc cggggacgtg ctctcggagg agcagatcaa cgagtgccag 1080
gagctcgggg tgctcgtqga cagggatgac cagggqgtgt tgctccagat cttcaccaag 1140
ccagtaggag acaggccaac ctttttcttg gagatgatac aaaggattgg gtgcatggag 1200
aaggatgaga gtgggcagga gtaccagaag ggcggctgcg gcgggtttgg gaagggcaac 1260
ttctcggagc tgttcaagtc cattgaggag tatgagaaat cccttgaagc caagcaagcc 1320
cctacagttc aaggatccta g 1341
<210> 4
<211> 446
<212> PRT
<213> Oryza sativa
<400> 4
Met Pro Pro Thr Pro Thr Pro Thr Ala Thr Thr Gly Ala Val Ser Ala
1 5 10 15
Ala Ala Ala Ala Gly Glu Asn Ala Gly Phe Arg Lou Val Gly His Arg
20 25 30

CA 02808152 2013-03-27
111d
Arg Phe Val Arg Ala Asn Pro Arg Ser Asp Arg Phe Gln Ala Leu Ala
35 40 45
Phe His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly
50 55 60
Arg Phe Ala Phe Ala Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu
65 70 75 80
Ser Thr Gly Asn Ser Ala His Ala Ser Leu Leu Leu Arg Ser Ala Ser
85 90 95
Val Ala Phe Leu Phe Thr Ala Pro Tyr Gly Gly Asp His Gly Val Gly
100 105 110
Ala Asp Ala Ala Thr Thr Ala Ser Ile Pro Ser Phe Ser Pro Gly Ala
115 120 125
Ala Arg Arg Phe Ala Ala Asp His Gly Leu Ala Val His Ala Val Ala
130 135 140
Leu Arg Val Ala Asp Ala Ala Asp Ala Phe Arg Ala Ser Val Ala Ala
145 150 155 160
Gly Ala Arg Pro Ala Phe Gln Pro Ala Asp Leu Gly Gly Gly Phe Gly
165 170 175
Leu Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu Arg Phe Val Ser
180 185 190
His Pro Asp Gly Ala Asp Ala Pro Phe Leu Pro Gly Phe Glu Gly Val
195 200 205
Ser Asn Pro Gly Ala Val Asp Tyr Gly Leu Arg Arg Phe Asp His Val
210 215 220
Val Gly Asn Val Pro Glu Leu Ala Pro Val Ala Ala Tyr Ile Ser Gly
225 230 235 240
Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp Val Gly
245 250 255
Thr Ala Glu Ser Gly Leu Asn Ser Val Val Leu Ala Asn Asn Ala Glu
260 265 270
Thr Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg
275 280 285
Ser Gln Ile Gln Thr Tyr Leu Asp His His Gly Gly Pro Gly Val Gln
290 295 300
His Ile Ala Leu Ala Ser Asp Asp Val Lau Gly Thr Leu Arg Glu Met
305 310 315 320
Arg Ala Arg Ser Ala Met Gly Gly Phe Glu Phe Leu Ala Pro Pro Pro
325 330 335
Pro Asn Tyr Tyr Asp Gly Val Arg Arg Arg Ala Gly Asp Val Leu Ser
340 345 350
Glu Glu Gln Ile Asn Glu Cys Gln Glu Leu Gly Val Leu Val Asp Arg
355 360 365
Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp
370 375 380
Arg Pro Thr Phe Phe Leu Glu Met Ile Gln Arg Ile Gly Cys Met Glu
385 390 395 400
Lys Asp Glu Ser Gly Gln Glu Tyr Gln Lys Gly Gly Cys Gly Gly Phe
405 410 415
Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu
420 425 430
Lys Ser Leu Glu Ala Lys Gln Ala Pro Thr Val Gln Gly Ser
435 440 445
<210> 5
<211> 1281

SLT OLT G91
b fle nTD ATO ETV PTV ATO dsy oaa aAy JeS TEA aAL ay a TPA TEA dsV
091 881 OgT G6I
ATO JAI nai nTO TPA nTD eIV na7 6IV aqd AID blci aqd PTV dgV nTD
OT GET OEI
PTV dsy 2TV TEA ETV naq PTV TPA PTV 6IV TPA PIV ne7 ATE) sTH dsy
SZI OZT GTT
ETV ETV 214d bali 6JV eTV PTV eTV eTV leS aqd ass oad neq PTV PTV
OTT SOT 001
J1-11, erd EDI ciGV uTV AID sTH eTV JAI 0.1d eTV qL qd naq aqd asS
86 06 58
naq las ATE) aes bay naq narl neri aes eTV sTH ETV IBS nsV AID agI
08 SL OL 89
las nari dsv las 61V PTV PTV nal Old PTV AID nP7 ATD a-Td 19S GU
09 GS OS
SIV ATE) PTV PTV JaS ETV ETV dsv sTy sA3 dai neq GTE) TEA STH Sill
cf7 017 ge
e-Td PTV nal 6LGTH Pqd 6.71/ ciski Jas 6.1v ()ad usv aqa 611/ WA sqd
OE SZ
usy bJv sTH ATD TPA naq 6av aqd PTV eTV nie nTD PTV PTV aaS uTV
SI OT
ETV ETV TEA ETV ETV ATE) ETV ETV ETV ETV aql, Ol J1L Old Old ;aW
9 <00>
eiTem eez <ETz>
LEd <ZTZ>
9Zt <VIZ>
9 <OTZ>
18ZI b eqoo4ebbbe
o4obeo54ob
09ZT eoftobaoba obeeobeepo buebqq000q bre5e54e4-4 ebbeb24epo 4b8e244b4o
00ZI bP05040143 882556eecb boT4656365 0640bbqbb6 82E83O2428 6e2056b6ee
OPTI 6854Ebbee6 2bbqp0b466 bo4ebbeee0 oqe04ee266 .440446q0b0 2e33bbe026
0901 66664152006
2200E,0440; ppe33q0640 64636bbv30 26qebbbe3e bbq56-40646
00-[ 6b6u405ef.6 epobqbebbe eqlebeopob eebbneoqob qboebbbboo 606366056e
096 b4bo5bDe5l
240e4De5o0 4e0e0o3qo0 bobbqeo4ab ebo;4obbob bbleoobbaq
006 obababOeob
qpbe666rE; oboebbeoao 6450260860 6e00664obo 66qe0e06eo
08 6460E6000o
563560epou 03ebb40344 608280E286 P006206306 0622302356
QSL 0e364bb336
ebouu34060 3343540645 3e8beb5340 2v38203534 061564u334
OZL peepqDpbbo
5268560600 eobbb45oe5 fiebboeboeo 1.q.be5Do5ol abeftepoqg
099 585502oqq.0
EtooLoqwe 4op5o5oo5 popbobbwb e563364602 236634E104e
009 oeopeboqab
5e36264356 50e-4086005 606E1563000 60554605 bbrboqqbbb
06G 63364034.10
30bpbobb60 6036056025 b3o0u43be6 qbop466004 3646346086
08V 356peq3436
eb3qb6ebo3 63qopelpoq4 0663633443 3613-ebbu600 53'96336346
017 oboowbobo
qboobibobq bbobowobb ovoceboobe oboai.obobb oboboDboob
09E o0b03q3443 0-
4.0005463 5306=2=6 l0boph0060 65oe0En50e qopobobboe
ou 34.4p4004qo
oloapoolob boowbopao baobabooqb obbeaboboo aoveDbbboe
0D,z. 334oqopeb0
owb0e0b00 6340633606 3666433660 41.paioqqob 3366636306
081 opqopbbpbo
8600606466 434.36266.46 3e30e33446 3bo4o6383e 3044353386
OZT 0343635000
ee344015334 boagoeepbo peoobbbgbo qoobooggbo bbobbeobeb
09 bobeobeoge
0653653564 6 06=8356 3062350363 obeoeoppop eboopoobae
G <006>
sAem 882 <-2>
VNO <ZTZ>
ITT
/2-E0-ETOZ Z5T808Z0

CA 02808152 2013-03-27
111f
Pro Phe Leu Pro Gly She Glu Gly Val Ala Ser Pro Gly Ala Ala Asp
180 185 190
Tyr Gly Leu Ser Arg Phe Asp His Ile Val Gly Asn Val Pro Glu Leu
195 200 205
Ala Pro Ala Ala Ala Tyr Phe Ala Gly She Thr Gly Phe His Glu Phe
210 215 220
Ala Glu Phe Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn
225 230 235 240
Ser Met Val Leu Ala Asn Asn Ser Glu Asn Val Leu Leu Pro Leu Asn
245 250 255
Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu
260 265 270
Asp His His Gly Gly Pro Gly Val Gin His Met Ala Leu Ala Ser Asp
275 280 285
Asp Val Leu Arg Thr Leu Arg Glu Met Gin Ala Arg Ser Ala Met Gly
290 295 300
Gly She Glu She Met Ala Pro Pro Thr Ser Asp Tyr Tyr Asp Gly Val
305 310 315 320
Arg Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gin Ile Lys Glu Cys
325 330 335
Gin Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu
340 345 350
Gin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Lou Phe Leu Glu
355 360 365
Ile Ile Gin Arg Ile Gly Cys Met Glu Lys Asp Glu Lys Gly Gin Glu
370 375 380
Tyr Gin Lys Gly Gly Cys Gly Gly She Gly Lys Gly Asn Phe Ser Gin
385 390 395 400
Leu She Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin
405 410 415
Ala Ala Ala Ala Ala Ala Ala Gin Gly Ser
420 425
<210> 7
<211> 1323
<212> DNA
<213> Avena sativa
<400> 7
atgccgccca cccccgccac cgccaccggc gccgccgcgg ccgccgtgac tccagagcac 60
gcggcccgga gctttccccg agtggtccgc gtcaacccgc gcagcgaccg attcbccgtg 120
ctctccttcc accacgtcga gctctggtgc gccgacgccg cctcagcggc cggacgcttc 180
tccttcgcgc tcggcgcgcc gctcgccgcc cggtccgacc tctccacggg gaactccgcg 240
cacgcctccc tcctgctccg ctcgggcgcc ctcgccttcc tcttcacggc gccctacgcg 300
ccgccgccgc aggaggccgc cacggccgca gccaccgcct ccatcccctc cttctccgcc 360
gacgccgcgc ggacgttcgc cgccgcccac ggcctcgcgg tgcgctccgt cggggtbcgc 420
gtcgctgacg ccgccgaggc cttccgcgtc agcgtagccg gcggcgctcg cccggccttc 480
gccccagccg acctcggcca tggcttcggc ctcgccgagg tcgagctcta cggcgacgtc 540
gtgctacgct tcgtcagcta cccggacgag acagacctgc cattcctgcc agggttcgag 600
cgcgtgagca gccccggcgc cgtggactac ggcctcacgc ggttcgacca cgtcgtgggc 660
aacgtcccgg agatggcccc ggtcatagac tacatgaaag gcttcttggg gttccacgag 720
ttcgccgagt tcaccgccga ggacgtgggc acgaccgaga gcgggctcaa ctcggtggtg 780
ctcgccaaca actccgaggc cgtgctgctg ccgctcaacg agcccgtgca cggcacaaag 840
cgacggagcc agatacagac gtacctggag tatcacggcg ggcccggcgt gcagcacatc 900
gcgctcgcca gcaacgacgt gctcaggacg ctcagggaga tgcgggcgcg cacgcccatg 960

CA 02808152 2013-03-27
111g
ggcggcttcg agttcatggc gccaccgcag gcgaaatact atgaaggcgt gcggcgcatc 1020
gcaggtgacg tgctctcgga agagcagatc aaggaatgcc aggagctggg ggtgctagtc 1080
gacagggatg atcaaggggt gttgctccaa atcttcacca agccagtagg ggacaggcca 1140
acgtttttcc tggagatgat ccaaagaatc gggtgcatgg agaaggacga ggtcgggcaa 1200
gagtaccaga agggtggctg cggcgggttt ggcaagggca atttctccga gctgttcaag 1260
tccattgagg actatgagaa atcccttgag gtcaagcaat ctgttgtagc tcagaaatcc 1320
tag 1323
<210> 8
<211> 440
<212> PRT
<213> Avena sativa
<400> 8
Met Pro Pro Thr Pro Ala Thr Ala Thr Gly Ala Ala Ala Ala Ala Vol
1 5 10 15
Thr Pro Glu His Ala Ala Arg Ser Phe Pro Arg Vol Val Arg Val Asn
20 25 30
Pro Arg Ser Asp Arg Phe Pro Val Leu Ser Phe His His Val Glu Leu
35 40 45
Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Leu
50 55 60
Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala
65 70 75 80
His Ala Ser Leu Leu Leu Arg Ser Gly Ala Leu Ala Phe Leu Phe Thr
85 90 95
Ala Pro Tyr Ala Pro Pro Pro Gin Glu Ala Ala Thr Ala Ala Ala Thr
100 105 110
Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Ala Arg Thr Phe Ala Ala
115 120 125
Ala His Gly Leu Ala Val Arg Ser Val Gly Val Arg Val Ala Asp Ala
130 135 140
Ala Glu Ala Phe Arg Val Ser Val Ala Gly Gly Ala Arg Pro Ala Phe
145 150 155 160
Ala Pro Ala Asp Leu Gly His Gly Phe Gly Leu Ala Glu Vol Glu Leu
165 170 175
Tyr Gly Asp Vol Val Leu Arg Phe Vol Ser Tyr Pro Asp Glu Thr Asp
180 185 190
Leu Pro Phe Leu Pro Gly Phe Glu Arg Val Ser Ser Pro Gly Ala Val
195 200 205
Asp Tyr Gly Leu Thr Arg Phe Asp His Val Val Gly Asn Vol Pro Glu
210 215 220
Met Ala Pro Vol Ile Asp Tyr Met Lys Gly Phe Leu Gly Phe His Gin
225 230 235 240
Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Leu
245 250 255
Asn Ser Val Vol Leu Ala Asn Asn Ser Glu Ala Val Leu Leu Pro Leu
260 265 270
Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Tyr
275 280 285
Leu Glu Tyr His Gly Gly Pro Gly Val Gln His Ile Ala Leu Ala Ser
290 295 300
Asn Asp Vol Leu Arg Thr Leu Arg Glu Met Arg Ala Arg Thr Pro Met
305 310 315 320

CA 02808152 2013-03-27
111h
Gly Gly Phe Glu Phe Met Ala Pro Pro Gin Ala Lys Tyr Tyr Glu Gly
325 330 335
Val Arg Arg Ile Ala Gly Asp Val Leu Ser Glu Glu Gin Ile Lys Glu
340 345 350
Cys Gin Glu Leu Gly Val Lou Val Asp Arg Asp Asp Gin Gly Val Leu
355 360 365
Leu Gin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Phe Phe Lou
370 375 380
Glu Met Ile Gin Arg Ile Gly Cys Net Giu Lys Asp Glu Val Gly Gin
385 390 395 400
Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser
405 410 415
Clu Lou Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Lou Glu Val Lys
420 425 430
Gin Ser Val Val Ala Gin Lys Ser
435 440
<210> 9
<211> 1077
<212> DNA
<213> Pseudomonas fluorescens
<400> 9
atggcagatc tatacgaaaa cccaatgggc ctgatgggct ttgaattcat cgaattcgcg 60
tcgccgacgc cgggtaccct ggagccgatc ttcgagatca tgggcttcac caaagtcgcg 120
acccaccgtt ccaagaacgt gcacctgtac cgccagggcg agatcaacct gatcctcaac 180
aacgagccca acagcatcgc ctcctacttt gcggccgaac acggcccgtc ggtgtgcggc 240
atggcgttcc gcgtgaagga ctcgcaaaag gcctacaacc gcgccctgga actcggcgcc 300
cagccgatcc atattgacac cgggccgatg gaattgaacc tgccggcgat caagggcatc 360
ggcggcgcgc cgttgtacct gatcgaccgt ttcggcgaag gcagctcgat ctacgacatc 420
gacttcgtgt acctcgaagg tgtggagcgc aatccggtcg gtgcaggtct caaagtcatc 480
gaccacctga cccacaacgt cLatcgcgyc cycaLggtct actgggccaa cttctacgag 540
aaattgttca acttccgtga agcgcgttac ttcgatatca agggcgagta caccggcctg 600
acttccaagg ccatgagtgc gccggacggc atgatccgca tcccgctgaa cgaagagtcg 660
tccaagggcg cggggcagat cgaagagttc ctgatgcagt tcaacggcga aggcatccag 720
cacgtggcgt tcctcaccga cgacctggtc aagacctggg acgcgttgaa gaaaatcggc 780
.atgcgcttca tgaccgcgcc gccagacact tattacgaaa tgctcgaagg ccgcctgcct 840
gaccacggcg agccggtgga tcaactgcag gcacgcggta tcctgctgga cggatcttcc 900
gtggaaggcg acaaacgcct gctgcLgcag atcttctcgg aaaccctgat gggcccggtg 960
ttcttcgaat tcatccagcg caagggcgac gatgggtttg gcgagggcaa cttcaaggcg 1020
ctgttcgagt ccatcgaacg tgaccaggtg cgtcgtggtg tattgaccgc cgattaa 1077
<210> 10
<211> 358
<212> PRT
<213> Pseudomonas fluorescens
<400> 10
Met Ala Asp Leu Tyr Glu Asn Pro Met Gly Lou Met Cly Phe Glu Phe
1 5 10 15
Ile Glu Phe Ala Ser Pro Thr Pro Gly Thr Lou Glu Pro Ile Phe Glu
20 25 30
Ile Met Gly Phe Thr Lys Val Ala Thr His Arg Ser Lys Asn Val His
35 40 45

CA 02808152 2013-03-27
111i
Leu Tyr Arg Gin Gly Glu Ile Asn Leu Ile Leu Asn Asn Glu Pro Asn
50 55 '60
Ser Ile Ala Ser Tyr Phe Ala Ala Glu His Gly Pro Ser Val Cys Gly
65 70 75 20
Met Ala Phe Arg Val Lys Asp Ser Gin Lys Ala Tyr Asn Arg Ala Leu
85 90 95
Glu Leu Gly Ala Gin Pro Ile His Ile Asp Thr Gly Pro Met Glu Leu
100 105 110
Asn Leu Pro Ala Ile Lys Gly Ile Gly Gly Ala Pro Leu Tyr Leu Ile
115 120 125
Asp Arg Phe Gly Glu Gly Ser Ser Ile Tyr Asp Ile Asp Phe Val Tyr
130 135 140
Leu Glu Gly Val Glu Arg Asn Pro Val Gly Ala Gly Leu Lys Val Ile
145 150 155 160
Asp His Leu Thr His Asn Val Tyr Arg Gly Arg Met Val Tyr Trp Ala
165 170 175
Asn Phe Tyr Glu Lys Leu Phe Asn Phe Arg Glu Ala Arg Tyr Phe Asp
180 185 190
Ile Lys Gly Glu Tyr Thr Gly Leu Thr Ser Lys Ala Met Ser Ala Pro
195 200 205
Asp Gly Met Ile Arg Ile Pro Leu Asn Glu Glu Ser Ser Lys Gly Ala
210 215 220
Gly Gin Ile Glu Glu Phe Leu Met Gin Phe Asn Gly Glu Gly Ile Gin
225 230 235 240
His Val Ala Phe Leu Thr Asp Asp Leu Val Lys Thr Trp Asp Ala Leu
245 250 255 =
Lys Lys Ile Gly Met Arg Phe Met Thr Ala Pro Pro Asp Thr Tyr Tyr
260 265 270
Glu Met Leu Glu Gly Arg Leu Pro Asp His Gly Glu Pro Vol Asp Gin
275 280 285
Leu Gin Ala Arg Gly Ile Leu Leu Asp Gly Ser Ser Val Glu Gly Asp
290 295 300
Lys Arg Leu Leu Leu Gin Ile Phe Ser Glu Thr Leu Met Gly Pro Val
305 310 315 320
Phe Phe Glu Phe Ile Gin Arg Lys Gly Asp Asp Gly Phe Gly Glu Gly
325 330 335
Asn Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Asp Gin Val Arg Arg
340 345 350
Gly Val Leu Thr Ala Asp
355
<210> 11
<400> 11
000
<210> 12
<400> 12
000

CA 02808152 2013-03-27
111j
<210> 13
<211> 1053
<212> DNA
<213> Synechococcus sp.
<400> 13
atgaacccgt ccattcgaat tgtccaaggg atccaccacc tgcacttcta cctttgggat 60
ctgccccgtt ggcgggaaca cttttgtcgg qtttqqggct tccgggtggc aagcgacgcc 120
ggcaacaccc tggagctgga gcagggatcc ctgcgcttgc gcctgtctca gccggcacgg 180
gcgggggacg aggtggaccg ccatttgcag cggcaLgggc cgggggtggt ggatgtggcc 240
ttggcggtgg gagagcagga gctaccggcc ttggcggagc tgttgcgggg ccgaggcgcc 300
caactggcgt ggatcccggc agcagcggcg ctctgcctcc acacccccta cgggatCcgg 360
cattctctga tocctggccc cttggatgcc gcccctgccg aagcgggcct gttttcccac 420
tgggatcacg tggtgttgaa cgtggagcag ggatccctgc aggcggcagc cgactggtat 480
gggcgggtgc tgggctggcg gcggctgtac cgctacagca tcggcaccgc cacctccggc 540
ctggaaagcg tggtggtggg ggatccggaa gcggggatcc aatgggccat caacgagccc 600
acctgtgccg ettcccagat tcaggagttt ttgcatgccc atggcggccc gggcattcag 660
cacgcggcgc tgcacagctc agacattgtt gccagcctgc gccggttgcg gcagggggga 720
gtggactttt tgcaagtggc gccgcagtac tacaccagcc tggaaaggga gctggggttg 780
gcgctccgtt ctgcccttgg gcaggccatc tcctggcaag acctggtgga gcagcagatc 840
cttctggatg ctaccctgcc cgcttctgat ggccaggatc gcccccttct gctgcagacc 900
tttacccagc ccctctttgg tcggcccacc tttttctttg aagtcattca acggctaggc 960
ggggccacgg gctttggcga ggccaatttt caggctttgt tcgaggccct ggaacggcaa 1020
cagcgacagc gacaccaqgc gctgacccct tag 1053
<210> 14
<211> 350
<212> PRT
<213> Synechococcus sp.
<400> 14
Net Asn Pro Ser Ile Arg Ile Vol Gin Gly Ile His His Leu His Phe
1 5 10 15 =
Tyr Leu Trp Asp Leu Pro Arg Trp Arg Glu His Phe Cys Arg Val Trp
20 25 30
Gly Phe Arg Val Ala Ser Asp Ala Gly Asn Thr Leu Glu Leu Glu Gin
35 40 45
Gly Ser Leu Arg Leu Arg Leu Ser Gin Pro Ala Arg Ala Gly Asp Glu
50 55 60
Vol Asp Arg His Leu Gin Arg His Gly Pro Gly Val Val Asp Val Ala
65 70 75 80
Leu Ala Val Gly Glu Gin Glu Leu Pro Ala Leu Ala Glu Leu Leu Arg
85 90 95
Gly Arg Gly Ala Gin Leu Ala Trp Ile Pro Ala Ala Ala Ala Leu Cys
1.00 105 110
Leu His Thr Pro Tyr Gly Ile Arg His Ser Leu Ile Pro Gly Pro Leu
115 120 125
Asp Ala Ala Pro Ala Glu Ala Gly Leu Phe Ser His Trp Asp His Val
130 135 140
Val Leu Asn Vol Glu Gin Gly Ser Leu Gin Ala Ala Ala Asp Trp Tyr
145 150 155 160
Gly Arg Val Leu Gly Trp Arg Arg Leu Tyr Arg Tyr Ser Ile Gly Thr
165 170 175
Ala Thr Ser Gly Leu Glu Ser Val Val Vol Gly Asp Pro Glu Ala Gly
180 185 190

CA 02808152 2013-03-27
111k
Ile Gin Trp Ala Ile Asn Glu Pro Thr Cys Ala Ala Ser Gin Ile Gin
195 200 205
Glu Phe Leu His Ala His Gly Gly Pro Gly Ile Gin His Ala Ala Leu
210 215 220
His Ser Ser Asp Ile Val Ala Ser Leu Arg Arg Leu Arg Gin Gly Gly
225 230 235 240
Val Asp Phe Leu Gin Val Ala Pro Gin Tyr Tyr Thr Ser Leu Glu Arg
245 250 255
Glu Leu Gly Leu Ala Leu Arg Ser Ala Leu Gly Gin Ala Ile Ser Trp
260 265 270
Gin Asp Leu Val Glu Gin Gin Ile Leu Leu Asp Ala Thr Leu Pro Ala
275 280 285
Ser Asp Gly Gin Asp Arg Pro Lou Leu Leu Gin Thr Phe Thr Gin Pro
290 295 300
Leu Phe Gly Arg Pro Thr Phe Phe Phe Glu Val Ile Gin Arg Leu Gly
305 310 315 320
Gly Ala Thr Gly Phe Gly Glu Ala Asn Phe Gin Ala Leu She Glu Ala
325 330 335
Leu Glu Arg Gin Gin Arg Gin Arg His Gin Ala Leu Thr Pro
340 345 350
<210> 15
<211> 1149
<212> DNA
<213> Biepharisma japonicum
<400> 15
atgacttatt acgacaagca agaaacgcgt ccagatcttg gcgaattcta tggtttccat 60
cacgttcgtt tttacgtctc caactcagag caagccgctt cgttctacac atctcgcttt 120
gggttttctc cggttgccta tgaaggattg gaaacaggaa accaaaaatt ctgtaccaat 180
gtcgtccgaa gcaaccatgt agtcatcgct tttacctcag ctctcactcc tgaagacaat 240
yaagLydauc yLuauyLtyy uaaycaLayL
ydLygdyLLu auyauaLLyu uLLLugLyLa 300
agtgacgcaa gagggatgta tgagaaagcg atagctaaag gctgtaaaag cttccgtgag 360
ccacaggttt tacaagatca atttggatct gttataatag cgtctctcca gacttatgga 420
gacactgttc acacattagt ccaaaatgtc gactatacag gacccttttt gcctggcttc 480
agagcaatca caaaagatga tccattaaac tctgcctttc ctcaggtaaa ttatgacatt 540
attgatcatg ttgtaggaaa tcagcctggt ggcgatatga ctcctacagt agaatggtat 600
gagaaatatc tagaatttca tcgatattgg tctgctgatg agtctgtaat ccataccgat 660
tattcagcat taaggtctgt tgtggttgct gattgggatg aagtgatcaa aatgcctatt 720
aatgagcctg ctgatggact tagaaaaagt caaatccaag aatatgtcga atattatggt 780
ggagcaggcg tacaacatat tgccttaaaa gtcaatgata ttatttcagt aataagcacc 840
ttaagggcta gaggtgtgga attcttagaa gttcctccta aatattatga tagcttaaga 900
aaaagacttg cgcattctgc ggtacaaatt gaagaagact taaaaagaat tgaagacctt 960
catattttgg ttgactttga cgaccgtggg tatttacttc agattttcac aaaaccagta 1020
gaagacagac ctactctgtt ttatgaaatt attcaaagac ataataacaa tggattcgga 1080
attggaaatt ttaaagccct atttgaatca ttggaacaag agcaagaaag aagaggtaat 1140
ttgatctaa 1149
<210> 16
<211> 382
<212> PRT
<213> Blepharisma japonicum

CA 02808152 2013-03-27
1111
<400> 16
Met Thr Tyr Tyr Asp Lys Gin Glu Thr Arg Pro Asp Lou Gly Glu Phe
1 5 10 15
Tyr Gly Phe His His Val Arg Phe Tyr Val Ser Asn Ser Glu Gin Ala
20 25 30
Ala Ser Phe Tyr Thr Ser Arg Phe Gly Phe Ser Pro Val Ala Tyr Glu
35 40 45
Gly Leu Glu Thr Gly Asn Gin Lys Phe Cys Thr Asn Val Val Arg Ser
50 55 60
Asn His Val Val Ile Ala Phe Thr Ser Ala Leu Thr Pro Glu Asp An
65 70 75 80
Glu Val Asn Arg His Val Gly Lys His Ser Asp Gly Val Gin Asp Ile
85 90 95
Ala Phe Ser Val Ser Asp Ala Arg Gly Met Tyr Glu Lys Ala Ile Ala
100 105 110
Lys Gly Cys Lys Ser Phe Arg Glu Pro Gin Val Leu Gin Asp Gin Phe
115 120 125
Gly Ser Val Ile Ile Ala Ser Leu Gin Thr Tyr Gly Asp Thr Val His
130 135 140
Thr Leu Val Gin Asn Val Asp Tyr Thr Gly Pro Phe Leu Pro Gly Phe
145 150 155 160
Arg Ala Ile Thr Lys Asp Asp Pro Leu Asn Ser Ala Phe Pro Gin Val
165 170 175
Asn Tyr Asp Ile Ile Asp His Val Val Gly Asn Gin Pro Gly Gly Asp
180 185 190
Net Thr Pro Thr Val Glu Trp Tyr Glu Lys Tyr Leu Glu Phe His Arg
195 200 205
Tyr Trp Ser Ala Asp Glu Ser Val Ile His Thr Asp Tyr Ser Ala Leu
210 215 220
Arg Ser Val Val Val Ala Asp Trp Asp Glu Val Ile Lys Net Pro Ile
225 230 235 240
Asn Glu Pro Ala Asp Gly Lou Arg Lys Ser Gin Ile Gln Glu Tyr Val
245 250 255
Glu Tyr Tyr Gly Gly Ala Gly Val Gin His Ile Ala Lou Lys Val Asn
260 265 270
Asp Ile Ile Ser Val Ile Ser Thr Leu Arg Ala Arg Gly Val Glu Phe
275 280 285
. Leu Glu Val Pro Pro Lys Tyr Tyr Asp Ser Leu Arg Lys Arg Leu Ala
290 295 300
His Ser Ala Val Gin Ile Glu Glu Asp Leu Lys Arg Ile Glu Asp Leu
305 310 315 320
His Ile Leu Val Asp Phe Asp Asp Arg Gly Tyr Leu Leu Gin Ile Phe
325 330 335
Thr Lys Pro Val Glu Asp Arg Pro Thr Leu Phe Tyr Glu Ile Ile Gin
340 345 350
Arg His Asn Asn Asn Gly Phe Gly Ile Gly Asn Phe Lys Ala Lou Phe
355 360 365
Glu Ser Leu Glu Gin Glu Gin Glu Arg Arg Gly Asn Leu Ile
370 375 380
<210> 17
<211> 1206
<212> DNA
<213> Rhodococcus sp.

CA 02808152 2013-03-27
11 lm
<400> 17
atgacgatcg agcagactct caccgacaag gaacgcctgg caggtctcga cctcggccag 60
ctcgagcagt tggtcgggct cgtcgagtac gacggcaccc gcgacccgtt cccggtcagc 120
ggctgggatg ccgtcgtctg ggtggtcggc aacgccaccc agaccgccca ctacttccag 180
tccgcgttcg ggatgaccct cgtcgcctac tccggaccca ccaccggcaa ccgcgaccac 240
cacagcttcg tcctcgaatc cggggccgtc cgcttcgtca tcaaaggcgc cgtgaacccg 300
gacagccccc tgatcgacca ccaccgcacc cacggcgacg gcgtcgtcga catcgccctc 360
gccgtccccg acgtcgacaa gtgcatcgcc cacgcccgcg cccagggcgc caccgtcctc 420
gacgaacccc acgacgtgac cgacgaccac ggcaccgtcc gcctcgccgc gatcgccacc 480
tacggcgaca cccgccacac cctcgtcgac cgcagccact acaccggccc ctacctgccc 540
ggctacaccg cccgcacctc cggccacacc aaacgggacg gggcacccaa gcgcctgttc 600
caggccctcg accacgtcgt cggcaacgtc gaactcggca agatggacca ctgggtcgac 660
ttctacaacc gggtcatggg ctttacgaac atggccgagt tcgtcggcga ggacatcgcc 720
accgactact ccgcgctgat gagcaaggtc gtctccaacg gcaaccaccg ggtcaagttc 780
cccctcaacg aacccgccct cgccaagaaa cgctcgcaga tcgacgaata cctcgacttc 840
taccgcggcc ccggcgccca gcacctggcc ctggccacca atgacatcct caccgccgtc 900
gaccagctga ccgccgaggg cgtcgagttc ctggccaccc ccgactccta ctacgaggac 960
cccgaactgc gggcccggat cggcaacgtc cgcgccccca tcgccgaact gcagaaacgc 1020
ggcatcctcg tcgaccgcga cgaagacggc tacctgctgc agatcttcac caaacccctc 1080
gtcgaccggc ccaccgtgtt cttcgaactc atcgaacgcc acggctccct cggcttcggc 1140
atcggcaact tcaaagccct cttcgaggcc atcgaacgcg aacaagccgc ccgcggaaac 1200
ttctga 1206
<210> 18
<211> 401
<212> PRT
<213> Rhodococcus sp.
<400> 18
Met Thr Ile Glu Gin Thr Leu Thr Asp Lys Glu Arg Leu Ala Gly Leu
1 5 10 15
Asp Leu Gly Gin Leu Glu Gin Leu Val Gly Leu Val Glu Tyr Asp Gly
20 25 30
Thr Arg Asp Pro Phe Pro Val Ser Gly Trp Asp Ala Val Val Trp Val
35 40 45
Val Gly Asn Ala Thr Gin Thr Ala His Tyr Phe Gin Ser Ala Phe Gly
50 55 60
Met Thr Leu Val Ala Tyr Ser Gly Pro Thr Thr Gly Asn Arg Asp His
65 70 75 80
His Ser Phe Val Leu Glu Ser Gly Ala Val Arg Phe Val Ile Lys Gly
85 90 95
Ala Val Asn Pro Asp Ser Pro Leu Ile Asp His His Arg Thr His Gly
100 105 110
Asp Gly Val Val Asp Ile Ala Leu Ala Val Pro Asp Val Asp Lys Cys
115 120 125
Ile Ala His Ala Arg Ala Gin Gly Ala Thr Val Leu Asp Glu Pro His
130 135 140
Asp Val. Thr Asp Asp His Gly Thr Val Arg Leu Ala Ala Ile Ala Thr
145 150 155 160
Tyr Gly Asp Thr Arg His Thr Leu Val Asp Arg Ser His Tyr Thr Gly
165 170 175
Pro Tyr Leu Pro Gly Tyr Thr Ala Arg Thr Ser Gly His Thr Lys Arg
180 185 190
Asp Gly Ala Pro Lys Arg Leu Phe Gin Ala Leu Asp His Val Val Gly
195 200 205

CA 02808152 2013-03-27
111n
Asn Val Glu Leu Gly Lys Met Asp His Trp Val Asp Phe Tyr Asn Arg
210 215 220
Val Met Gly Phe Thr Asn Met Ala Glu Phe Val Gly Glu Asp Ile Ala
225 230 235 240
Thr Asp Tyr Ser Ala Leu Met Ser Lys Val Val Ser Asn Gly Asn His
245 250 255
Arg Val Lys Phe Pro Lou Asn Glu Pro Ala Lou Ala Lys Lys Arg Ser
260 265 270
Gln Ile Asp Glu Tyr Leu Asp Phe Tyr Arg Gly Pro Gly Ala Gln His
275 280 285
Leu Ala Leu Ala Thr Asn Asp Ile Leu Thr Ala Val Asp Gln Leu Thr
290 295 300
Ala Glu Gly Val Glu Phe Leu Ala Thr Pro Asp Ser Tyr Tyr Glu Asp
305 310 315 320
Pro Glu Leu Arg Ala Arg Ile Gly Asn Val Arg Ala Pro Ile Ala Glu
325 330 335
Leu Gln Lys Arg Gly Ile Leu Val Asp Arg Asp Glu Asp Gly Tyr Leu
340 345 350
Lou Gln Ile Phe Thr Lys Pro Leu Val Asp Arg Pro Thr Val Phe Phe
355 360 365
Glu Leu Ile Glu Arg His Gly Ser Leu Gly Phe Gly Ile Gly Asn Phe
370 375 380
Lys Ala Leu Phe Glu Ala Ile Glu Arg Glu Gln Ala Ala Arg Gly Asn
385 390 395 400
Phe
<210> 19
<211> 1107
<212> DNA
<213> Picrophilus torridus
<400> 19
atgtatggca aaaatttaat ctcagaacta agggaaaagg agatctttaa acgattacat 60
cacgtggaat tttacgttag cagtgccaaa acatggtcat atttcatgaa caggggtctt 120
ggatttaaaa cagtggcata tgccggtcca gaaaccggga taagggacaa gatatcctat 180
gttatgtccc agggcactgc aaggatatct tttacatcat caatgaatga tgatagctat 240
atatcgaatc atgttaaaaa acacggggat ggcgtaaagg atatagcact tgaggtcgat 300
gatctggacg aggcaaaaag cctgatagaa aagtatggaa caaaggtttc aaaaataaat 360
gaaataaagg atggaaatgg aaagataaga actgcagaga taaaaacgta cggtgaaacc 420
gttcatacat taatagaaac cggggattac aatggcgtat tcatgcccgg ttatgaggaa 480
tctgaaataa attcaaaaaa cactgggata aaaaagatcg atcatatagt tggaaatgtc 540
tatgagggcg agatggatag ctqggttaat ttttacatag aaaaacttgg ctttgagcat 600
ttaataacct ttgatgataa agatataaga actgattaca gcgcattaag atcaaaggtt 660
gtaaaataca atgacgatat cgtatttcca ataaatgagc ctgcaaaggg cttaagaaaa 720
tcacagatag aggaatatct tgactattac aggtctgagg gcgttcagca catagcactg 780
ttaactgatg atataataaa aactgtatcc atgatggagg aaaacggcat agaattttta 840
aaaacaccag gatcatacta tgaatcccta tcatcaagga taggctcaat agacgaggat 900
ttaaatgaaa tagagaaaca taacatactt gtggatcgtg atgagaacgg atacctatta 960
cagatcttca caaagcctgt tactgacagg ccaacgttct tctttgaggt catacagaga 1020
aagggtgcaa ggtcattcgg caacggtaac tttaaggcac tttttgaggc gatagaaagg 1080
gagcaggcaa agagaggaaa cctatga 1107
<210> 20
<211> 368

CA 02808152 2013-03-27
1110
<212> PRT
<213> Picrophilus torridus
<400> 20
Met Tyr Gly Lys Asn Leu Ile Ser Glu Leu Arg Glu Lys Glu Ile Phe
1 5 10 15
Lys Arg Leu His His Val Glu Phe Tyr Val Ser Ser Ala Lys Thr Trp
20 25 30
Ser Tyr Phe Met Asn Arg Gly Leu Gly Phe Lys Thr Val Ala Tyr Ala
35 40 45
Gly Pro Glu Thr Gly Ile Arg Asp Lys Ile Ser Tyr Val Met Ser Gin
50 55 60
Gly Thr Ala Arg Ile Ser Phe Thr Ser Ser Met Asn Asp Asp Ser Tyr
65 70 75 80
Ile Ser Asn His Val Lys Lys His Gly Asp Gly Val Lys Asp Ile Ala
85 90 95
Leu Glu Val Asp Asp Leu Asp Glu Ala Lys Ser Leu Ile Glu Lys Tyr
100 105 110
Gly Thr Lys Val Ser Lys Ile Asn Glu Ile Lys Asp Gly Asn Gly Lys
115 120 125
Ile Arg Thr Ala Glu Ile Lys Thr Tyr Gly Glu Thr Val His Thr Leu
130 135 140
Ile Glu Thr Gly Asp Tyr Asn Gly Val Phe Met Pro Gly Tyr Glu Clu
145 150 155 160
Ser Glu Ile Asn Ser Lys Asn Thr Gly Ile Lys Lys Ile Asp His Ile
165 170 175
Val Gly Asn Val Tyr Glu Gly Glu Met Asp Ser Trp Val Asn Phe Tyr
180 185 190
Ile Glu Lys Leu Gly Phe Glu His Leu Ile Thr Phe Asp Asp Lys Asp
195 200 205
Ile Arg Thr Asp Tyr Ser Ala Leu Arg Ser Lys Val Val Lys Tyr Asn
210 215 220
Asp Asp Ile Val Phe Pro Ile Asn Glu Pro Ala Lys Gly Leu Arg Lys
225 230 235 240
Ser Gin Ile Glu Glu Tyr Leu Asp Tyr Tyr Arg Ser Glu Gly Val Gin
245 250 255
His Ile Ala Leu Leu Thr Asp Asp Ile Ile Lys Thr Val Ser Met Met
260 265 270
Glu Glu Asn Gly Ile Glu Phe Leu Lys Thr Pro Gly Ser Tyr Tyr Glu
275 280 285
Ser Leu Ser Ser Arg Ile Gly Ser Ile Asp Glu Asp Leu Asn Glu Ile
290 295 300
Glu Lys His Asn Ile Leu Val Asp Arg Asp Glu Asn Gly Tyr Leu Leu
305 310 315 320
Gin Ile Phe Thr Lys Pro Val Thr Asp Arg Pro Thr Phe Phe Phe Glu
325 330 335
Val Ile Gin Arg Lys Gly Ala Arg Ser Phe Gly Asn Gly Asn Phe Lys
340 345 350
Ala Leu Phe Glu Ala Ile Glu Arg Glu Gin Ala Lys Arg Gly Asn Leu
355 360 365
<210> 21
<211> 1164
<212> DNA
<213> Kordia algicida

CA 02808152 2013-03-27
111p
<400> 21
atggcagcag aaataaaaaa cttaaaagat ttacaaaata cagaatacgg actcaaaaaa 60
ttatttgacg aagcagaaga ctttettcca cttttaggaa cagactacgt agaattatac 120
gtcgggaacg ccaaacaatc ggcacatttc tacaaaacgg cttttggttt tcaatcagaa 180
gcttacgcag gattggaaac aggattaacc gacagagttt catacgtatt aaaacaagat 240
aaaattcgct tggtcttaac aacaccatta ggaaaaggtg gcgaaatcaa tgagcatatc 300
gatttacacg gcgatggcgt aaaagtagta gcactttggg tagaagatgc tadaaaagcc 360
tttgaagaaa cgaccaaaag aggcgcaaaa ccgtacatgg aaccaacaaa agaagaagat 420
gaaaacggat atgtaattcg ctcaggaatc tatacgtacg gagaaacggt tcatgttttt 480
gtagaacgta aaaactataa cggagtottt ttaccaggat atcaaagatg ggaatctcac 540
tacaatccgg agccagttgg cttaaaattc atcgatcaca tggtaggaaa tgtaggttgg 600
ggagaaatga aagaatggtg tgaattctac gcgaaagtaa tgggatttgc gcaaattatc 660
tcctttacag atgatgatat ttctaccgat tttactgcgt tgatgagtaa agtaatgagt 720
aatggaaatg gtagaatcaa atttccaatc aatgaacccg cagaaggaaa aaagaaatcg 780
caaattgaag aatatctaga cttttacaat ggttcaggag tacaacatat tgcggttgct 840
acagacaata ttattgatac ggtttcgcaa atgcgcgaac gtggagtaga attcttatac 900
gttccagata catattatga tgacttgtta gaacgtgttg gcgacatcga tgaagatgta 960
gaagaactca aaaaacacgg aatcttaatt gatcgtgatg aagaaggata cttattgcag 1020
ttatttacca aaaccattgt agacagacca acaatgttct ttgaagtcat tcagcgtaaa 1080
ggcgcacaat catttggagt aggaaacttt aaagctttat ttgaagcgat agaaagagaa 1140
caagctgctc gcggaacatt gtaa 1164
<210> 22
<211> 387
<212> PRT
<213> Kordia algicida
<400> 22
Met Ala Ala Glu Ile Lys Asn Leu Lys Asp Lou Gin Asn Thr Glu Tyr
1 5 10 15
Gly Leu Lys Lys Leu Phe Asp Glu Ala Glu Asp Phe Leu Pro Leu Leu
20 25 30
Gly Thr Asp Tyr Val Glu Leu Tyr Val Gly Asn Ala Lys Gin Ser Ala
35 40 45
His Phe Tyr Lys Thr Ala Phe Gly Phe Gin Ser Glu Ala Tyr Ala Gly
50 55 60
Leu Glu Thr Gly Leu Thr Asp Arg Val Ser Tyr Val Leu Lys Gin Asp
65 70 75 80
Lys Ile Arg Leu Val Leu Thr Thr Pro Leu Gly Lys Gly Gly Glu Ile
85 90 95
Asn Glu His Ile Asp Leu His Gly Asp Gly Val Lys Val Val Ala Leu
100 105 110
Trp Val Glu Asp Ala Thr Lys Ala Phe Glu Glu Thr Thr Lys Arg Gly
115 120 125
Ala Lys Pro Tyr Met Glu Pro Thr Lys Glu Glu Asp Glu Asn Gly Tyr
130 135 140
Val Ile Arg Ser Gly Ile Tyr Thr Tyr Gly Glu Thr Val His Val Phe
145 150 155 160
Val Glu Arg Lys Asn Tyr Asn Gly Val Phe Leu Pro Gly Tyr Gin Arg
165 170 175
Trp Glu Her His Tyr Asn Pro Glu Pro Val Gly Leu Lys Phe Ile Asp
180 185 190
His Met Val Gly Asn Val Gly Trp Gly Glu Met Lys Glu Trp Cys Glu
195 200 205

EZET fm4
0zEi obbbepq a6vofmob4o oeeobeeiob ee.644oso46 esbeb4sq4s .6.6r5q4gool
ogn epeoprqbiob epop43qqop pebbbepobb 44455.5affq. bqobbibboe p8poopqer6
003I 2vnbbbbee5 ebqebbevele E64e0b4abb 04effeeeo4 4eoDrebebb4 4044b44bDp
orn epoo5epeb5 f6b4beoabe eooeDq4oqv emoD436405 qbobbbeooe b4?be69o96
0801 b4U4ob465 EtieqoePbele oq6q5e6brE 44r5vaeobb ebboeoqobq barbbbboob
OZOI o6o65066.6 qboaboebqe ququeboyo bobb33qoab obbi,e344bu boT4o6bobb
06 bquoDbboqD
b3bobse3b4 eb72666e5qo 63EMEoqp6 qboQbaebob eoobb4o5ob
006 b4v3robe35
qbobb00006 babOoppoeo 3ebb.44op4b opbpopqebp pobpoboono
068 beeaDeubbo
epb4.55opbe boeeD4peoo p4o5;DEILo sebsbboboe eoeeooboqo
08L 5q664p5ow
Reo4obbbob ei5ebboeope oElbeq6oee6 ebbobboso4 qbebcoboqq
OZL bEbE00445
bbbovOlgob bgaboq4oeq pobpabbobb pobabb4obe Elb3o6gb3ee
099 obbpqfoqpo
t3oeboqqb6 e65e6qobbb pe4De5bobb obobb0000b pooebqbobb
009 El4bo4qb6bb
pobqoosqoo qboboebbob peboPMpoo e4o5e5-4boe -41163o4o548
0pg oqbov5o6f,o
eqoqoftbo4 fieeb0000go o5ooq.4obbo qoDaboqobe bogb000but
OBV oq4bobb3op
bobpbobboo bEoboq6obv pobobooggo 3baP6bebb3 borboo65#,
OZP obooqobobo
lboobo6o.64 bboboloobb oeopebsobb obo;gpbobb obobbv5pob
ogE oabopqaqqo
74oDobwbo 1DDEl3upp15 opOppboobo 66ovo6D5oe lo3D6D66oe
00E 04q343o445
obo4opo5o6 6.6o4o600qo h4obqoopqb 3boeo5oboo epee6,655ov
or,F, oo4oqopebo
o4b5o5obso bo4oboo5ob obbo4obbbo 4goo434gob oobbbobo36
081 ooqoobt.obo
sE3obo61.66 loqobaSfy# oeooPoo.446 obo4oboaos oog4oboovb
OZT 004obobo3o
ev645o600q Boq4oesobo ouoobbb4bo 400b3oq4bo bbobEreo5u6
09 Bo6poqeobb
obbobbqboo oobo6floo2 poBooboobp pepoepoopo nboopoobqe
CZ <OOP>
Joioo-Fq wntguos <ETZ>
VNG <ZTZ>
CUT <ITZ>
CZ <WIZ>
flE S8E
Itu kip
08E GLE
64V ETV ETV uT9 nTO 61V nT9 91I eTV TITO 94d n97 ETV 9k1 agd USV
G9E 09E SSE
AID TPA 4T9 .1aS eTD PTV AM
sArl b1v uTo aTI TPA nTD 94d 94d
OGE SPE OP
49w 211,1 cud Bay cley Ten 9T1 141 ski iqj eqd naq uTD nei naq aAI
SEC OEE SZE
ATO nTD nTD dslar by dev au nal aTI ATO a71.1 ski ski naq nT9 nTO
OZC GTE OIC SOC
TEA dsV nID dsv aTI dsv An TEA b4v rr nor' neq dsv dsv JAI JAI
00 56Z 06?
2111 dsT cud TpA JAI tier' eqa nTO TEA An faid nTD ELIV 4EN uTO JES
883 083 812
TA d3V aTITI LISV
dsV 3q1 eTV TeA PTV ell eTH eTD TPA ATD
OLZ G93 093
aes AID usig airy atd dslar naa iAI nTD niD eTI eID aes ski ski ski
SGZ OGZ GPZ
AIDnID eTV 01ci nT0 US aTI Old Nd ski en i bay AID uGv /CTD UGV
06Z SEZ OEZ GZZ
is qaw TeA sAq aas iaw naq ETV 114I 94a dgV I4I 19S air dsV dsV
OZZ STZ OIZ
dEV atid, eqd 19S 91I eTI eTD eTV e4d AID 4aW TPA sArl eTV 1A1 04d
bITT
/2-E0-ETOZ ZST80830 VJ

CA 02808152 2013-03-27
111r
<210> 24
<211> 440
<212> PRT
<213> Sorghum bicolor
<400> 24
Met Pro Pro Thr Pro Thr Thr Ala Ala Ala Thr Gly Ala Ala Val Ala
1 5 10 15
Ala Ala Ser Ala Glu Gln Ala Ala Phe Arg Leu Val Gly His Arg Asn
20 25 30
Phe Val Arg Val Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ala Phe
35 40 45
His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg
50 55 60
Phe Ser Phe Gly Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser
65 70 75 80
Thr Gly Asn Thr Ala His Ala Ser Leu Leu Leu Arg Ser Cly Ala Leu
85 90 95
Ala Phe Leu Phe Thr Ala Pro Tyr Ala His Gly Ala Asp Ala Ala Thr
100 105 110
Ala Her Leu Pro Ser Phe Ser Ala Ala Glu Ala Arg Arg Phe Ala Ala
115 120 125
Asp His Gly Leu Ala Val Arg Ala Val Ala Leu Arg Val Ala Asp Ala
130 135 140
Glu Asp Ala Phe Arg Ala Ser Val Ala Ala Gly Ala Arg Pro Ala Phe
145 150 155 160
Glu Pro Val Glu Leu Gly Leu Gly She Arg Leu Ala Glu Val Glu Leu
165 170 175
Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Pro Asp Asp Ala Asp
180 185 190
Ala Her Phe Leu Pro Gly Phe Val Gly Val Thr Ser Pro Gly Ala Ala
195 200 205
Asp Tyr Gly Leu Arg Arg Phe Asp His Ile Vol Gly An Vol Pro Glu
210 215 220
Leu Ala Pro Ala Ala Ala Tyr Phe Ala Gly Phe Thr Gly Phe His Glu
225 230 235 240
Phe Ala Glu She Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Leu
245 250 255
Asn Ser Met Val Leu Ala Asn Asn Ala Glu Asn Val Leu Leu Pro Leu
260 265 270
Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr Tyr
275 280 285
Leu Asp His His Gly Gly Pro Gly Val Gln His Met Ala Leu Ala Ser
290 295 300
Asp Asp Val Leu Arg Thr Leu Arg Glu Met Gln Ala Arg Ser Ala Met
305 310 315 320
Gly Gly Phe Glu Phe Met Ala Pro Pro Ala Pro Glu Tyr Tyr Asp Gly
325 330 335
Val Arg Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gln Ile Lys Glu
340 345 350
Cys Gln G1u Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu
355 360 365
Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu
370 375 380
Glu Ile Ile Gln Arg Ile Gly Cys Met Glu Lys Asp Glu Lys Gly Gln
385 390 395 400

CA 02808152 2013-03-27
ills
Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Cly Asn Phe Ser
405 410 415
Gin Leu Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys
420 425 430
Gin Ala Ala Ala Ala Gin Gly Ser
435 440
<210> 25
<211> 1311
<212> DNA
<213> Triticum aestivum
<400> 25
atgccgccca cccccaccac ccccgcagcc accggcgccg gcgctgccgc cgcggtgacg 60
ccggagcacg cgcggccgcg ccgaatggtc cgcttcaacc cgcgcagcga ccgcttccac 120
acgctatcct tccaccacgt cgagttctgg tgcgcggacg ccgcctccgc cgccggccgc 180
ttcgccttcg cgctcggcgc gccgctcgcc gccaggtccg acctctccac ggggaactcc 240
gtgcacgcct cccagctgct ccgctcgggc aacctcgcct tcctcttcac cgcgccctac 300
gcgaacggct gcgacgccgc caccgcctcc ctgccctcct tctccgccga cgccgcgcgc 360
cggttctccg cggaccacgg gctcgcagtg cgctccatag cactgcgcgt cgcagacgcc 420
gcagaggcct tccgcgccag cgtcgacgga ggcgcgcgcc cggccttcag ccccgtggac 480
ctcggccgcg gotteggctt cgcggaggtc gagctctacg gcgacgtcgt gctccgcttc 540
gtcagtcacc cggatgacac ggacgtgccc ttcttgccgg ggttcgaggg cgtgagcaac 600
ccggatgccg tggactacgg cctgacgcgg ttcgaccacg tcgtcggcaa cgteccggag 660
cttgcccccg ccgccgcata cgtcgccggg ttcgcggggt tccacgagtt cgccgagttc 720
acgacggagg acgtgggcac ggccgagagc gggctcaant cgatggtgct cgccaacaan 780
tcggagggcg tgctgctgcc gctcaacgag ccggtgcacg gcaccaagcg ccggagccag 840
atacagacgt tcctggaaca ccacggcggc tcgggcgtgc agcacatcgc ggtggccagc 900
agcgacgtgc tcaggacgct cagggagatg cgtgcgcgct ccgccatggg cggcttcgac 960
ttcctgccac cccgctgccg aaagtactac gaaggcgtgc ggcgcatcgc cggggatgtg 1020
ctctcggagg cgcagatcaa ggaatgccag gagctggggg tgctcgtcga cagggacgac 1080
caaggggtgt tgctacaaat cttcacaaag ccagtggggg acaggccaac gctgttcctg 1140
gagatgatcc aaaggatcgg gtgcatggag aaggacgaga gaggggaaga gtaccagaag 1200
ggtggctgcg gcgggttcgg caaaggcaac ttctccgagc tgttcaagtc cattgaagat 1260
tacgagaagt cccttgaagc caagcaatct gctgcagttc agggatcata g 1311
<210> 26
<211> 436
<212> PRT
<213> Triticum aestivum
<400> 26
Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Gly Ala Ala
1 5 10 15
Ala Ala Val Thr Pro Glu His Ala Arg Pro Arg Arg Met Val Arg Phe
20 25 30
Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu
35 40 45
Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala
50 55 60
Lela Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser
65 70 75 80
Val His Ala Ser Gin Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe
85 90 95

CA 02808152 2013-03-27
lilt
Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Lela Pro
100 105 110
Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu
115 120 125
Ala Vol Arg Ser Ile Ala Leu Arg Vol Ala Asp Ala Ala Glu Ala Phe
130 135 140
Arg Ala Ser Val Asp Gly Gly Ala Arg Pro Ala Phe Ser Pro Vol Asp
145 150 155 160
Leu Gly Arg Gly Phe Gly Phe Ala Glu Val Glu Leu Tyr Gly Asp Val
165 170 175
Vol Leu Arg Phe Val Ser His Pro Asp Asp Thr Asp Val Pro Elle Leu
180 185 190
Pro Gly Phe Glu Gly Val Ser Asn Pro Asp Ala Val Asp Tyr Gly Leu
195 200 205
Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala
210 215 220
Ala Ala Tyr Val Ala Gly Phe Ala Gly Phe His Glu Phe Ala Glu Phe
225 230 235 240
Thr Thr Glu Asp Vol Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Vol
245 250 255
Leu Ala Asn Asn Ser Glu Gly Vol Leu Leu Pro Leu Asn Glu Pro Val
260 265 270
His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His
275 280 285
Gly Gly Ser Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Vol Leu
290 295 300
Arg Thr Len Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp
305 310 315 320
Phe Leu Pro Pro Arg Cys Arg Lys Tyr Tyr Glu Gly Val Arg Arg Ile
325 330 335
Ala Gly Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu
340 345 350
Gly Vol Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe
355 360 365
Thr Lys Pro Vol Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin
370 375 380
Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys
385 390 395 400
Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys
405 410 415
Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ser Ala Ala
420 425 430
Val Gin Gly Ser
435 =
<210> 27
<211> 1305
<212> DNA
<213> Hordeum vulgare
<400> 27
atgccgccca cccccaccac ccccgcggct accggcgccg ccgccgcggt gacgccggag 60
cacgcgcgac cgcaccgaat ggtccgcttc aacccgcgca gcgaccgctt ccacacgctc 120
tccttccacc acgtcgagtt ctggtgcgcg gacgccgcct ccgccgccgg ccgcttcgcg 180
ttcgcgctcg gcgcgccgct cgccgccagg tccgacctct ccacggggaa ctccgcgcac 240

CA 02808152 2013-03-27
111u
gcctcccagc tgctccgctc gggctccctc gccttcctct tcaccgcgcc ctacgccaac 300
ggctgcgacg ccgccaccgc ctccctgccc tccttctccg ccgacgccgc gcgccggttc 360
tccgccgacc acgggatcgc ggtgcgctcc gtagcgctgc gcgtcgcaga cgccgccgag 420
gccttccgcg ccagtcgtcg acggggcgcg cgcccggcct tcgcccccgt ggacctcggc 480
cgcggcttcg cgttcgcgga ggtcgagctc tacggcgacg tcgtgctccg cttcgtcagc 540
cacccggacg gcacggacgt gcccttcttg ccggggttcg agggcgtaac caacccggac 600
gccgtggact acggcctgac gcggttcgac cacgtcgtcg gcaacgtccc ggagcttgcc 660
cccgccgcag cctacatcgc cgggttcacg gggttccacg agttcgccga gttcacggcg 720
gaggacgtgg gcacgaccga gagcgggctc aactcggtgg tgctcgccaa caactcggag 780
ggcgtgctgc tgccgctcaa cgagccggtg cacggcacca agcgccggag ccagatacag 840
acgttcctgg aacaccacgg cggcccgggc gtgcagcaca tcgcggtggc cagcagtgac 900
gtgctcagga cgctcaggaa gatgcgtgcg cgctccgcca tgggcggctt cgacttcctg 960
ccacccccgc tgccgaagta ctacgaaggc gtgcgacgcc ttgccgggga tgtcctctcg 1020
gaggcgcaga tcaaggaatg ccaggagctg ggtgtgctcg tcgataggga cgaccaaggg 1080
gtgttgctcc aaatcttcac caagccagta ggggacaggc cgaccttgtt cctggagatg 1140
atccagagga tcgggtgcat ggagaaggac gagagagggg aagagtacca gaagggtggc 1200
tgcggcgggt tcggcaaagg caacttctcc gagctgttca agtccattga agattacgag 1260
aagtcccttg aagccaagca atctgctgca gttcagggat catag 1305
<210> 28
<211> 434
<212> PRT
<213> Hordeum vulgare
<400> 28
Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala
1 5 10 15
Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro
20 25 30
Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Tip
35 40 45
Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly
50 55 60
Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His
65 70 75 SO
Ala Ser Gin Leu Leu Arg Ser Gly Ser Lou Ala Phe Lou Phe Thr Ala
85 90 95
Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Lau Pro Ser Phe
100 105 110
Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile Ala Val
115 120 125
Arg Set Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Ala
130 135 140
Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp Leu Gly
145 150 155 160
Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu
165 170 175
Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu Pro Gly
180 185 190
Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Lou Thr Arg
195 200 205
Phe Asp His Val Val Gly Asn Val Pro Glu Lou Ala Pro Ala Ala Ala
210 215 220
Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala
225 230 235 240

CA 02808152 2013-03-27
111v
Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Val Leu Ala
245 250 255
Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val His Gly
260 265 270
Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His Gly Gly
275 280 285
Pro Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr
290 295 300
Leu Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu
305 310 315 320
Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu Ala Gly
325 330 335
Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu Gly Val
340 345 350
Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe Thr Lys
355 360 365
Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin Arg Ile
370 375 380
Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys Gly Gly
385 390 395 400
Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile
405 410 415
Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ser Ala Ala Val Gin
420 425 430
Gly Ser
<210> 29
<211> 1209
<212> DNA
<213> Rhodococcus sp.
<400> 29
atgactaccg ccgacattcg cctgacgccc cgcgaggtgg ccgcacatct ggagaccgac 60
gagctccggc agttggtcgg gctcgtcgaa cacgacgacg cgtcggatcc gtttcccgtg 120
gtcgcgatgg atgccgtggt gttcgtgtgc ggcaacgcga cgcagagcac gcagtacttc 180
gtctccacgt ggggcatgac cctcgtcgcc tacgccgggc cggagaccgg tcagcgctcg 240
cacaagtcct tcgtcctcga gtcggggtcg gcacggttcg tgctgcacgg cgccgtcgat 300
ccgaagagcc cgctcgcgga ccatcaccgg gcgcacggcg acggcgtggt ggacctggcg 360
atggaagttc tcgacgtcga ccgctgcatc gcgcatgcac gctcgcaggg ggccaccatt 420
ctcgaggagc cgcgcgacgt cacggatcag ttcggcaccg tgcggctcgc ggcgatcgcc 480
acgtacggca gcacccggca caccatcgtc qaccgaagcc gatacgacgg cccctacctc 540
cccggattcg tcgcgcgctc cagcggtttc gcggcgcgac cgggtaaacc cccgcgattg 600
ttccaggcgc tcgaccacgc cgtcggcaac gtcgagatgg gccggatgga tcactgggtc 660
cggttctaca accgcgtcat gggcttcacg aacatggccg aattcgtcgg cgacgacatc 720
gccacggagt actcggcgct gatgtcgaag gtcgtggcga acggcaatca ccgggtgaag 780
ttcccgctca acgaacccgc ggtgggaaag aagaagtcgc agatcgacga atatctcgag 840
ttctacggtg agccgggctg ccagcatctg gccctcgcga ccggagacat cctcgcgacg 900
gtggacgcgt tgcgggccga gggtgLcgaa ttcctgaaca cacccgacgc gtactacgag 960
gacccacagc tgcgcgcccg gatcggcagg gtgcgggtgc cggtggagga actgcagaag 1020
cgcggaatcc tcgtcgaccg cgacgaggac ggatacctcc tgcagatctt caccaaaccg 1080
ctcggcgacc ggccgaccgt gttcttcgag gtgatcgaac ggcacggttc gctcgggttc 1140
ggggcgggta acttccaggc cctgttcgaa tccatcgagc gtgaggaggc ggcgcgcggc 1200
aatctgtga 1209

CA 02808152 2013-03-27
111w
<210> 30
<211> 402
<212> PRT
<213> Rhodococcus sp.
<400> 30
Met Thr Thr Ala Asp Ile Arg Lou Thr Pro Arg Glu Val Ala Ala His
1 5 10 15
Leu Glu Thr Asp Glu Leu Arg Gin Leu Val Gly Leu Val Glu His Asp
20 25 30
Asp Ala Ser Asp Pro Phe Pro Vol Val Ala Met Asp Ala Vol Val Phe
35 40 45
Val Cys Gly Asn Ala Thr Gin Ser Thr Gin Tyr Phe Val Ser Thr Trp
50 55 60
Gly Met Thr Leu Val Ala Tyr Ala Gly Pro Glu Thr Gly Gin Arg Ser
65 70 75 80
His Lys Ser Phe Val Leu Glu Ser Gly Her Ala Arg Phe Val Leu His
85 90 95
Gly Ala Val Asp Pro Lys Ser Pro Lou Ala Asp His His Arg Ala His
100 105 110
Gly Asp Gly Val Vol Asp Leu Ala Met Glu Vol Leu Asp Val Asp Arg
115 120 125
Cys Ile Ala His Ala Arg Ser Gin Gly Ala Thr Ile Leu Glu Glu Pro
130 135 140
Arg Asp Val Thr Asp Gin Phe Gly Thr Val Arg Leu Ala Ala Ile Ala
145 150 155 160
Thr Tyr Gly Ser Thr Arg His Thr Ile Val Asp Arg Ser Arg Tyr Asp
165 170 175
Gly Pro Tyr Leu Pro Gly Phe Val Ala Arg Ser Ser Gly Phe Ala Ala
180 185 190
Arg Pro Gly Lys Pro Pro Arg Leu Phe Gin Ala Leu Asp His Ala Vol
195 200 205
Gly Asn Val Glu Met Gly Arg Met Asp His Trp Val Arg Phe Tyr Asn
210 215 220
Arg Val Met Gly Phe Thr Asn Met Ala Glu Phe Vol Gly Asp Asp Ile
225 230 235 240
Ala Thr Glu Tyr Ser Ala Leu Met Her Lys Vol Val Ala Asn Gly Asn
245 250 255
His Arg Val Lys Phe Pro Leu Asn Glu Pro Ada Val Gly Lys Lys Lys
260 265 270
Ser Gin Ile Asp Glu Tyr Leu Glu Phe Tyr Gly Glu Pro Gly Cys Gin
275 280 285
His Leu Ala Leu Ala Thr Gly Asp Ile Lou Ala Thr Val Asp Ala Leu
290 295 300
Arg Ala Glu Gly Val Glu Phe Leu Asn Thr Pro Asp Ala Tyr Tyr Glu
305 310 315 320
Asp Pro Gin Lou Arg Ala Arg Ile Gly Arg Val Arg Val Pro Val Glu
325 330 335
Glu Leu Gin Lys Arg Gly Ile Leu Val Asp Arg Asp Glu Asp Gly Tyr
340 345 350
Leu Lou Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Val Phe
355 360 365
Phe Glu Val Ile Glu Arg His Gly Ser Leu Gly Phe Gly Ala Gly Asn
370 375 380

CA 02808152 2013-03-27
4
111x
Phe Gin Ala Leu Phe Glu Ser Ile Glu Arg Glu Gin Ala Ala Arg Gly
385 390 395 400
Aon Leu
<210> 31
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> Nucleic acid sequence encoding Arabidopsis HPPD plus containing
at the 5 end a nucleic acid encoding an Ala and 6 His amino
acids
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for an Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 31
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgoggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgabctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc yUculAutt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtage ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gaqtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 32
<211> 450
<212> PRT
<213> Artificial sequence

CA 02808152 2013-03-27
lily
<220>
<223> encoded by SEQ ID No. 31
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 32
Met Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Pile Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Her Pro Her Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gln Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335

CA 02808152 2013-03-27
111z
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450
<210> 33
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> miscjeature
<222> (7)..(24)
<223> sequence coding an His Tag containing 6 His
<400> 33
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ciateccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagagg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga aggagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aatgcggcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140

CA 02808152 2013-03-27
111aa
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 34
<211> 450
<212> PET
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 33
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISCJEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 34
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu the Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Se/ Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg the Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe the Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser the Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gil, Asn Val Pro Glu Leu Gly
225 230 235 240

CA 02808152 2013-03-27
111bb
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ala
260 265 270
Ala Vol Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Giy Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450
<210> 35
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant IIPPD polypeptide
<220>
<221> misc_feature
<222> (4)¨(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 35
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420

CA 02808152 2013-03-27
Mcc
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggettgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattgc cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 36
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 35
<220>
<221> MISCJEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 36
Met Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Lau Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140

CA 02808152 2013-03-27
ill dd
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Vol Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Vol Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser ?he Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Lou Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Vol Leu Leu Pro Ile Ala Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Lou Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arq Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Vol Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly lie Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Lou Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Ary Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Vol Gly
450
<210> 37
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala

CA 02808152 2013-03-27
Mee
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 37
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctoc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga aggagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcatc tttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgettc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 38
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 37
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 38
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Sex Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45

CA 02808152 2013-03-27
111ff
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Lou Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arc Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala He Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arq Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Ser Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly =
450
<210> 39
<211> 1353

Ca 02808152 2013-03-27
111gg
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 39
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg goggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg cgctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 40
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 39
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala

CA 02808152 2013-03-27
111 hh
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 40
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pio Gly Phe Glu A/A- Val Glu Asp Ala Su/ Her Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Ala Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
lie Phe Arg Thr Lou Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400

CA 02808152 2013-03-27
111ii
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Lou
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450
<210> 41
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequebc encoding mutant HOOD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 41
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtogctog tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg qaaccgccga qaqcqqttta aattcagogg tcctqqctaq caatqatgaa 840
atggttcttc tagcgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 42
<211> 450

. CA 02808152 2013-03-27
111jj
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 41
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 42
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu She Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Lou Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly She Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Lou Ala Ser Asn Asp Glu Met Val Leu Leu Ala Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300

= CA 02808152 2013-03-27
111 kk
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu Lou Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Lou
435 440 445
Val Gly
450
<210> 43
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 43
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctatatccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttcattatt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagattggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900

, = . CA 02808152 2013-03-27
11111
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattatgg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcaaagg caatttctct gagotottca agtccattga agaatacgaa 1320
aagactattg aagccaaaca gttagtggga tga 1353
<210> 44
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 43
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 44
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met A/9- Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Clu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205

, CA 02808152 2013-03-27
111mm
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Giu Cys Glu
355 360 365
Glu Leu Gly Ile Met Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
365 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Cly Phe Cly Lys Cly Asia Phe Ser Glu Lou
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450
<210> 45
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)¨(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 45
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggegotg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180

=. = CA 02808152 2013-03-27
111nn
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctottacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctettgtc gttcottatt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggygttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttqg aacataacqa aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcattgg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 46
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 45
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC_FEATORE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 46
Net Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile (flu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leta Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110

, . CA 02808152 2013-03-27
11100
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Lou Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Clu Leu Gly Ile Lou Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
365 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Ile Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450
<210> 47
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature

, CA 02808152 2013-03-27
"pp
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> raise feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 47
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840
atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggccaggg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 48
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 47
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala
<220>
<221> MISC FEATURE
<222> (3).7(8)
<223> His Tag made of 6 His
<400> 48
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15

. CA 02808152 2013-03-27
4 =
=
111qg
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val
20 25 30
Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Her Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Lou Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser lie
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Cly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala
245 250 255
Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Gin Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Lou
435 440 445
Val Gly
450

CA 02808152 2013-03-27
111rr
<210> 49
<211> 1353
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mutant HPPD polypeptide
<220>
<221> misc_feature
<222> (4)..(6)
<223> sequence coding for Ala
<220>
<221> misc_feature
<222> (7)..(24)
<223> sequence coding for an His Tag containing 6 His
<400> 49
atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60
gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120
aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180
gacgcaacca acgtcgctcg tcacttctcc tggggtctgg ggatgagatt ctccgccaaa 240
tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300
cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360
accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420
ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480
agtgtagcta atggcgctat tcattcgtcg cctcctatcg tcctcaatga aggagttacg 540
atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600
gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660
ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720
ccggctttaa cttatgtagc gaggttcact ggttttcacc aattcgcaga gttcacagca 780
gacgacgttg gaaccgccga gagcggttta aattcagagg tcctggctag caatgatgaa 840
atggttottc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900
acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960
atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020
cottctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080
gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140
acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200
atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260
tgtggtggtt ttggcgtggg caatttctct gagctcttca agtccattga agaatacgaa 1320
aagactcttg aagccaaaca gttagtggga tga 1353
<210> 50
<211> 450
<212> PRT
<213> Artificial sequence
<220>
<223> encoded by SEQ ID No. 49
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Ala

* CA 02808152 2013-03-27
hiss
<220>
<221> MISC_FEATURE
<222> (3)..(8)
<223> His Tag made of 6 His
<400> 50
Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn
1 5 10 15
Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly She Lys Leu Val
20 25 30
Gly She Ser Lys She Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys
35 40 45
Val Lys Arg Phe His His Ile Glu She Trp Cys Gly Asp Ala Thr Asn
50 55 60
Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg She Ser Ala Lys
65 70 75 80
Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr
85 90 95
Ser Gly Asp Leu Arg Phe Leu She Thr Ala Pro Tyr Ser Pro Ser Leu
100 105 110
Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe
115 120 125
Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val
130 135 140
Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala She Ser Ile
145 150 155 160
Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Tie Val Leu Asn
165 170 175
Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu
180 185 190
Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu
195 200 205
Pro Gly Phe Glu Ay Vul Glu Asp Ala Ser Ser She Pro Leu Asp Tyr
210 215 220
Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly
225 230 235 240
Pro Ala Leu Thr Tyr Val Ala Gly She Thr Gly Phe His Gin She Ala
245 250 255
Giu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser
260 265 270
Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu
275 280 285
Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu
290 295 300
His Ann Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp
305 310 315 320
Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly
325 330 335
She Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys
340 345 350
Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu
355 360 365
Glu Lou Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin
370 375 380
Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile
385 390 395 400

= CA 02808152 2013-03-27
Hitt
Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr
405 410 415
Gin Ser Gly Gly Cys Gly Gly Phe Gly Val Gly Asn Phe Ser Glu Leu
420 425 430
Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu
435 440 445
Val Gly
450

112
The following embodiments are provided:
Embodiment 1. An isolated nucleic acid comprising a nucleotide sequence
encoding a
mutated HPPD protein,
wherein said mutated HPPD protein has HPPD activity, and
wherein said mutated HPPD protein comprises a replacement of the amino acid at
the
position corresponding to position 421 of SEQ ID No. 2 with Asp or Asn.
Embodiment 2. The isolated nucleic acid of embodiment 1, wherein said HPPD
protein
is derived from Streptomyces avermitilis (Genebank SAV11864), Daucus carota
(Genebank DCU 87257), Arabidopsis thaliana (Genebank AF047834), Mycosphaerella
graminicola (Genebank AF038152), oryza sativa / rice [BAD26248], Zea mays /
corn
[ACN36372], Avena sativa [ABZ23427], Pseudomonas fluorescens [ABF50055],
Synechococcus sp. [YP 473959], Blepharisma japonicum [BAF91881], Rhodococcus
RHA1 sp. ro0240 [YP_702005], Rhodococcus RHA1 sp. ro0341 [YP_703002],
Picrophilus torridus [YP_024147], Kordia algicida [ZP_02161490], Sorghum
bicolor
[XP_002453359], Triticum aestivum / wheat [AAZ67144], or Hordeum vulgare /
barley
[048604].
Embodiment 3. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 4 that comprises Asp or Asn, at position 418 of the amino acid sequence
of SEQ
ID No. 4 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 4. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 6 that comprises Asp or Asn at position 395 of the amino acid sequence
of SEQ
ID No. 6 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 5. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
Date Recue/Date Received 2022-12-02

113
ID No. 8 that comprises Asp or Asn at position 412 of the amino acid sequence
of SEQ
ID No. 8 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 6. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 10 that comprises Asp or Asn at position 335 of the amino acid sequence
of SEQ
ID No. 10 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 7. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 14 that comprises Asp or Asn at position 327 of the amino acid sequence
of SEQ
ID No. 14 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 8. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 16 that comprises Asp or Asn at position 361 of the amino acid sequence
of SEQ
ID No. 16 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 9. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 18 that comprises Asp or Asn at position 381 of the amino acid sequence
of SEQ
ID No. 18 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 10. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 30 that comprises Asp or Asn at position 382 of the amino acid sequence
of SEQ
ID No. 30 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 11. The isolated nucleic acid of embodiment 1, wherein the amino
acid
sequence of said mutated HPPD protein is a variant of the amino acid sequence
of SEQ
Date Recue/Date Received 2022-12-02

114
ID No. 20 that comprises Asp at position 348 of the amino acid sequence of SEQ
ID No.
20 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 12. The isolated nucleic acid of embodiment 1, wherein said the
amino
acid sequence of mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 22 that comprises Asp or Asn at position 367 of the amino acid sequence
of SEQ
ID No. 22 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 13. The isolated nucleic acid of embodiment 1, wherein said the
amino
acid sequence of mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 24 that comprises Asp or Asn at position 412 of the amino acid sequence
of SEQ
ID No. 24 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 14. The isolated nucleic acid of embodiment 1, wherein said the
amino
acid sequence of mutated HPPD protein is a variant of the amino acid sequence
of SEQ
ID No. 26 that comprises Asp or Asn at position 408 of the amino acid sequence
of SEQ
ID No. 26 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 15. The nucleic acid of any one of embodiments Ito 14, wherein said
mutated HPPD protein increases the tolerance of a plant to at least one
herbicide acting
on HPPD.
Embodiment 16. A protein encoded by the isolated nucleic acid of any one of
embodiments 1 to 15.
Embodiment 17. A gene or a nucleic acid comprising a coding sequence
comprising the
nucleic acid of any one of embodiments 1 to 15 operably linked to a plant-
expressible
promoter and optionally a transcription termination and polyadenylation
region.
Date Recue/Date Received 2022-12-02

115
Embodiment 18. A method of obtaining a mutated HPPD protein modulating the
tolerance of a plant to at least one herbicide acting on HPPD, wherein said
mutated
HPPD protein has HPPD activity, the method comprising
i) providing an HPPD protein;
ii) replacing the amino acid at position 421 of SEQ ID NO. 2 in said HPPD
protein so that the resulting amino acid sequence comprises Asp, Phe, or Asn
at the position corresponding to position 421 of the amino acid sequence of
SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one
herbicide acting on HPPD,
wherein an inhibition of the resulting protein of less or more than that
observed with a
reference HPPD protein is indicative that the resulting protein modulates the
tolerance
of a plant to said herbicide.
Embodiment 19. The method of embodiment 18, wherein said mutated HPPD protein
increases the tolerance of a plant to at least one HPPD inhibitor herbicide.
Embodiment 20. The nucleic acid of embodiment 15 or the method of embodiment
19,
wherein the herbicide acting on HPPD is tembotrione.
Embodiment 21. A method of producing a transgenic plant comprising introducing
into
said plant the nucleic acid of any one of embodiments 1 to 15 and 20, operably
linked to
a plant expressible promoter or the chimeric gene of embodiment 17.
Embodiment 22. The method of embodiment 21, wherein the nucleic acid of
embodiment 16 or a nucleic acid identified by the method of embodiment 18 or
19, both
operably linked to a plant expressible promoter, or the chimeric gene of
embodiment 17
comprising the nucleic acid of embodiment 15 is introduced into said plant.
Embodiment 23. A plant cell comprising the isolated nucleic acid of any one of
embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic
information.
Date Recue/Date Received 2022-12-02

116
Embodiment 24. A cell of a plant, a cell of a part thereof or a cell of plant
tissue
comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the
chimeric
gene of embodiment 17 in its genetic information.
Embodiment 25. A cell of a plant comprising the isolated nucleic acid of any
one of
embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic
information
obtained from the method of embodiment 21 or 22.
Embodiment 26. The cell of embodiment 24 or 25 which is a cell comprising the
isolated
nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of
embodiment 17
in its genetic information of a plant selected from wheat, cotton, canola,
rice, corn,
soybean, and sorghum.
Embodiment 27. A cell of a seed of a plant comprising the isolated nucleic
acid of any
one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its
genetic
information as defined in any one of embodiments 24 to 26.
Embodiment 28. A cell of progeny of a plant cell comprising the isolated
nucleic acid of
any one of embodiments Ito 15 or the chimeric gene of embodiment 17 in its
genetic
information as defined in any one of embodiments 23 to 26 or a cell of progeny
of a
seed cell as defined in embodiment 27.
Embodiment 29. A method of modulating a plant's tolerance to at least one
herbicide
acting on HPPD or of obtaining a plant tolerant to an HPPD inhibitor herbicide
comprising introducing the isolated nucleic acid of any one of embodiments 1
to 15
operably linked to a plant expressible promoter or the chimeric gene of
embodiment 17
into a plant's genome.
Embodiment 30. A method for controlling weeds comprising spraying at least one
HPPD
inhibitor herbicide on or around a crop plant, wherein said crop plant
contains the
nucleic acid according to embodiment 15 operably linked to a plant expressible
Date Recue/Date Received 2022-12-02

117
promoter or the chimeric gene of embodiment 17 comprising the nucleic acid of
embodiment 15.
Embodiment 31. Use of a chimeric gene of embodiment 17 or the nucleic acid of
any
.. one of embodiments 1 to 15 operably linked to a plant expressible promoter
for
modulating the tolerance of a plant to at least one HPPD inhibitor herbicide
applied for
controlling weeds.
Embodiment 32. The method of any one of embodiments 22, 29 or 30 or the use of
.. embodiment 31, wherein the tolerance of a plant to at least one HPPD
inhibitor
herbicide is increased.
Date Recue/Date Received 2022-12-02