U.S. patent application number 12/391343 was filed with the patent office on 2010-03-25 for o-methyltransferases of tetrahydrobenzylisoquinoline alkaloid biosynthesis in papaver somniferum.
This patent application is currently assigned to DONALD DANFORTH PLANT SCIENCE CENTER. Invention is credited to Susanne Frick, Stefanie Haase-Fernando, Toni M. Kutchan, Anan Ounaroon.
Application Number | 20100075385 12/391343 |
Document ID | / |
Family ID | 34130124 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075385 |
Kind Code |
A1 |
Kutchan; Toni M. ; et
al. |
March 25, 2010 |
O-Methyltransferases of Tetrahydrobenzylisoquinoline Alkaloid
Biosynthesis in Papaver Somniferum
Abstract
The present invention relates to methyl transfer enzymes
involved in alkaloid biosynthesis in opium poppy. More
particularly, the invention relates to proteins having
(R,S)-reticuline 7-O-methyltransferase activity, to proteins having
(R,S)-norcoclaurine 6-O-methyltransferase activity and to
derivatives and analogues of these proteins. The invention also
relates to nucleic acid molecules encoding the proteins, and their
derivatives and analogues, and to their use in the production of
methylated catechols and tetrahydrobenzylisoquinolines.
Inventors: |
Kutchan; Toni M.; (St.
Louis, MO) ; Ounaroon; Anan; (Bangkok, TH) ;
Frick; Susanne; (Augsburg, DE) ; Haase-Fernando;
Stefanie; (Calgary, CA) |
Correspondence
Address: |
THOMPSON COBURN LLP
ONE US BANK PLAZA, SUITE 3500
ST LOUIS
MO
63101
US
|
Assignee: |
DONALD DANFORTH PLANT SCIENCE
CENTER
St. Louis
MO
|
Family ID: |
34130124 |
Appl. No.: |
12/391343 |
Filed: |
February 24, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10888656 |
Jul 8, 2004 |
7514251 |
|
|
12391343 |
|
|
|
|
Current U.S.
Class: |
435/122 ;
435/193; 435/254.2; 536/23.2 |
Current CPC
Class: |
C12N 9/1007 20130101;
C12N 15/8243 20130101 |
Class at
Publication: |
435/122 ;
435/193; 536/23.2; 435/254.2 |
International
Class: |
C12P 17/12 20060101
C12P017/12; C12N 9/10 20060101 C12N009/10; C07H 21/04 20060101
C07H021/04; C12N 1/19 20060101 C12N001/19 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 2003 |
EP |
EP 03020023.2 |
Claims
1.-18. (canceled)
19. An isolated or purified protein comprising the amino acid
sequence of FIG. 13 (SEQ ID NO: 21) or a variant of the amino acid
sequence of FIG. 13 (SEQ ID NO: 21), said variant having at least
70% identity with the PSOMT2 amino acid sequence of FIG. 13 (SEQ ID
NO: 21) over a length of at least 300 amino acids and, said variant
having 0-methyltransferase activity.
20. The protein according to claim 19 wherein the variant has at
least 80% identity with the PSOMT2 amino acid sequence of FIG. 13
(SEQ ID NO: 21) over a length of at least 300 amino acids.
21. The protein according to claim 19 wherein the variant has at
least 80% identity with the PSOMT2 amino acid sequence of FIG. 13
(SEQ ID NO: 21) over a length of at least 300 amino acids.
22. The protein according to claim 21, wherein the variant has at
least 95% identity with the PSOMT2 amino acid sequence of FIG. 13
(SEQ ID NO: 21) over a length of at least 300 amino acids and
wherein said variant contains one or more conserved amino acid
motifs, said motifs comprising amino acid sequence LVDVGGG,
PXXDAXXMK, XGKVI, DLPHV, HVGGDMF, or GKERT, wherein X represents
any amino acid.
23. The protein according to claim 22, wherein the variant has at
least 97% identity with the PSOMT2 amino acid sequence of FIG. 13
(SEQ ID NO: 21) over a length of at least 300 amino acids.
24. The protein according to claim 23 comprising the PSOMT2 amino
acid sequence illustrated in FIG. 3 (SEQ ID NO: 3).
25. The protein according to claim 23 comprising the PSOMT2a amino
acid sequence illustrated in FIG. 13 (SEQ ID NO: 13).
26. A protein or peptide comprising of a fragment of the protein
illustrated in FIG. 16 (SEQ ID NO: 25), said fragment having a
length of 100 to 345 amino acids, including the portion spanning at
least one of positions 93, 150, 233, 245 and 274, wherein X.sub.93,
X.sub.150, X.sub.233, X.sub.245, X.sub.274 are chosen from the
following amino acids: TABLE-US-00011 X.sub.93: Pro, Val X.sub.150:
Val, Glu X.sub.233: Ser, Pro X.sub.245: Ala, Gly; X.sub.274: Gly,
Val,
with the proviso that X.sub.93 is not Pro when X.sub.150,
X.sub.233, X.sub.245, X.sub.274 have the following meanings:
Xaa.sub.150 is Glu, Xaa.sub.233. is Ser, Xaa.sub.245 is Ala and
Xaa.sub.274 is Gly, said protein or peptide having
O-methyltransferase activity.
27. The protein or peptide according to claim 26, comprising or
consisting of a fragment of the PSOMT2 protein illustrated in FIG.
3 (SEQ ID NO: 3), said fragment having a length of 100 to 345 amino
acids, including the portion spanning at least one of positions 93,
150, 233, 245 and 274 of the sequence illustrated in FIG. 3 (SEQ ID
NO: 3).
28. The protein or peptide according to claim 26, comprising or
consisting of a fragment of the PSOMT2a protein illustrated in FIG.
13 (SEQ. ID n.degree. 21), said fragment having a length of 100 to
345 amino acids, including the portion spanning at least one of
positions 93, 150, 233, 245 and 274 of the sequence illustrated in
FIG. 13 (SEQ ID NO: 21).
29. The protein or peptide according to claim 27 having 150 to 300
amino acids.
30. The protein according to claim 19, which is a dimer comprising
two protein sub-units, each sub-unit being chosen from any one of
proteins as defined in claim 19.
31. The protein according to claim 19, having (R,S)-norcoclaurine
6-O-methyltransferase activity.
32. An isolated nucleic acid molecule encoding a protein according
to claim 19.
33.-45. (canceled)
46. A cell transformed or transfected by a nucleic acid molecule
according to claim 19.
47. The cell according to claim 46 which is a prokaryotic cell.
48. (canceled)
49. Cell according to claim 46 which is a eukaryotic cell.
50. Cell according to claim 49 which is a yeast cell.
51.-64. (canceled)
65. Method for the production of methylated
tetrahydrobenzylisoquinolines, said method comprising the steps of:
i) contacting in vitro a protein having norcoclaurine
6-O-methyltransferase activity with a substrate chosen from
(R,S)-norcoclaurine, (R,S)-isoorientaline, (R)-norprotosinomenine
and (S)-norprotosinomenine at pH 6.0 to 9.0, wherein the protein
having norcoclaurine 6-O-methyltransferase activity is a protein
according to claim 19; ii) recovering the methylated
tetrahydrobenzylisoquinolines thus produced.
66. (canceled)
67. A method for producing a protein having norcoclaurine
6-O-methyltransferase activity, said method comprising i)
transforming or transfecting a cell with a nucleic acid molecule
according to claim 32, in conditions permitting the expression of
the protein having norcoclaurine 6-O-methyltransferase activity,
ii) propagating the said cell, and iii) recovering the
thus-produced protein having norcoclaurine 6-O-methyltransferase
activity.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/888,656 filed on Jul. 8, 2004, which is
incorporated by reference herein in its entirety and which claims
priority to European Patent Application Serial No. No. 03020023.2,
filed Sep. 3, 2003.
[0002] The present invention relates to methyl transfer enzymes
involved in alkaloid biosynthesis in opium poppy. More
particularly, the invention relates to proteins having
(R,S)-reticuline 7-O-methyltransferase activity, to proteins having
(R,S)-norcoclaurine 6-O-methyltransferase activity and to
derivatives and analogues of these proteins. The invention also
relates to nucleic acid molecules encoding the proteins, and their
derivatives and analogues, and to their use in the production of
methylated catechols and tetrahydrobenzylisoquinolines.
[0003] Enzymatic methylation is a ubiquitous reaction occurring in
diverse organisms including bacteria, fungi, plants and animals,
and resulting in the modification of acceptor molecules for
different functional and regulatory purposes. Enzymatic
O-methylation is catalyzed by O-methyltransferases [E.C.
2.1.1.6.x], and involves the transfer of the methyl group of
S-adenosyl-L-methionine (AdoMet) to the hydroxyl group of an
acceptor molecule. S-Adenosylmethionine (AdoMet).sup.1-dependent
O-methyltransferases (OMTs) are important components of plant
natural product biosynthesis, yielding methyl ether derivatives of
hydroxylated polycyclic aromatic low molecular weight compounds.
Regiospecific oxygen methylation significantly contributes to the
vast metabolic diversity of plant secondary metabolism.
[0004] Over the past few years, the structural genes of several
plant OMTs have been isolated, often using homology-based cloning
techniques which exploit the high amino acid sequence similarity
observed between plant OMTs, and the presence of conserved sequence
motifs (Refs 1-3). However, whilst amino acid sequence comparison
can assist in the isolation of the genes, it cannot be used to
reliably predict the in vivo function of plant OMTs because of the
broad substrate specificities that can be found for closely related
enzymes. Indeed, it has become clear that substrate discrimination
by plant O-methyltransferases can vary among the same enzyme from
different species, for example the different substrate specificity
of coclaurine 6-O-methyltransferase of tetrahydrobenzylisoquinoline
alkaloid biosynthesis from Thalictrum tuberosum (4) and from Coptis
japonica (5). This can also occur within one species, as for
caffeic acid 3-O-methyltransferase from Nicotiana tabacum (6). In
addition, many metabolic pathways in plants are only putatively
elucidated, further complicating the assignment of a function to an
isolated OMT gene. Functional characterisation of the enzymes is
thus not trivial.
[0005] O-Methyltransferases of phenylpropanoid and of alkaloid
biosynthesis are probably the biochemically best studied in the
plant natural product field. They play a particularly important
role in the opium poppy, Papaver somniferum, which produces more
than eighty tetrahydrobenzylisoquinoline-derived alkaloids,
including the narcotic analgesic phenanthrene alkaloids codeine and
morphine, and the antitussive phthalidisoquinoline noscapine, the
vasodilator papaverine and the antimicrobial benzo[c]phenanthridine
sanguinarine.
[0006] As shown in FIG. 1, in Papaver somniferum a central
biosynthetic pathway leads from two molecules of L-tyrosine to
(S)-reticuline (reviewed in 7). The pathway then bifurcates as the
(S)-reticuline molecule is regio- and stereospecifically
transformed into committed isoquinoline subclass intermediates. Two
classes of enzyme effectuate this diversification--oxidoreductases
and O-methyltransferases. The latter enzymes catalyze two steps in
the formation of (S)-reticuline, prior to the branch point of the
morphine and sanguinarine pathways. Then in the specific pathway
that leads to morphine, (S)-reticuline is oxidized by
(S)-reticuline oxidase to form the dehydroreticulinium ion, which
is then stereospecifically reduced to (R)-reticuline. To enter the
sanguinarine pathway, the N-methyl group of (S)-reticuline is
oxidatively cyclized by the berberine bridge enzyme to the bridge
carbon (C-8) of (S)-scoulerine.
[0007] Requisite to metabolic engineering of commercial varieties
of P. somniferum is the understanding of the alkaloid biosynthetic
pathways at the molecular genetic level. However, of the enzymes
involved in alkaloid biosynthesis in P. somniferum, genes encoding
only six of them have been isolated to date. One of the first to be
isolated was a cDNA encoding the cytochrome P-450-dependent
monooxygenase (S)--N-methylcoclaurine 3'-hydroxylase (8,9) and the
corresponding cytochrome P-450 reductase (10). This enzyme is
common to the biosynthetic pathways of all the P. somniferum
alkaloids. Specific to the sanguinarine pathway is the cDNA
encoding the berberine bridge enzyme (9, 11, 12). Finally, specific
to morphine biosynthesis are the cDNAs for salutaridinol
7-O-acetyltransferase (13) that results in the formation of the
five-ring system of the morphinans and for codeinone reductase, the
penultimate enzyme of the morphine pathway that reduces codeinone
to codeine (14).
[0008] With regard to the O-methyl transferases involved in P.
somniferum alkaloid biosynthesis, very little is known to date.
Norcoclaurine 6-O-methyltransferase activity and
(S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase activity
have been detected in protein extracts of P. somniferum (29).
Recently, Facchini and Park published the mRNA and amino acid
sequence of a putative norcoclaurine 6-O-methyltransferase from P.
somniferum (31) However the function of the enzyme was not
investigated by these authors. Decker (30) carried out a study
aimed at characterizing proteins in the latex of P. somniferum
using two-dimensional gel electrophoresis, and demonstrated the
presence of spots, which, once excised and micro-sequenced were
seen to have homology with a maize O-methyl transferase. To date,
however, no O-methyl transferases involved in P. somniferum
alkaloid biosynthesis have been cloned and fully characterised.
Moreover, to date no reports of (R,S)-reticuline
7-O-methyltransferase activity in P. somniferum have ever been made
in the literature.
[0009] It is thus an object of the present invention to identify
and characterise both at the protein and nucleic acid levels, and
at the functional level, 0-methyl transferases involved in P.
somniferum alkaloid biosynthesis.
[0010] More specifically, the present invention relates to the
isolation and characterization of cDNAs encoding
O-methyltransferases of tetrahydrobenzylisoquinoline alkaloid
biosynthesis in P. somniferum, namely (R,S)-reticuline
7-O-methyltransferase and (R,S)-norcoclaurine
6-O-methyltransferase.
[0011] In the framework of the present invention, the inventors
have isolated S-Adenosyl-L-methionine:(R,S)-reticuline
7-O-methyltransferase, which converts reticuline to laudanine in
tetrahydrobenzylisoquinoline biosynthesis in Papaver somniferum. A
proteomic analysis of P. somniferum latex indicated the presence of
protein(s) showing homology to a maize O-methyltransferase (30),
but gave no indication as to whether the fragments were from a
single protein, and no indication of the possible function of the
protein. The cDNA was amplified from P. somniferum RNA by reverse
transcription PCR using primers based on the internal amino acid
sequences. The recombinant protein was expressed in Spodoptera
frugiperda Sf9 cells in a baculovirus expression vector. Steady
state kinetic measurements with the heterologously expressed enzyme
and mass spectrometric analysis of the enzymic products suggest
that the enzyme is capable of carry through sequential
O-methylations, first on the isoquinoline-, then on the benzyl
moiety of several substrates. The tetrahydrobenzylisoquinolines
(R)-reticuline (4.20 s.sup.-1mM.sup.-1), (S)-reticuline (4.50),
(R)-protosinomenine (1.67), and (R,S)-isoorientaline (1.44) as well
as guaiacol (5.87) and isovanillic acid (1.21) are O-methylated by
the enzyme with the ratio k.sub.cat/K.sub.m shown in parentheses. A
phylogenetic comparison of the amino acid sequence of this
O-methyltransferase to those from forty-three other plant species
suggests that this enzyme groups more closely to isoquinoline
biosynthetic O-methyltransferases from Coptis japonica than to
those from Thalictrum tuberosum. In addition, P. somniferum cDNAs
encoding two (R,S)-norcoclaurine 6-O-methyltransferases have been
isolated and similarly characterized. The present inventors have
thus surprisingly discovered that different alleles of
(R,S)-norcoclaurine 6-O-methyltransferase exist in P.
somniferum.
[0012] More specifically, the invention concerns a first protein,
comprising or consisting of the Papaver somniferum (R,S)-reticuline
7-O-methyltransferase protein illustrated in FIG. 9 (SEQ ID NO: 2),
(hereafter designated the PSOMT1 sequence), or fragments or
variants of the illustrated PSOMT1 sequence. The PSOMT1 proteins of
the invention thus comprise or consist of: [0013] i) the amino acid
sequence illustrated in FIG. 9 (SEQ ID NO: 2) ("PSOMT1") or, [0014]
ii) a fragment of the amino acid sequence illustrated in FIG. 9
(SEQ ID NO: 2), said fragment having at least 100 amino acids
("i.e. fragments of PSOMT1"), or [0015] iii) a variant of the amino
acid sequence of FIG. 9 (SEQ ID NO: 2), said variant having at
least 70% identity with the amino acid sequence of FIG. 9 (SEQ ID
NO: 2) over a length of at least 300 amino acids (i.e. "variants of
PSOMT1").
[0016] The fragments or variants of the PSOMT1 protein as defined
above will be collectively referred to herein as the "PSOMT1
derivatives".
[0017] Preferably, the PSOMT1 protein and derivatives are in
dimeric form, i.e. the protein is a dimer comprising two protein
sub-units, each sub-unit being chosen from any one of proteins (i),
(ii) or (iii) as defined above. Both homodimers and heterodimers
are within the scope of the invention. In the context of the
invention, the designation "PSOMT1 proteins" includes dimeric forms
of said proteins. The proteins may be purified from natural
sources, or made by chemical or recombinant techniques.
[0018] According to the invention, the PSOMT1 protein and
derivatives, and dimers thereof, generally have O-methyltransferase
activity, particularly (R,S)-reticuline 7-O-methyltransferase
activity. In the context of the invention, "(R,S)-reticuline
7-O-methyltransferase activity" signifies the capacity of a protein
to methylate (R) or (S) or (R,S)-reticuline at the 7-hydroxyl
group, forming (R)-7-O-methylreticuline, (S)-7-O-methylreticuline,
and (R,S)-7-O-methylreticuline, respectively. The proteins of the
invention catalyse this reaction both in vivo and in vitro.
Preferably, the enzymes of the invention methylate (R) or
(S)-reticuline with equal efficiency, as shown by substantially
equal k.sub.cat/K.sub.m ratios. The 7-O-methylation by PSOMT1 and
derivatives preferably has a pH optimum of approximately pH 8.0,
and a temperature optimum of 37.degree. C. The (R,S)-reticuline
7-O-methyltransferase activity in vitro is measured using the
experimental protocols described in the Examples below on purified
enzyme as obtained from a eukaryotic cell, for example further to
heterologous expression in a eukaryotic host, or any other suitable
technique.
[0019] The PSOMT1 protein of the invention also has the capacity to
methylate substrates other than (R) and (S)-reticuline. In
particular, the PSOMT1 protein has the capacity to methylate in
vitro the following substrates, in addition to (R)-reticuline,
(S)-reticuline, at the 7-hydroxy position: guaiacol, isovanillic
acid, (R,S)-orientaline, (R)-protosinomenine and
(R,S)-isoorientaline. Optimal pH for these methylations are
isovanillic acid: pH 7.5: (R)-protosinomenine pH 9.0; guaiacol: pH
8.0; (R,S)-isoorientaline: pH 7.5-9.0.
[0020] The PSOMT1 protein derivatives of the invention may also
exhibit this capacity to methylate the above substrates in
vitro.
[0021] A first preferred embodiment of the invention thus comprises
the full length PSOMT1 (R,S)-reticuline 7-O-methyltransferase
protein whose amino acid sequence is shown in FIG. 9 (PSOMT1) (SEQ
ID NO: 2). The protein of the invention as illustrated in FIG. 9
has 355 amino acids, and a molecular weight of approximately 43 kDa
(Genebank accession number AY268893). According to this embodiment
of the invention, the full length P. somniferum enzyme may be
obtained by isolation and purification to homogeneity from cell
suspension culture, or from plant parts of P. somniferum, at any
stage of development, and from latex of mature or immature plants.
Alternatively, the enzyme may be produced by recombinant means in
suitable host cells such as plant cells or insect cells. The
protein may consist exclusively of those amino acids shown in FIG.
9 (SEQ ID NO: 2), or may have supplementary amino acids at the N-
or C-terminus. For example, tags facilitating purification may be
added. The protein may also be fused at the N- or C-terminus to a
heterologous protein. A particularly preferred embodiment of the
invention is a protein comprising a homodimer of the PSOMT1
sequence of FIG. 9, having an Mr of approximately 85 kDa.
[0022] According to a second embodiment of the invention, the
PSOMT1 protein may comprise or consist of a fragment of the amino
acid sequence illustrated in FIG. 9 (SEQ ID NO: 2), wherein said
fragment has a length of at least 20 amino acids, for example at
least 40 amino acids and preferably a length of 150 to 354 amino
acids.
[0023] By protein "fragment" is meant any segment of the full
length sequence of FIG. 9 (SEQ ID NO: 2) which is shorter than the
full length sequence. The fragment may be a C- or N-terminal
fragment having for example approximately 20 or 60 or 175 or 250 or
amino acids, or may be an internal fragment having 20 to
approximately 250 amino acids. Preferably the protein fragments
have a length of 200 to 350 amino acids, for example 250 to 320
amino acids, or 275 to 300 amino acids. Particularly preferred are
fragments having a length of between 255 and 350 amino acids, such
as the FIG. 9 (SEQ ID NO: 2) sequence having undergone truncation
at the C- or N-terminal, or short peptides having a length of 20 to
65 amino acids, for example 35 to 50 amino acids.
[0024] The protein fragments of the invention may or may not have
(R,S)-reticuline 7-O-methyltransferase activity. Normally,
fragments comprising at least 250, or at least 300 consecutive
amino acids of the protein shown in FIG. 9 (SEQ ID NO: 2) are
enzymatically active, i.e. have O-methyltransferase activity,
particularly (R,S)-reticuline 7-O-methyltransferase activity.
[0025] A particularly preferred class of peptides according to the
invention are peptides which comprise or consist of a stretch (or
"tract") of at least 8, preferably at least 10, and most preferably
at least 25 amino acids unique to the (R,S)-reticuline
7-O-methyltransferase (PSOMT1) illustrated in FIG. 9 (SEQ ID NO:
2). By "unique to PSOMT1" is meant a tract of amino acids which is
not present in other plant O-methyltransferases as listed in Table
II below. These PSOMT1-specific peptides typically have a length of
10 to 100 amino acids, for example 12 to 70 amino acids, or 18 to
50 amino acids. Such peptides can be used for generation of
PSOMT1-specific antibodies for immunodetection and
immunopurification techniques.
[0026] In general, the PSOMT1 fragments of the invention may
consist exclusively of part of the FIG. 9 (SEQ ID NO: 2) sequence.
Alternatively, they may additionally comprise supplementary amino
acids which are heterologous to the illustrated P. somniferum
enzyme, for example N- and/or C-terminal extensions. Such
supplementary amino acids may be amino acids from
O-methyltransferase enzymes from species other than P. somniferum,
thus providing a chimeric (R,S)-reticuline 7-O-methyltransferase
enzyme, or may be purification tags, fusion proteins etc.
[0027] According to a third preferred embodiment of the invention,
the protein comprises or consists of a "variant" of the amino acid
sequence of FIG. 9 (SEQ ID NO: 2). By "variant" is meant a protein
having at least 70% identity with the amino acid sequence of FIG. 9
(SEQ ID NO: 2) over a length of at least 300 amino acids, and
preferably at least 80%, 85% or 90% identity with the amino acid
sequence of FIG. 9, over a length of at least 300 amino acids.
Particularly preferred are variants having at least 90% or at least
95% identity, for example 95.5 to 99.9% identity. Preferred
variants have sequences which differ from the amino acid sequence
illustrated in FIG. 9 (SEQ ID NO: 2) by insertion, replacement
and/or deletion of at least one amino acid, for example insertion,
replacement and/or deletion of one to 10 amino acids, or one to
five amino acids. Variants differing from the FIG. 9 (SEQ ID NO: 2)
sequence by one to ten amino acid replacements are particularly
preferred, for example two, three, four or five amino acid
substitutions. Such variants may or may not have (R,S)-reticuline
7-O-methyltransferase activity, as defined previously. Preferably,
the variants have this activity.
[0028] Particularly preferred "variant" proteins of the invention
are allelic variants of PSOMT1, or PSOMT1 proteins arising from
expression of other members of a PSOMT1 gene family. For example,
there may exist within a given species of Papaver, or within a
given genotype of P. somniferum, variants of the PSOMT1 gene
containing a number of single point polymorphisms, some of which
may give rise to changes in amino acid sequence. Typically, these
variants contain one to fifteen amino acid substitutions, for
example one to ten, or one to six, with respect to the FIG. 9 (SEQ
ID NO: 2) sequence. Amino acid changes are usually conservative,
with a neutral amino acid such as isoleucine or serine being
replaced by another neutral amino acid such as valine or alanine,
or an acidic amino acid such as aspartic acid being replaced by
another acidic amino acid such as glutamic acid etc.
(R,S)-reticuline 7-O-methyltransferase activity is usually
conserved.
[0029] Other PSOMT1 variants of the invention include proteins
which again have at least 70% identity with the amino acid sequence
of FIG. 9 (SEQ ID NO: 2) over a length of at least 300 amino acids,
and which contain at least part of one or more of the conserved
amino acid motifs shown as shaded boxes (Motifs A, J, K, B, C and
L) in FIG. 3 (PSOMT1 sequence). In accordance with this variant of
the invention, the partial motifs which are conserved are as
follows:
TABLE-US-00001 Part of Motif A: LVDVGGG (SEQ. ID NO: 26) Part of
Motif B: PXXDAXXMK (SEQ. ID NO:27) Part of Motif C: XGKVI (SEQ. ID
NO: 28) Part of Motif J: DLPHV (SEQ. ID NO: 29) Part of Motif K:
HVGGDMF (SEQ. ID NO: 30) Part of Motif L: GKERT (SEQ. ID NO:
31)
using the one-letter amino acid code, and wherein "X" represents
any amino acid.
[0030] The invention thus also includes variants of the FIG. 9 (SEQ
ID NO: 2) protein having the required degree of identity with the
FIG. 9 protein (at least 70%) and including for example the
LVDVGGGTG motif (SEQ ID NO: 32) and the AGKERTEAE (SEQ ID NO: 33)
motif.
[0031] The PSOMT1 proteins of the invention can be used for the
production of methylated catechols or methylated
tetrahydrobenzylisoquinolines. An example of such a method
comprises the steps of: [0032] i) contacting in vitro a PSOMT2
protein having (R,S)-reticuline 7-O-methyltransferase activity with
a substrate chosen from guaiacol, isovanillic acid, (R)-reticuline,
(S)-reticuline, (R,S)-orientaline, (R)-protosinomenine and
(R,S)-isoorientaline at a pH between 7.5 to 9, [0033] ii)
recovering the methylated catechols or methylated
tetrahydrobenzylisoquinolines thus produced.
[0034] The PSOMT1 proteins used in this in vitro method are
generally used in purified, dimeric form.
[0035] In addition to the proteins described above, the invention
also relates to nucleic acid molecules encoding the PSOMT1
proteins, for example cDNA, single and double stranded DNA and RNA,
genomic DNA, synthetic DNA, or to their complementary
sequences.
[0036] Examples of particularly preferred nucleic acid molecules
are molecules comprising or consisting of: [0037] i) the nucleic
acid sequence illustrated in FIG. 8 (SEQ ID NO: 1), or [0038] ii) a
fragment of the nucleic acid sequence illustrated in FIG. 8 (SEQ ID
NO: 1), said fragment having a length of at least 60 nucleotides,
or [0039] iii) a variant of the sequence illustrated in FIG. 8 (SEQ
ID NO: 1), said variant having at least 70% identity with the
sequence of FIG. 8 (SEQ ID NO: 1) over a length of at least 900
bases, or [0040] iv) a sequence complementary to sequences (i),
(ii) or (iii), or [0041] v) any one of sequences (i), (ii) or (iii)
in double-stranded form, or [0042] vi) the RNA equivalent of any of
sequences (i), (ii), (iii), (iv) or (v).
[0043] The nucleic acid molecules (i), (ii), (iii), (iv), (v) and
(vi) are also referred to herein collectively as "(R,S)-reticuline
7-O-methyltransferase gene or derivatives thereof".
[0044] The sequence of FIG. 8 (SEQ ID NO: 1) indicates the coding
region of the full length cDNA of P. somniferum (R,S)-reticuline
7-O-methyltransferase. The invention encompasses any nucleic acid
molecule which consists of this coding sequence, or which
additionally includes further nucleotides at either the 5' and/or
3' extremities, for example, the full sequence shown in FIG. 8 (SEQ
ID NO: 1), which includes 5' and 3' untranslated regions. The
additional nucleotides may be other untranslated regions, or
endogenous or exogenous regulatory sequences, or fusions to other
coding regions.
[0045] Also within the scope of the invention are molecules
comprising or consisting of fragments of the nucleic acid sequence
illustrated in FIG. 8 (SEQ ID NO: 1), said fragments having a
length of at least 25 nucleotides, preferably 30 nucleotides, and
most preferably at least 60 nucleotides In the context of the
invention, a nucleic acid "fragment" signifies any segment of the
full length sequence of FIG. 10 (SEQ ID NO: 15) which is shorter
than the full length sequence. Preferred fragments of the invention
have a length of 60 to 1430 nucleotides, and encode an
enzymatically active (R,S)-reticuline 7-O-methyltransferase.
[0046] Other fragments include 5'- or 3'-terminal truncations, or
an internal fragment, of the sequence of FIG. 8, for example a
fragment of approximately 75 to 1400 nucleotides. Preferred
fragments have a length of 80 to 1300 nucleotides, for example 90
to 1200 or 100 to 1000 nucleotides. Shorter fragments having a
length of 18 or 30 to 150 nucleotides can be used as primers in
nucleic acid amplification reactions, enabling the isolation of
related O-methyltransferases of species other than P. somniferum,
or of different lines within a given species of Papaver. When the
nucleic acid fragment of the invention is relatively short, i.e.
between approximately 18 to 50 nucleotides, it usually comprises a
stretch (or tract) of at least 18 nucleotides which is unique to
the (R,S)-reticuline 7-O-methyltransferase. Such unique tracts may
for example encode protein fragments which do not occur in other
plant O-methyltransferases as shown in Table II, or may be chosen
from the untranslated regions shown in FIG. 8. These fragments, or
their complementary sequences, are useful in amplification
reactions.
[0047] Molecules comprising fragments of the FIG. 8 (SEQ ID NO: 1)
sequence also include genomic DNA which may contain at least one
intron, and which can thus be considered to be an assembly of
fragments linked by one or more intronic sequences. Such a genomic
molecule may further comprise the endogenous (R,S)-reticuline
7-O-methyltransferase regulatory sequences.
[0048] The nucleic acid molecules of the invention may also be a
variant of the nucleotide sequence illustrated in FIG. 8 (SEQ ID
NO: 1), wherein said variant has at least 70% identity with the
sequence of FIG. 8 (SEQ ID NO: 1) over a length of at least 900
bases, and preferably at least 80%, or at least 90% or at least 95%
identity with the sequence of FIG. 8 (SEQ ID NO: 1), over a length
of at least 900 bases. Particularly preferred variants show 95 to
99.9% identity for example 96 to 99.5% identity. Most preferred
variants differ from the sequence of FIG. 8 (SEQ ID NO: 1) by
insertion, replacement and/or deletion of at least one nucleotide,
for example replacement of one to two hundred nucleotides, or
insertion of a total of 2 or more nucleotides, for example an
insertion of 3 to 100 nucleotides, whilst conserving at least 70%
identity with the FIG. 8 (SEQ ID NO: 1) sequence. An example of a
sequence variant is a sequence that is degenerate with respect to
the sequence illustrated in FIG. 8 (SEQ ID NO: 1).
[0049] Typically, nucleic acid variants of the invention have the
capacity to hybridise to the sequence illustrated in FIG. 8 (SEQ ID
NO: 1) in stringent conditions, particularly to the coding sequence
illustrated in FIG. 8. Stringent conditions are for example those
set out in Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA,
1989 pages 387-389, paragraph 11.
[0050] Particularly preferred nucleic acid variants of the
invention are variants of the (R,S)-reticuline
7-O-methyltransferase gene occurring within a given species of P.
somniferum, such as allelic variants or gene family members.
Allelic variants usually have up to 1% difference in nucleotide
sequence with respect to the full length coding sequence, for
example with respect to the coding sequence shown in FIG. 8, and
usually share the same chromosomal location. Such allelic variants
thus show at least 99% identity with the coding sequence shown in
FIG. 8 (SEQ ID NO: 1), for example at least 99.3 or at least 99.7%
identity, and comprise at least one nucleic acid substitution with
respect to this sequence, for example 2 to 10 base changes. The
changes are usually single base changes and may be silent or may
give rise to amino acid differences.
[0051] According to the invention, members of a gene family usually
differ by up to 5% with respect to the full length coding sequence,
for example with respect to the coding sequence shown in FIG. 8,
and need not share the same chromosomal location. Such family
members thus show at least 95% identity with the coding sequence
shown in FIG. 8 (SEQ ID NO: 1), for example at least 97% identity,
and comprise at least one nucleic acid substitution with respect to
this sequence, for example 2 to 50 base changes, more particularly
2 to 30 changes. Again, the changes are usually single base changes
and may be silent or may give rise to amino acid differences.
[0052] Nucleic acid variants and fragments of the invention may
encode an enzymatically active protein or not. Preferred variants
encode proteins having (R,S)-reticuline 7-O-methyltransferase
activity, as defined previously.
[0053] Further variants of the nucleic acid sequences of the
invention include mutants obtained for example, by mutagenesis,
either directed or random, producing new enzymes with modified
specificities. For example, mutants capable of methylating a
broader range of substrates, or capable of methylating substrates
totally different from the natural substrates can be generated, for
example mutants capable of methylating morphine to produce codeine.
Variants generated in such directed evolution methods generally
differ by up to 5% with respect to the full length coding sequence,
for example with respect to the coding sequence shown in FIG. 8
(SEQ ID NO: 1), showing at least 95% identity with the coding
sequence shown in FIG. 8, for example at least 97% identity, and
comprise at least one nucleic acid substitution, insertion or
deletion with respect to this sequence, for example 2 to 50 base
changes, more particularly 2 to 15 changes. The changes usually
give rise to amino acid differences.
[0054] A further major aspect the invention relates to a second
methyl transferase enzyme involved in alkaloid biosynthesis in
opium poppy, namely (R,S)-norcoclaurine 6-O-methyltransferase. In
the framework of the invention, it has been discovered that
different allelic variants of the gene encoding this protein exist
in P. somniferum. The invention thus relates to the different
variants of this protein, and to the corresponding genes and
derivatives thereof.
[0055] More particularly, the invention concerns the so-called
PSOMT2 proteins illustrated in FIG. 3 (SEQ ID NO: 3), and FIG. 13
(SEQ ID NO: 21), and variants and derivatives thereof. These PSOMT2
proteins are allelic variants of the P. somniferum
(R,S)-norcoclaurine 6-O-methyltransferase.
[0056] According to a preferred embodiment, the invention thus
concerns a protein having O-methyltransferase activity,
particularly (R,S)-norcoclaurine 6-O-methyltransferase activity,
said protein comprising or consisting of: [0057] i) the PSOMT2
amino acid sequence illustrated in FIG. 3 (SEQ ID NO: 3) or, [0058]
ii) the PSOMT2a amino acid sequence illustrated in FIG. 13 (SEQ ID
NO: 21) or [0059] iii) a fragment of the PSOMT2 or PSMOT2a amino
acid sequences illustrated in FIG. 3 (SEQ ID NO: 3), and FIG. 13
(SEQ ID NO: 21),said fragment having at least 100 amino acids, or
[0060] iv) a variant of the PSOMT2 or PSMOT2a amino acid sequence
of FIG. 3 (SEQ ID NO: 3), or FIG. 13 (SEQ ID NO: 21), said variant
having at least 70% identity, and preferably at least 80% or 90%
identity, most preferable at least 97% identity, for example at
least 99% identity, with the PSOMT2 amino acid sequence of FIG. 3
(SEQ ID NO: 3) or FIG. 13 (SEQ ID NO: 21), over a length of at
least 300 amino acids.
[0061] The fragments or variants of the PSOMT2 and PSOMT2a protein
as defined above will be collectively referred to herein as the
"PSOMT2 derivatives".
[0062] Again, as with the PSOMT1 proteins, the PSOMT2 protein and
derivatives are preferably in dimeric form, i.e. the protein is a
dimer comprising two protein sub-units, each sub-unit being chosen
from any one of proteins (i), (ii), (iii) or (iv) as defined above.
Both homodimers and heterodimers of the PSOMT2 proteins and
derivatives are within the scope of the invention. In the context
of the invention, the designation "PSOMT2 proteins" includes
dimeric forms of said proteins. The proteins may be purified from
natural sources, or made by chemical or recombinant techniques.
[0063] According to a preferred embodiment of the invention, the
protein comprises or consists of a variant of the amino acid
sequence illustrated in FIG. 14 (SEQ ID NO: 23). Such a variant has
from 1 to 10 amino acid substitutions, deletions and/or insertions
with respect to the amino acid sequence illustrated in FIG. 14 (SEQ
ID NO: 23), and has not more than 99.8% identity with the full
length sequence of FIG. 14. The said variant has
O-methyltransferase activity, particularly (R,S)-norcoclaurine
6-O-methyltransferase activity.
[0064] The invention thus encompasses allelic variants of the FIG.
14 (SEQ ID NO: 23) sequence, which preferably have between 97% and
99.7% identity with the full length sequence of FIG. 14, for
example between 98.5% and 99.5% identity. Such variants include
those having from 1 to 5 amino acid substitutions with respect to
the FIG. 14 sequence, particularly 2 to four amino acid
substitutions.
[0065] It has been established by the inventors that the naturally
occurring variants of the P. somniferum (R,S)-norcoclaurine
6-O-methyltransferase are particularly susceptible to have
variation at any one of amino acid positions 93, 150, 233, 245 and
274, wherein the amino acid positions referred to are those
illustrated in FIGS. 14 (SEQ ID NO: 23) and 16. Consequently, the
invention includes PSOMT2 proteins wherein at least one amino acid
substitution, deletions or insertion occurs at a position chosen
from positions 93, 150, 233, 245 and 274, as illustrated in FIG.
14. Preferably, the variation is a single amino acid substitution,
occurring at one or more of positions 93, 150, 233, 245 and 274,
for example at positions 93, 235 and 245.
[0066] Typically, the PSMOT2 proteins of the invention comprise or
consist of the sequence illustrated in FIG. 16, (SEQ ID NO: 25)
wherein "X" at positions 93, 150, 233, 245 and 274 represents the
occurrence of any amino acid, but are preferably chosen from the
following amino acids:
TABLE-US-00002 X.sub.93: Pro, Val X.sub.150: Val, Glu X.sub.233:
Ser, Pro X.sub.245: Ala, Gly; X.sub.274: Gly, Val,
[0067] Advantageously, X.sub.93 is not Pro when X.sub.150,
X.sub.233, X.sub.245, X.sub.274 together represent the following
amino acids: Xaa.sub.150 is Glu, Xaa.sub.233 is Ser, Xaa.sub.245 is
Ala and Xaa.sub.274 is Gly.
[0068] According to one embodiment of this mode of the invention,
the methyl transferase enzyme thus comprises the full length PSOMT2
(R,S)-norcoclaurine 6-O-methyltransferase protein whose amino acid
sequence is shown in FIG. 3 (PSOMT2) (SEQ ID NO: 3 and SEQ ID NO:
19), or the full length PSOMT2a (R,S)-norcoclaurine
6-O-methyltransferase protein whose amino acid sequence is shown in
FIG. 13 (PSOMT2) (SEQ ID NO: 21). These proteins have 346 amino
acids, and a molecular weight of approximately 43 kDa (Genebank
accession number AY268894). According to this embodiment of the
invention, the full length PSOMT2 enzymes may be obtained by
isolation and purification to homogeneity from cell suspension
culture, or from plant parts of P. somniferum, at any stage of
development, and from latex of mature or immature plants.
Alternatively, the enzyme may be produced by recombinant means in
suitable host cells such as plant cells or insect cells. The
protein may consist exclusively of those amino acids shown in FIG.
3 or 13, or may have supplementary amino acids at the N- or
C-terminus. For example, tags facilitating purification may be
added. The protein may also be fused at the N- or C-terminus to a
heterologous protein. A particularly preferred embodiment of the
invention is a protein comprising a homodimer of the PSOMT2
sequence of FIG. 3 or 13, having an Mr of approximately 85 kDa.
[0069] The PSOMT2 proteins and derivatives as defined above, and
dimers thereof, generally have O-methyltransferase activity,
particularly (R,S)-norcoclaurine 6-O-methyltransferase activity. In
the context of the invention, "(R,S)-norcoclaurine
6-O-methyltransferase activity" signifies the capacity of a protein
to carry out methylation of (R,S)-norcoclaurine, (S)-norcoclaurine,
and/or (R)-norcoclaurine at the 6-hydroxyl group, forming
(R,S)-coclaurine, forming (S)-coclaurine, and (R)-coclaurine,
respectively. The proteins of the invention catalyse this reaction
both in vivo and in vitro. The 6-O-methylation by PSOMT2 and
derivatives preferably occurs over a wide range of pH (pH 6.0 to
9.0), and a temperature optimum of 37 to 41.degree. C. The
(R,S)-norcoclaurine 6-O-methyltransferase activity in vitro is
measured using the experimental protocols described in the Examples
below on purified enzyme as obtained from a eukaryotic cell, for
example further to heterologous expression in a eukaryotic host, or
any other suitable technique.
[0070] The PSOMT2 proteins and derivatives of the invention also
have the in vitro capacity to methylate substrates other than
(R,S)-norcoclaurine, (S)-norcoclaurine, and/or (R)-norcoclaurine.
In particular, the PSOMT2 proteins have the capacity to methylate
in vitro the following substrates, in addition to (R)-reticuline,
(S)-reticuline, at the 6-hydroxy position: (R)-norprotosinomenine,
(S)-norprotosinomenine and (R,S)-isoorientaline. Optimal pH for
these 6-O-methylations is at pH 7.5, with a temperature optima
again at 37 to 41.degree. C.
[0071] In accordance with another embodiment of the PSOMT2 aspect
of the invention, the protein or peptide may be comprise or consist
a portion or fragment of the full length protein illustrated in
FIG. 16. Such a fragment generally has a length of 25 to 345 amino
acids, for example 100 to 340 amino acids, or 150 to 300 amino
acids, and spans that part of the protein which encompasses at
least one of positions 93, 150, 233, 245 and 274, wherein X has the
previously ascribed meaning.
[0072] By PSOMT2 protein "fragment" is meant any segment of the
full length sequence of FIG. 16 which is shorter than the full
length sequence. The fragment may be a C- or N-terminal fragment
having for example approximately 25 or 60 or 175 or 250 or amino
acids, or may be an internal fragment having 30 to approximately
250 amino acids. Preferably the PSOMT2 protein fragments have a
length of 200 to 350 amino acids, for example 250 to 320 amino
acids, or 275 to 300 amino acids. Particularly preferred are
fragments having a length of between 255 and 350 amino acids, such
as the FIG. 3 or FIG. 13 sequence having undergone truncation at
the C- or N-terminal.
[0073] Examples of PSOMT2 protein fragments and peptides thus
include proteins comprising or consisting of amino acids 1 to 150
of the FIG. 3 or FIG. 13 sequence, or amino acids 139 to 250, or
230 to 346 of the FIG. 3 or FIG. 13 sequence.
[0074] The PSOMT2 protein fragments of the invention may or may not
have (R,S)-norcoclaurine 6-O-methyltransferase activity. Normally,
fragments comprising at least 250, or at least 300 consecutive
amino acids of the protein shown in FIG. 9 (SEQ ID NO: 2) are
enzymatically active, i.e. have O-methyltransferase activity,
particularly (R,S)-norcoclaurine 6-O-methyltransferase
activity.
[0075] A particularly preferred class of PSOMT2 peptides according
to the invention are peptides which comprise or consist of a
stretch (or "tract") of at least 8, preferably at least 10, and
most preferably at least 25 amino acids unique to the
(R,S)-norcoclaurine 6-O-methyltransferase (PSOMT2) illustrated in
FIG. 3 (SEQ ID NO: 3) or 13 (SEQ ID NO: 21). By "unique to PSOMT2"
is meant a tract of amino acids which is not present in other plant
O-methyltransferases as listed in Table II below. These
PSOMT2-specific peptides typically have a length of 10 to 100 amino
acids, for example 12 to 70 amino acids, or 18 to 50 amino acids.
Such peptides can be used for generation of PSOMT2-specific
antibodies for immunodetection and immunopurification
techniques.
[0076] Other PSOMT2 variants of the invention include proteins
which again have at least 95 or 97% identity with the amino acid
sequence of FIG. 3 (SEQ ID NO: 3) or FIG. 13 (SEQ ID NO: 21) over a
length of at least 300 amino acids, and which contain at least part
of one or more of the conserved amino acid motifs shown as shaded
boxes (Motifs A, J, K, B, C and L) in FIG. 3 (PSOMT2 sequence). In
accordance with this variant of the invention, the partial motifs
which are conserved are as follows:
TABLE-US-00003 Part of Motif A: LVDVGGG (SEQ. ID NO: 26) Part of
Motif B: PXXDAXXMK (SEQ. ID NO:27) Part of Motif C: XGKVI (SEQ. ID
NO: 28) Part of Motif J: DLPHV (SEQ. ID NO: 29) Part of Motif K:
HVGGDMF (SEQ. ID NO: 30) Part of Motif L: GKERT (SEQ. ID NO:
31)
using the one-letter amino acid code, and wherein "X" represents
any amino acid.
[0077] The PSOMT2 proteins of the invention can be used for the
production of methylated tetrahydrobenzylisoquinolines. An example
of such a method comprises the steps of: [0078] i) contacting in
vitro a protein having norcoclaurine 6-O-methyltransferase
activity, for example a PSOMT2 protein or derivative as defined
above, with a substrate chosen from (R,S)-norcoclaurine,
(R,S)-isoorientaline, (R)-norprotosinomenine and
(S)-norprotosinomenine at pH 6.0 to 9.0, [0079] ii) recovering the
methylated tetrahydrobenzylisoquinolines thus produced.
[0080] The PSOMT2 proteins used in this in vitro method are
generally used in purified, dimeric form.
[0081] In addition to the PSOMT2 proteins described above, the
invention also relates to nucleic acid molecules encoding the
PSOMT2 proteins, for example cDNA, single and double stranded DNA
and RNA, genomic DNA, synthetic DNA, or to their complementary
sequences.
[0082] Examples of particularly preferred nucleic acid molecules
are molecules comprising or consisting of: [0083] i) the nucleic
acid sequence illustrated in FIG. 10 (SEQ ID NO: 18), or [0084] ii)
the nucleic acid sequence illustrated in FIG. 11 (SEQ ID NO: 20),
or [0085] iii) a fragment of the nucleic acid sequence illustrated
in FIG. 10 or 11, said fragment having a length of at least 60
nucleotides, or [0086] iv) a variant of the sequence illustrated in
FIG. 10 or 11, said variant having at least 70% identity, for
example at least 80% or 90% identity, and preferably at least 99 to
99.9% identity, with the sequence of FIG. 10 or 11 over a length of
at least 900 bases, or [0087] v) a sequence complementary to
sequences (i), (ii), (iii), or (iv), [0088] vi) any one of
sequences (i), (ii), (iii), (iv) or (v) in double-stranded form, or
[0089] vii) the RNA equivalent of any of sequences (i), (ii),
(iii), (iv), (v) or (vi).
[0090] The nucleic acid molecules (i), (ii), (iii), (iv), (v), (vi)
and (vi) are also referred to herein collectively as the
"norcoclaurine 6-O-methyltransferase gene or derivatives
thereof".
[0091] Preferred nucleic acid molecule of the invention are
variants of the sequence illustrated in FIG. 12 (SEQ ID NO: 22).
Such variants comprise or consist of a sequence having from 1 to 10
nucleotide insertions, substitutions or deletions with respect to
the nucleic acid sequence illustrated in FIG. 12, and have not more
than 99.9% identity, preferably not more than 99.5% identity with
the full length sequence of FIG. 12. These variants include the
different norcoclaurine 6-O-methyltransferase gene alleles, and
preferably differ from the FIG. 12 sequence by 1 to 5 single
nucleotide substitutions, which may or may not give rise to amino
acid changes.
[0092] Such variants include the nucleic acid molecule comprising
or consisting of the PSOMT2 coding sequence illustrated in FIG. 10,
or the PSOMT2a coding sequence illustrated in FIG. 11. The
invention encompasses any nucleic acid molecule which consists of
either one of the coding sequences illustrated in FIGS. 10 and 11,
or which additionally includes further nucleotides at either the 5'
and/or 3' extremities, for example, the full sequence shown in FIG.
10 (SEQ ID NO: 18), which includes 5' and 3' untranslated regions.
The additional nucleotides may be other untranslated regions, or
endogenous or exogenous regulatory sequences, or fusions to other
coding regions.
[0093] Also within the scope of the invention are molecules
comprising or consisting of fragments of the nucleic acid sequence
illustrated in FIG. 10 or 11. Such fragments having a length of at
least 25 nucleotides, preferably 30 nucleotides, and most
preferably at least 60 nucleotides In the context of the invention,
a PSOMT2 nucleic acid "fragment" signifies any segment of the full
length sequence of FIG. 10 or 11 which is shorter than the full
length sequence. Preferred fragments of the invention have a length
of 60 to 1040 nucleotides, and encode an enzymatically active
norcoclaurine 6-O-methyltransferase.
[0094] Particularly preferred PSOMT2 nucleic acid fragments
comprise or consist of a stretch (or tract) of the sequence
illustrated in FIG. 10 (SEQ ID NO: 18) or FIG. 11 (SEQ ID NO: 20),
said fragment having from 60 to 1000 nucleotides, and spans that
part of the molecule which encodes at least one of amino acids 93,
150, 233, 245 and 274. Typical fragment lengths are from 100 to 500
bases.
[0095] Other PSOMT2 fragments include 5'- or 3'-terminal
truncations, or an internal fragment, of the sequence of FIG. 10 or
11, for example a fragment of approximately 75 to 1400 nucleotides.
Preferred fragments have a length of 80 to 1300 nucleotides, for
example 90 to 1200 or 100 to 1000 nucleotides. Shorter fragments
having a length of 15 or 18 to 150 nucleotides can be used as
primers in nucleic acid amplification reactions, enabling the
isolation of related O-methyltransferases of species other than P.
somniferum, or of different lines within a given species of
Papaver. Examples of such sequences are molecules having a length
of 15 to 300 nucleotides, for example 20 to 50 nucleotides; and
comprising at least 15 consecutive nucleotides of the 5' sequence
from nucleotide 1 to nucleotide 31 of the sequence illustrated in
FIG. 10 (SEQ ID NO: 18). A further example is a molecule having a
length of 15 to 300 nucleotides, for example 20 to 50 nucleotides,
and comprising at least 15 consecutive nucleotides of the 3'
extremity of the sequence illustrated in FIG. 10 (SEQ ID NO: 18),
extending from nucleotide 1210 to nucleotide 1320.
[0096] When the nucleic acid fragment of the invention is
relatively short, i.e. between approximately 18 to 50 nucleotides,
it usually comprises a stretch (or tract) of at least 18
nucleotides which is unique to the PSOMT2 gene. Such unique tracts
may for example encode protein fragments which do not occur in
other plant O-methyltransferases as shown in Table II, or may be
chosen from the untranslated regions shown in FIG. 10. These
fragments, or their complementary sequences, are useful in
amplification reactions.
[0097] Molecules comprising fragments of the FIG. 10 or FIG. 11
sequence also include genomic DNA which may contain at least one
intron, and which can thus be considered to be an assembly of
fragments linked by one or more intronic sequences. Such a genomic
molecule may further comprise the endogenous norcoclaurine
6-O-methyltransferase regulatory sequences.
[0098] Typically, nucleic acid variants of the invention have the
capacity to hybridise to the sequence illustrated in FIG. 10 or 11
in stringent conditions, particularly to the coding sequence
illustrated in FIG. 10 or 11. Stringent conditions are for example
those set out in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
USA, 1989 pages 387-389, paragraph 11.
[0099] Nucleic acid variants and fragments of the invention may
encode an enzymatically active protein or not. Preferred variants
encode proteins having O-methyltransferase activity, particularly
norcoclaurine 6-O-methyltransferase activity, as defined
previously.
[0100] Further variants of the PSOMT2 nucleic acid sequences of the
invention include mutants obtained for example, by mutagenesis,
either directed or random, producing new enzymes with modified
specificities. For example, mutants capable of methylating a
broader range of substrates, or capable of methylating substrates
totally different from the natural substrates can be generated, for
example mutants capable of methylating morphine to produce codeine.
Variants generated in such directed evolution methods generally
differ by up to 5%, for example by up to 2 or 3% with respect to
the full length coding sequence, for example with respect to the
coding sequence shown in FIG. 10 or 11, showing at least 95%
identity with the coding sequence shown in FIG. 10 or 11, for
example at least 97% identity, and comprise at least one nucleic
acid substitution, insertion or deletion with respect to this
sequence, for example 2 to 50 base changes, more particularly 2 to
15 changes. The changes usually give rise to amino acid
differences.
[0101] In a more general context, the invention also encompasses
nucleic acid molecules that are complementary to any of the
foregoing molecules, variants and fragments, both PSOMT1 and PSOMT2
derivatives. In the context of the invention, "complementary" means
that Watson-Crick base-pairs can form between a majority of bases
in the complementary sequence and the reference sequence.
Preferably, the complementarity is 100%, but one or two mismatches
in a stretch of twenty or thirty bases can be tolerated.
Additionally, complementary stretches may be separated by
non-complementary stretches. Examples of nucleic acids of the
invention which comprise sequences complementary to the PSOMT1 and
PSOMT2 derivatives include primers, ribozymes, deoxyribozymes,
antisense sequences, and interfering RNA.
[0102] The nucleic acid molecules of the invention may contain at
least one nucleotide analogue in replacement of, or in addition to,
a naturally occurring nucleotide. Ribonucleotide and
deoxyribonucleotide derivatives or modifications are well known in
the art, and are described, for example, in Principles of Nucleic
Acid Structure (Ed, Wolfram Sanger, Springer-Verlag, New York,
1984), particularly pages 159-200), and in the CRC Handbook of
Biochemistry (Second edition, Ed, H. Sober, 1970). A large number
of modified bases are found in nature, and a wide range of modified
bases have been synthetically produced. For example, amino groups
and ring nitrogens may be alkylated, such as alkylation of ring
nitrogen atoms or carbon atoms such as N1 and N7 of guanine and C5
of cytosine; substitution of keto by thioketo groups; saturation of
carbon.dbd.carbon double bonds. Bases may be substituted with
various groups, such as halogen, hydroxy, amine, alkyl, azido,
nitro, phenyl and the like. Examples of suitable nucleotide
analogues are listed in Table I below. In accordance with this
embodiment of the invention, synthetic genes comprising one or more
nucleotide analogues, for example methylated bases, are made, for
example by chemical synthesis, and can be introduced into cells for
a transient expression process in vivo.
TABLE-US-00004 TABLE 1 Nucleotide Analogs Abbreviation Description
ac4c 4-acetylcytidine chm5u 5-(carboxyhydroxylmethyl)uridine cm
2'-O-methylcytidine cmnm5s2u
5-carboxymethylaminomethyl-2-thiouridine d dihydrouridine fm
2'-O-methylpseudouridine galq .beta.,D-galactosylqueosine gm
2'-O-methylguanosine I inosine i6a N6-isopentenyladenosine m1a
1-methyladenosine m1f 1-methylpseudouridine m1g 1-methy[guanosine
ml1 1-methylinosine m22g 2,2-dimethylguanosine m2a
2-methyladenosine m2g 2-methylguanosine m3c 3-methylcytidine m5c
5-methylcytidine m6a N6-methyladenosine m7g 7-methylguanosine mam5u
5-methylaminomethyluridine mam5s2u
5-methoxyaminomethyl-2-thiouridine manq
.beta.,D-mannosylmethyluridine mcm5s2u
5-methoxycarbonylmethyluridine mo5u 5-methoxyuridine ms2i6a
2-methylthio-N6-isopentenyladenosine ms2t6a
N-((9-.beta.-D-ribofuranosyl-2-methylthiopurine-6-
yl)carbamoyl)threonine mt6a
N-((9-.beta.-D-ribofuranosylpurine-6-yl)N-methyl-
carbamoyl)threonine mv uridine-5-oxyacetic acid methylester o5u
uridine-5-oxyacetic acid (v) osyw wybutoxosine p pseudouridine q
queosine s2c 2-thiocytidine s2t 5-methyl-2-thiouridine s2u
2-thiouridine s4u 4-thiouridine t 5-methyluridine t6a
N-((9-.beta.-D-ribofuranosylpurine-6-yl)carbamoyl)threoninetm
2'-O-methyl-5-methyluridine um 2'-O-methyluridine yw wybutosine x
3-(3-amino-3-carboxypropyl)uridine, (acp3)u araU
.beta.,D-arabinosyl araT .beta.,D-arabinosyl
[0103] The nucleic acid molecules of the invention can be used to
transform or transfect eukaryotic and prokaryotic cells. To this
end, the sequences are usually operably linked to transcription
regulatory sequences such as promoters, transcription terminators,
enhancers etc. The operable link between the (R,S)-reticuline
7-O-methyltransferase-derived coding sequence or the norcoclaurine
6-O-methyltransferase coding sequence, and the regulatory
sequence(s) may be direct or indirect, i.e. with or without
intervening sequences. They may also contain internal ribosome
entry sites (IRES). The regulatory sequences may be endogenous to
the coding sequence, i.e. they are the regulatory sequences
naturally associated with the (R,S)-reticuline
7-O-methyltransferase gene or the norcoclaurine
6-O-methyltransferase gene in the genome of the plant.
Alternatively, the regulatory sequences may be heterologous to the
(R,S)-reticuline 7-O-methyltransferase sequence or the
norcoclaurine 6-O-methyltransferase sequence. In this latter case
the resulting construct forms a chimeric gene, comprising a coding
sequence derived from the methyltransferase gene, operably linked
to at least one heterologous transcription regulatory sequence. In
the context of the invention, the term "coding sequence" signifies
a DNA sequence that encodes a functional RNA molecule. The RNA
molecule may be untranslated, or may encode an enzymatically-active
protein, or enzymatically-inactive protein.
[0104] The invention also relates to eukaryotic and prokaryotic
cells transformed or transfected by the nucleic acid sequences
derived from the (R,S)-reticuline 7-O-methyltransferase gene, and
from the norcoclaurine 6-O-methyltransferase gene. An example of a
suitable prokaryotic cell is a bacterial cell. Examples of suitable
eukaryotic cells are yeast cells, vertebrate cells such as
mammalian cells, for example mouse, monkey, or human cells, or
invertebrate cells such as insect cells. Plant cells are
particularly preferred. In the context of the present invention,
the term "plant" is to be understood as including mosses and
liverworts. The plant cells can be any type of plant cells,
including monocotyledonous or dicotyledonous plant cells. The cells
may be differentiated cells or callus for example suspension
cultures. Cells of the genus Papaver are particularly
preferred.
[0105] According to the invention, cells are transfected or
transformed using techniques conventional in the art, in conditions
allowing expression of the (R,S)-reticuline 7-O-methyltransferase
gene or derivatives, or norcoclaurine 6-O-methyltransferase or
derivatives. A number of transformation techniques have been
reported for Papaver. For example, microprojectile bombardment of
cell suspension cultures may be used. Transformation may also be
effected using Agrobacterium tumefaciens, or Agrobacterium
rhizogenes, using either cell suspension cultures or tissue
explants. A number of further techniques are available and are
known to the skilled man.
[0106] When transforming cells with the methyltransferase genes or
derivatives of the invention, the choice of cell is made depending
upon the objective to be achieved.
[0107] One objective is to produce recombinant (R,S)-reticuline
7-O-methyltransferase enzyme, or derivatives thereof. A preferred
method for producing proteins having this activity comprises:
[0108] i) transforming or transfecting a cell with a
(R,S)-reticuline 7-O-methyltransferase gene or derivatives as
defined above, in conditions permitting the expression of the
protein having (R,S)-reticuline 7-O-methyltransferase activity,
[0109] ii) propagating the said cells, and [0110] iii) recovering
the thus-produced protein having (R,S)-reticuline
7-O-methyltransferase activity.
[0111] A further objective is to produce recombinant norcoclaurine
6-O-methyltransferase enzyme, or derivatives thereof. A preferred
method comprises the steps of: [0112] i) transforming or
transfecting cells with a (R,S)-norcoclaurine 6-O-methyltransferase
gene or derivatives thereof, as defined above, in conditions
permitting the expression of the protein having norcoclaurine
6-O-methyltransferase activity, [0113] ii) propagating the said
cells, and [0114] iii) recovering the thus-produced protein having
norcoclaurine 6-O-methyltransferase activity.
[0115] For the purpose of producing recombinant enzyme, any of the
above listed cell-types can be used. Plant cells such as cells of a
Papaver species, or insect cells, as demonstrated in the examples
below, are particularly suitable. Bacterial cells, such as E. coli,
can also be used.
[0116] The enzymes of the invention, and their derivatives and
variants, can also be used in semi-synthetic drug preparation,
where necessary in association with other enzymes involved in
alkaloid biosynthesis, for example in the preparation of the
analgesics codeine and morphine, and the antitussive noscapine, the
vasodilator papaverine and the antimicrobial benzo[c]phenanthridine
sanguinarine.
[0117] The (R,S)-reticuline 7-O-methyltransferase genes and
derivatives of the invention can also be used for producing
7-O-methylreticuline. Such a method comprises the steps of: [0118]
i) introducing a nucleic acid molecule encoding a protein of the
invention having (R,S)-reticuline 7-O-methyltransferase activity
into a plant cell which is capable of expressing (R)-reticuline or
(S)-reticuline, [0119] ii) propagating said plant cell in
conditions wherein the (R,S)-reticuline 7-O-methyltransferase and
the (R)-reticuline or (S)-reticuline are expressed, thereby
producing a multiplicity of cells, [0120] iii) recovering
7-O-methylreticuline from said multiplicity of cells.
[0121] Likewise, the (R,S)-norcoclaurine 6-O-methyltransferase
genes and derivatives of the invention can also be used for
producing (R) or (S)-coclaurine. Such a method comprises the steps
of: [0122] i) introducing an exogenous nucleic acid molecule
encoding a protein having norcoclaurine 6-O-methyltransferase
activity into a plant cell which is capable of expressing
(S)-norcoclaurine, [0123] ii) propagating said plant cell in
conditions wherein the norcoclaurine 6-O-methyltransferase activity
and the (S)-norcoclaurine are expressed, thereby producing a
multiplicity of cells, [0124] iii) recovering (S)-coclaurine from
said multiplicity of cells.
[0125] In such methods the multiplicity of cells is preferably a
cell culture of differentiated or undifferentiated cells.
[0126] Various aspects of the invention are illustrated in the
Figures:
[0127] FIG. 1. Schematic biosynthetic pathway leading from
(S)-norcoclaurine to (S)-scoulerine, (R)-reticuline and laudanine
in P. somniferum. The pathway from (S)-norcoclaurine to
(S)-reticuline is central to the isoquinoline alkaloids accumulated
in opium poppy. (S)-Reticuline is a branch point intermediate that
is subsequently oxidized at C-1-N to lead into the morphinan
pathway, or at N--CH.sub.3 to proceed on to (S)-scoulerine--derived
alkaloids such as the benzo[c]phenanthridines. In addition,
reticuline can be 7-O-methylated to laudanine.
[0128] FIG. 2. Two-dimensional gel electrophoretic pattern of the
cytosolic fraction proteins of latex collected from P. somniferum
capsules. Fifty micrograms protein were loaded per gel and were
visualized by silver staining. The arrow points to the position of
the O-methyltransferase described in this work. Protein spots from
Coomassie Brilliant Blue R-250-stained gels were excised, the
proteins digested in situ with endoproteinase Lys-C and the
peptides resolved and sequenced according to (24).
[0129] FIG. 3 Amino acid sequence comparison of PSOMT1 (SEQ ID
NO:2) and PSOMT2 (SEQ ID NO:3) from P. somniferum. The shaded
motifs are conserved regions motif A, J, K, B, C and L indicative
of plant methyltransferases according to Joshi and Chiang (2).
These sequence signatures were based upon plant methyltransferase
amino acid sequence comparisons and were not functionally defined.
They are mainly used to indicate whether unidentified proteins may
be O-methyltransferases.
[0130] FIG. 4. Phylogenetic tree of plant methyltransferases of
defined and of unknown function. Forty-four amino acid sequences of
proteins of plant origin were compared to generate a tree that
indicates the phylogenetic relationship between PSOMT1, PSOMT2 and
other putative and defined O-methyltransferases. PSOMT1 grouped
most closely to two putative methyltransferases from pine, while
PSOMT2 was most similar to (R,S)-norcoclaurine
6-O-methyltransferase of (S)-reticuline biosynthesis from C.
japonica (5). Two additional (R,S)-norcoclaurine
6-O-methyltransferases from T. tuberosum are clearly more related
to caffeic acid O-methyltransferases from a variety of plant
species than to either PSOMT1 or PSOMT2. The abbreviations and
accession numbers of the amino acid sequences referred to in FIG. 4
are shown in Table II:
TABLE-US-00005 TABLE II Database Abbreviation Plant Enzyme
accession CbrIEMT Clarkia breweri (Iso)eugenol O-methyltransferase
AAC01533 CbrCafOMT Clarkia breweri caffeic acid O-methyltransferase
AAB71141 TtuCatOMT4 Thalictrum tuberosum caffeic acid
O-methyltransferase AAD29845 Ttu6OMT1 Thalictrum tuberosum caffeic
acid O-methyltransferase AAD29841 Ttu6OMT2 Thalictrum tuberosum
caffeic acid O-methyltransferase AAD29842 TtuOMT3 Thalictrum
tuberosum caffeic acid/catechol O-methyltransferase AAD29843
TtuCafOMT5 Thalictrum tuberosum caffeic acid O-methyltransferase
AAD29845 PsoCatOMT Papaver somniferum catechol O-methyltransferas
AY268895 GecIli2OMT Glycyrrhiza echinata Isoliquiritigenin 2{grave
over ( )}OMT BAA13683 MsatIliOMT Medicago sativa isoliquiritigenin
2'-O-methyltransferase AAB48059 PtaCafOMT Pinus taeda caffeic acid
O-methyltransferase AAC49708 PraCafOMT Pinus radiata caffeic acid
O-methyltransferase AAD24001 Pso7OMT Papaver somniferum reticuline
7-O methyltransferase AY268893 Pso6OMT Papaver somniferum
norcoclaurine 6-O methyltransferase AY268894 Cj6OMT Coptis japonica
norcoclaurine 6-O methyltransferase BAB08004 Cj4{grave over ( )}OMT
Coptis japonica 3{grave over ( )}hydroxy-N-methylcoclaurine 4{grave
over ( )}O- BAB08005 methyltransferase TaOMT Triticum aestivum
o-methyltransferase AAD10485 ZmOMT Zea mays O-methyltransferase
P47917 HvF7OMT Hordeum vulgare S52015 RhybOOMT Rosa hybrida orcinol
O-methyltransferase AAM23004 RhybOOMT2 Rosa hybrida orcinol
O-methyltransferase AAM23005 PduOMT Prunus dulcis
O-methyltransferase CAA11131 ParOMT Prunus armeniaca
O-methyltransferase AAB71213 PpyOMT Pyrus pyrifolia
O-methyltransferase BAA86059 ObaCVOMT Ocimun basilicum chavicol
O-methyltransferase1 AF435007 ObaEOMT Ocimun basilicum eugenol
O-methyltransferase1 AF435008 Msat7-IOMT Medicago sativa
isoflavone-7-O-methyltransferase T09254 MsatOMT Medicago sativa
o-methyltransferase iomt2003 T09299 PsatHMOMT Pisum sativum
6a-hydroxymaackiain methyltransferase T06786 AthCatOMT Arabidopsis
thaliana catechol O-methyltransferas1 T04963 CrocafOMT Catharanthus
roseus caffeic acid O-methyltransferase AAK20170 ObaCafOMT Ocimun
basilicum caffeic acid O-methyltransferase1 AAD38189 ZelCafOMT
Zinnia elegans caffeic acid O-methyltransferase AAA86718 NtaCafOMT
Nicotiana tabacum catechol O-methyltransferase S36403 CanOMT
Capsicum annuum O-diphenol-O-methyltransferase T12259 PtoCafOMT
Populus tomentosa caffeic acid 3-O methyltransferase AAF63200
PtrCaf3OMT Populus tremuloides caffeic acid 3-O methyltransferase
Q00763 PbaCafOMT Populus balsamifera caffeic acid
O-methyltransferase CAA01820 PdulCafOMT Prunus dulcis caffeic acid
O-methyltransferase CAA58218 MsatCafOMT Medicago sativa caffeic
acid O-methyltransferase AAB46623 AthCafOMT Arabidopsis thaliana
caffeic acid O-methyltransferase1 AAB96879 CamCafOMT Chrysosplenium
americanum caffeic acid O-methyltransferase1 AAA86982 EglCafOMT
Eucalyptus globulus caffeic acid O-methyltransferase1 AAD50440
EguOMT Eucalyptus gunnii caffeic acid O-methyltransferase1
CAA52814
[0131] FIG. 5. RNA gel blot analysis of PSOMT1 and PSOMT2. Top
panel, PSOMT1 is expressed predominantly in bud and stem, and to a
much lesser degree, in leaf of P. somniferum. Middle panel, PSOMT2
is expressed in bud, stem, leaf and root, and to a lesser degree in
capsule. These results were obtained after blotting a P. somniferum
RNA gel and hybridizing to .sup.32P-labeled full-length PSOMT1 or
PSOMT2. Radioactivity was visualized by phosphorimagery. The bottom
panel is a photograph of ethidium bromide-visualized RNA in the gel
prior to blotting. This served as an RNA loading control.
[0132] FIG. 6. Chemical structures of the substrates methylated by
either PSOMT1 or PSOMT2.
[0133] FIG. 7. Mass spectrometric fragmentation of orientaline
transformed by PSOMT1. Each substrate and the corresponding enzymic
reaction products were analyzed by HPLC-MS. Orientaline is shown as
an example here due to the complex methylation patterns that
resulted after incubation with PSOMT1 in the presence of AdoMet.
Three products can be identified, resulting from monomethylation at
the isoquinoline moiety, monomethylation at the benzyl moiety and
double methylation. The main product is monomethylated at the free
isoquinoline hydroxyl at C-7.
[0134] FIG. 8. Nucleotide sequence of cDNA encoding
(R,S)-reticuline 7-O-methyltransferase from P. somniferum (PSOMT1;
(SEQ ID NO:1). The cDNA encoding PSOMT1 contains 1437 bp including
a 5' non-coding region of 40 bp, a 3' non-coding region of 329 bp
and a complete open reading frame of 1068 bp encoding 355 amino
acids. Location of the first and last nucleotide of the coding
sequence indicated in bold type.
[0135] FIG. 9: Amino acid sequence of (R,S)-reticuline
7-O-methyltransferase from P. somniferum (PSOMT1; SEQ ID NO:2).
[0136] FIG. 10: Nucleotide sequence of cDNA encoding
(R,S)-norcoclaurine 6-O-methyltransferase from P. somniferum
(PSOMT2; SEQ ID NO:18). The cDNA encoding PSOMT2 contains 1346 bp
including a 5' non-coding region of 59 bp, a 3' non-coding region
of 246 bp and a complete open reading frame of 1041 bp encoding 346
amino acids. Location of the first and last nucleotide of the
coding sequence indicated in bold type, "n" represents any
nucleotide A, C, T or G, preferably T.
[0137] FIG. 11: Nucleotide sequence of cDNA encoding
(R,S)-norcoclaurine 6-O-methyltransferase from P. somniferum
(variant PSOMT2a; SEQ OD MP:20), encompassing the reading frame
only. This sequence was generated by PCR with primers at the start
and stop codons. Nucleotides in bold type and singly underlined are
those that differ from the PSOMT2 sequence as illustrated in FIG.
10, and which give rise to amino acid changes. The nucleotide in
bold type and doubly underlined is the nucleotide which differs
from the PSOMT2 sequence as illustrated in FIG. 10 and which does
not lead to an amino acid change.
[0138] FIG. 12: Nucleotide sequence of cDNA encoding putative
(R,S)-norcoclaurine 6-O-methyltransferase from P. somniferum
(according to Facchini et al., GenBank accession AY217335; SEQ ID
NO:22): the coding sequence is from nucleotides 28 to 1068, as
numbered in FIG. 12.
[0139] FIG. 13: Amino acid sequence of (R,S)-norcoclaurine
6-O-methyltransferase from P. somniferum (variant PSOMT2a; SEQ ID
NO:21). In bold, underlined, are variable amino acids.
[0140] FIG. 14: Amino acid sequence of putative (R,S)-norcoclaurine
6-O-methyltransferase from P. somniferum (according to Facchini et
al., GenBank accession AY217335; SEQ ID NO:23).
[0141] FIG. 15: Alignment of amino acid sequences of variants of
the (R,S)-norcoclaurine 6-O-methyltransferase from P. somniferum:
Abbreviations: "AO" signifies the PSOMT2 sequence of the invention,
as illustrated in FIG. 3 (SEQ ID NO:3); "SH" signifies the PSOMT2a
variant of the invention, as illustrated in FIG. 13 (SEQ ID NO:21),
and "PF" signifies the putative (R,S)-norcoclaurine
6-O-methyltransferase according to Facchini et al (GenBank
AY217335; SEQ ID NO:23).
[0142] FIG. 16: Amino acid sequence of (R,S)-norcoclaurine
6-O-methyltransferase from P. somniferum (SEQ ID NO:25), wherein X
represents positions at which amino acid variation occurs, and may
be any amino acid.
EXAMPLES
[0143] The abbreviations used in the following Examples are:
AdoMet, S-adenosyl-L-methionine; COMT, catechol
O-methyltransferase; RT-PCR, reverse transcriptase-polymerase chain
reaction; HPLC, high performance liquid chromatography; RACE, rapid
amplification of DNA ends; MS, mass spectrometry; bp, base pairs;
OMT, O-methyltransferase; PSOMT, Papaver somniferum
O-methyltransferase
A. Experimental Procedures
[0144] Plant Material--P. somniferum seedlings were routinely grown
aseptically on Gamborg B5 medium (15) containing 0.8% agar in a
growth chamber at 22.degree. C., 60% relative humidity under cycles
of 16 h light/8 h dark with a light intensity of 85 .mu.mol
sec.sup.-1 m.sup.-2 per .mu.A. Differentiated P. somniferum plants
were grown either outdoors in Saxony-Anhalt or in a greenhouse at
24.degree. C., 18 h light and 50% humidity.
[0145] Generation of Partial cDNAs from P. somniferum-Partial cDNAs
encoding O-methyltransferases from P. somniferum were produced by
PCR using cDNA generated by reverse transcription of mRNA isolated
from floral stem. DNA amplification using either Taq or Pfu
polymerase was performed under the following conditions: 3 min at
94.degree. C., 35 cycles of 94.degree. C., 30 s; 50.degree. C., 30
s; 72.degree. C., 1 min. At the end of 35 cycles, the reaction
mixtures were incubated for an additional 7 min at 72.degree. C.
prior to cooling to 4.degree. C. The amplified DNA was resolved by
agarose gel electrophoresis, the bands of approximately correct
size (400 bp) were isolated and subcloned into pGEM-T Easy
(Promega) prior to nucleotide sequence determination. The specific
sequences of the oligodeoxynucleotide primers used are given in the
Results section.
[0146] Generation of Full-Length cDNAs-The sequence information
requisite to the generation of a full-length cDNA was derived from
the nucleotide sequence of the partial cDNA produced as described
in the Results section. The complete nucleotide sequence was
generated in two steps using one O-methyltransferase-specific PCR
primer (PSOMT1: 5'-AGT CAT TTC CAT CTG GTC GCA ACA-3' (SEQ. ID NO:
4) for 5'-RACE and 5'-ATG GAT ACT GCA GAA GAA AGG TTG-3' (SEQ. ID
NO: 5) for 3'-RACE; PSOMT2: 5'-ATA AGG GTA AGC CTC AAT TAC AGA
TTG-3' (SEQ. ID NO: 6) for 5'-RACE and 5'-GCT GCA GTG AAA GCC ATA
ATC T-3' (SEQ. ID NO: 7) for 3'-RACE) and one RACE-specific primer
as specified by the manufacturer. The 5'- and 3'-RACE-PCR
experiments were carried out using a SMART cDNA amplification kit
(Clontech). RACE-PCR was performed using the following PCR cycle: 3
min at 94.degree. C., 25 cycles of 94.degree. C., 30 s; 68.degree.
C., 30 s; 72.degree. C., 3 min. At the end of 25 cycles, the
reaction mixtures were incubated for an additional 7 min at
72.degree. C. prior to cooling to 4.degree. C. The amplified DNA
was resolved by agarose gel electrophoresis, the bands of the
expected size (PSOMT1: 990 bp for 5'-RACE and 1177 bp for 3'-RACE;
PSOMT2: 1124 bp for 5'-RACE and 671 bp for 3'-RACE) were isolated
and subcloned into pGEM-T Easy prior to sequencing.
[0147] The full-length clone was generated in one piece using the
primers PSOMT1: 5'-TAT CGG ATC CAT GGA TAC TGC AGA A-3' (SEQ. ID
NO: 8) and 5'-TTA GGC GGC CGC TTA TTC TGG AAA GGC-3' (SEQ. ID NO:
9) or PSOMT2: 5'-TAT CGG ATC CAT GGA AAC AGT AAG C-3' (SEQ. ID NO:
10) and 5'-TTA GGC GGC CGC TTA ATA AGG GTA AGC-3' (SEQ. ID NO: 11)
for PCR with P. somniferum floral stem cDNA as template. The final
primers used for cDNA amplification contained recognition sites for
the restriction endonucleases BamHI and NotI, appropriate for
subcloning into pFastBac HTa (Life Technologies) for functional
expression. DNA amplification was performed under the following
conditions: 3 min at 94.degree. C., 35 cycles of 94.degree. C., 30
s; 60.degree. C., 30 s; 72.degree. C., 2 min. At the end of 35
cycles, the reaction mixtures were incubated for an additional 7
min at 72.degree. C. prior to cooling to 4.degree. C. The amplified
DNA was resolved by agarose gel electrophoresis, the band of
approximately correct size (PSOMT1: 1068 bp; PSOMT2: 1041 bp) was
isolated and subcloned into pCR4-TOPO (Invitrogen) prior to
nucleotide sequence determination.
[0148] Heterologous Expression and Enzyme Purification-The
full-length cDNA generated by RT-PCR was ligated into pFastBac HTa
that had been digested with restriction endonucleases BamHI and
NotI. The recombinant plasmid was transposed into baculovirus DNA
in the Escherichia coli strain DH10BAC (Life Technologies) and then
transfected into Spodoptera frugiperda Sf9 cells according to the
manufacturer's instructions. The insect cells were propagated and
the recombinant virus was amplified according to (16, 17).
INSECT-XPRESS serum-free medium (Bio Whittaker) was used in the
enzyme expression experiments.
[0149] After infection of 20 ml suspension grown insect cells had
proceded for 3-4 days at 28.degree. C. and 130 rpm, the cells were
removed by centrifugation under sterile conditions at 900.times.g
for 5 min at 4.degree. C. All subsequent steps were performed at
4.degree. C. The pellet was discarded and to the medium was added
0.73 g NaCl, 2.5 ml glycerol and 50 .mu.l
.quadrature.-mercaptoethanol. The pH was adjusted to 7.0 with 1.0 M
NaOH. The His-tagged O-methyltransferase was then purified by
affinity chromatography using a cobalt resin (Talon, Clontech)
according to the manufacturer's instructions.
[0150] Enzyme assay and product identification: The O-methylation
reactions catalysed by the two O-methyltransferases were assayed at
least two times in duplicate according to Ruffer et al. (1983a;
1983b) as follows. Substrate (25 nmol), [methyl-3H]-AdoMet (20,000
dpm, 0.4 fmol), AdoMet (10 nmol) Tris/HC1 buffer pH 8.0 (10
.mu.mol), ascorbate (5 .mu.mol) and 5-10 .mu.g of enzyme were
incubated in a total volume of 150 .mu.l at 35.degree. C. for 5-60
min. The enzymic reaction was terminated by addition of 200 .mu.l
ethylacetate. The organic phase (300 .mu.l) was added to 3 ml high
flash point liquid scintillation cocktail (Packard) and the
radioactivity quantified with a Beckman LS6000TA liquid
scintillation counter. For Km determinations, substrate
concentration was varied from 0 to 400 .mu.m.
[0151] The identity of the enzymic reaction products was
ascertained by HPLC-MS using a Finnigan MAT TSQ 7000 (electrospray
voltage 4.5 kV, capillary temperature 220.degree. C., carrier gas
N.sub.2) coupled to a Micro-tech Ultra-Plus Micro-LC equipped with
an Ultrasep RP18 column; 5 .mu.m; 1.times.10 mm), Solvent system
(A) 99.8% (v/v) H.sub.2O, 0.2% HOAc (B) 99.8% CH.sub.3CN (v/v),
0.2% HOAc; gradient: 0-15 min 10-90% B, 15-25 min 90% B; flow 70
.mu.l min.sup.-1). The collision-induced dissociation (CID;
collision energy, -25 eV; collision gas, argon; collision pressure,
1.8.times.10.sup.-3 Torr) mass spectra for the
tetrahydrobenzylisoquinoline alkaloids were recorded.
[0152] General Methods--Total RNA was isolated and RNA gels were
run and blotted as described previously (20). Genomic DNA was
isolated and DNA gels were run and blotted according to (21). cDNA
clones were labeled by PCR labeling with
[.quadrature.-.sup.32P]dATP. Hybridized RNA on RNA gel blots and
DNA on DNA gel blots were visualized with a STORM phosphor imager
(Molecular Dynamics). The entire nucleotide sequence on both DNA
strands of the full-length clone was determined by dideoxy cycle
sequencing using internal DNA sequences for the design of
deoxyoligonucleotides as sequencing primers. Saturation curves and
double reciprocal plots were constructed with the Fig. P program
Version 2.7 (Biosoft, Cambridge, UK). The influence of pH on enzyme
activity was monitored in sodium citrate (pH 4-6), sodium phosphate
(pH 6-7.0) and Tris-HCl (pH 7.0-9), glycine/NaOH (pH 9-10.5)
buffered solutions.
B. Results
[0153] Amino Acid Sequence Analysis of a Putative
O-Methyltransferase and Isolation of the Corresponding cDNA--Latex
was harvested from field-grown P. somniferum by incising capsules
3-6 days after flower petal fall. The exuded latex was immediately
added to ice-cold potassium phosphate buffer containing 20 mM
sodium ascorbate and 500 mM mannitol, pH 7.2. The latex buffer
ratio was approximately 1:1. Particulates were removed by
centrifugation (22, 23) prior to two-dimensional polyacrylamide gel
electrophoretic resolution of the proteins in the 1000.times.g
supernatant according to (24) (FIG. 2). Internal amino acid
microsequencing of proteins in the size range expected for plant
methyltransferase monomers (approximately 40 kDa) yielded five
peptides from a single protein that was homologous to
O-methyltransferases. The amino acid sequences of these five
peptides are as follows:
TABLE-US-00006 OMT-Pep 1 RTEAE (SEQ. ID NO: 24) OMT-Pep 2
VIIVDCVLRPDGNDL (SEQ. ID NO: 12) OMT-Pep 3 VGGDMFVDIPEADAV (SEQ. ID
NO: 13) OMT-Pep 4 ILLNNAGFPRYNVIRTPAFPcII (SEQ. ID NO: 14) EA
OMT-Pep 5 DGFSGIAGSLVDGG (SEQ. ID NO: 15)
[0154] Degenerated oligodeoxynucleotide primers were derived from
OMT-Pep 1 and OMT-Pep 5 as shown below:
TABLE-US-00007 OMT-Pep 5 sense primer: (SEQ. ID NO: 16) 5'-GCI GGI
A/T C/G I C/T TI GTI GAC/T GTI GGI GG-3' OMT-Pep 1 antisense
primer: (SEQ. ID NO: 17) 5'-C/T TC IGC C/T TC IGT ICG/T C/T TC
CTT-3'
PCR amplification of P. somniferum cDNA prepared from stem poly
(A).sup.+ RNA yielded a DNA band of the expected size
(approximately 400 bp) upon analysis by agarose gel
electrophoresis. Subcloning of the PCR product into pGEM-T Easy
followed by nucleotide sequence determination of randomly chosen
samples identified two independent O-methyltransferase-encoding
partial cDNA clones denoted PSOMT1 and PSOMT2. Each
O-methyltransferase partial sequence was used to design specific
oligodeoxynucleotide primers for RACE-PCR, by which cDNAs
containing the entire open reading frames for both
O-methyltransferases were generated. The details of these
experiments are provided in the Experimental Procedures
section.
[0155] Sequence Analyses of O-Methyltransferases--Translation of
the complete nucleotide sequences of PSOMT1 and PSOMT2 yielded
polypeptides of 356 and 347 amino acids, respectively. Amino acid
sequence alignment carried out using the program from Heidelberg
Unix Sequence Analysis Resources demonstrated 38.9% identity of the
two proteins. Amino acid sequences of O-methyl transfer enzymes
contain consensus sequences putatively involved in catalysis.
Conserved motifs A, B, C, J, K and L proposed by Joshi and Chiang
(2) are shown for PSOMT1 and PSOMT2 as shaded regions in FIG.
3.
[0156] A phylogenetic diagram of forty-four putative and defined
O-methyltransferase amino acid sequences from seventeen plants was
constructed using the Phylogeny Inference Package program (PHYLIP
Version 3.57c) (FIG. 4). Among these forty-four sequences, PSOMT1
showed the closest relationship to a catechol 3-O-methyltransferase
from Pinus taeda (loblolly pine) (32) and to a putative caffeic
acid O-methyltransferase from Monterey pine Pinus radiata. In
contrast, PSOMT2 grouped together with norcoclaurine
6-O-methyltransferase from C. japonica (5). The next most closely
related sequence was 3'-hydroxy-N-methylcoclaurine
4'-O-methyltransferase, also from C. japonica (5). These new P.
somniferum O-methyl transfer enzymes group more closely to
isoquinoline biosynthetic O-methyltransferases from C. japonica
than to those identified from T. tuberosum (4). Table III below
shows results of some of the sequence comparisons, indicating %
amino acid identity. Abbreviations are given in Table II above. The
results of the phylogenetic analysis formed the basis for the
enzymes assays that were later carried out with heterologously
expressed cDNAs as reported below.
TABLE-US-00008 TABLE III Amino Acid Sequence comparisons Ps6OMT
Ps7OMT ttu6OMT1 ttu6OMT2 Cj6OMT Cj4OMT PraCafOMT PtaOMT Ps6OMT --
36 29.1 28 63.4 52.2 Ps7OMT 36 -- 32.3 32 35.7 32.3 44.1 44.4
ttu6OMT1 29.1 32.3 -- 93.6 30.8 30.6 ttu6OMT2 28 32 93.6 -- 30 32
Cj6OMT 63.4 35.7 30.8 30 -- 50.4 Cj 4OMT 52.2 32.3 30.6 32 50.4
--
[0157] Gene Expression Analyses--RNA gel blot analysis suggests
that PSOMT1 is expressed predominantly in bud and stem, and to a
much lesser degree, in leaf of P. somniferum (FIG. 5). In contrast,
PSOMT2 transcript is detectable in bud, stem, leaf and root, and to
a lesser degree in capsule (FIG. 5). The distribution of PSOMT2
transcript parallels the distribution of transcript of several
other genes of tetrahydrobenzylisoquinoline biosynthesis in P.
somniferum. Cyp80b1 that encodes the cytochrome P-450-dependent
monooxygenase (S)--N-methylcoclaurine 3'-hydroxylase (8,9) common
to the biosynthetic pathways of all the P. somniferum alkaloids,
salAT that encodes salutaridinol 7-O-acetyltransferase (13) and
cor1 that encodes codeinone reductase (14), both specific to
morphine biosynthesis, are all expressed in bud, capsule, leaf,
root and stem. This gene transcript distribution of PSOMT2 taken
together with the results of the phylogenetic analysis is congruent
with PSOMT2 encoding norcoclaurine 6-O-methyltransferase of
(S)-reticuline biosynthesis (4,5).
[0158] The comparative transcript distribution and phylogenetic
analysis of PSOMT1 suggests that the gene product may be involved
in tetrahydrobenzylisoquinoline alkaloid formation, but not
directly in either the (S)-reticuline or the morphine biosynthetic
pathways.
[0159] Purification and Functional Characterization of Recombinant
Enzymes--The PSOMT1 and PSOMT2 cDNAs were each constructed to
express the recombinant proteins with six histidine residues
elongating the amino terminus. The proteins were then purified from
S. frugiperda Sf9 cell culture medium in one step by cobalt
affinity chromatography to yield electrophoretically homogeneous
proteins. PSOMT1 and PSOMT2 each have relative molecular masses of
43 kDa as determined by SDS-PAGE. This compares with the calculated
molecular masses of 39,841 and 38,510 based on the translation of
the nucleotide sequences. The native relative molecular masses were
determined by gel filtration on a calibrated Sephacryl 200 column
(Pharmacia). PSOMT1 and PSOMT2 are each homodimers with an Mr of 85
and 80 kDa, respectively. This is consistent with that observed for
norcoclaurine 6-O-methyltransferases of (S)-reticuline biosynthesis
in T. tuberosum (4).
[0160] Radioassay of pure, recombinant O-methyltransferases using
[methyl-.sup.3H]-AdoMet together with each of forty different
substrates demonstrated that PSOMT1 and PSOMT2 are relatively
substrate-specific (Table IV below). PSOMT1 methylates the simple
catechols guaiacol and isovanillic acid as well as the
tetrahydrobenzylisoquinolines (R)-reticuline, (S)-reticuline,
(R,S)-orientaline, (R)-protosinomenine and (R,S)-isoorientaline.
PSOMT2 is more specific, methylating only (R,S)-norcoclaurine,
(R)-norprotosinomenine, (S)-norprotosinomenine and
(R,S)-isoorientaline. The limited quantities of (R,S)-orientaline
prohibited further kinetic characterization of methylation of this
particular substrate.
[0161] PSOMT1 has a pH optimum at 8.0 for guaiacol, (R)-reticuline
and (S)-reticuline. The optimal pH for methylation of
(R)-protosinomenine and isovanillic acid are 9.0 and 7.5,
respectively, whereas the optimal pH for methylation of
(R,S)-isoorientaline ranges from 7.5-9.0. PSOMT2 methylates
(R,S)-norcoclaurine over a wide pH range (6.0-9.0). Methyl transfer
to (R)-norprotosinomenine, (S)-norprotosinomenine and
(R,S)-isoorientaline has an optimum at pH 7.5. The temperature
optima for PSOMT1 with various the substrates are: guaiacol,
(R)-reticuline and (S)-reticuline (37.degree. C.),
(R)-protosinomenine (39.degree. C.), (R,S)-isoorientaline and
isovanillic acid (37-41.degree. C.). PSOMT2 optimally methylated
all substrates at 37-41.degree. C.
[0162] The kinetic parameters determined for methylation of each
substrate of PSOMT1 and PSOMT2 are shown in Table V. As designated
by the ratio k.sub.cat/K.sub.m, PSOMT1 methylates (R)-reticuline
and (S)-reticuline with equal efficiency. Both substrates occur in
P. somniferum, but only (R)-reticuline is specific to morphine
biosynthesis. The high k.sub.cat/K.sub.m, ratio for guaiacol (135%
of those values determined for reticuline) does not correlate with
in vivo significance, since this simple catechol has not been
reported to occur in P. somniferum. Likewise, (R)-protosinomenine,
(R,S)-isoorientaline and isovanillic acid do not occur in this
plant. The highest k.sub.cat/K.sub.m, ratio for PSOMT2 was obtained
with (R,S)-norcoclaurine as substrate. The next best substrates are
(R)- and (S)-norprotosinomenine with values equal to 55% of that
obtained for norcoclaurine. However, norprotosinomenines do not
naturally occur in P. somniferum.
TABLE-US-00009 TABLE IV Substrate specificities of PSOMT1 and
PSOMT2 Substrate PSOMT1 PSOMT2 Phenolics: 1 Catechol 0 0 2
Protocatechuic acid 0 0 3 Dopamine 0 0 4 Caffeic acid 0 0 5
Guaiacol 242 0 6 Isovanillic acid 40 0 7 Vanillic acid 0 0
Isoquinoline alkaloids: 8 (R,S)-Norcoclaurine 0 100 .sup.a 9
(S)-Coclaurine 0 0 10 (R,S)-Isococlaurine 0 0 11 (R,S)-4{acute over
( )}-O-methylcoclaurine 0 0 12 (R,S)-Nororientaline 0 0 13
(R)-Norprotosinomenine 0 26 14 (S)-Norprotosinomenine 0 26 15
(R)-Norreticuline 0 0 16 (S)-Norreticuline 0 0 17
(R)-7-Dehydroxy-norreticuline 0 0 18 (S)-7-Dehydroxy-norreticuline
0 0 19 (R,S)-N-Methylcoclaurine 0 0 20
(R,S)-6-O-Methyllaudanosoline 0 0 21 (S)-4{acute over (
)}-O-Methyllaudanosoline 0 0 22 (R)-Reticuline 100 .sup.a 0 23
(S)-Reticuline 100 .sup.a 0 24 (R,S)-Orientaline 48 0 25
(R)-Protosinomenine 52 0 26 (R,S)-Isoorientaline 46 47 27
(R,S)-Laudanidine 0 0 28 (R,S)-Codamine 0 0 29 (S)-Scoulerine 0 0
30 (S)-Coreximine 0 0 31 Salutaridine 0 0 32 Codeine 0 0 33
Morphine 0 0 Flavonoids: 34 Quercetin 0 0 35
Quercetin-3-methylether 0 0 36 Quercetin-7-methylether 0 0 37
Luteolin 0 0 38 Morin 0 0 39 Cyanidin 0 0 Coumarin: 40 Esculetin 0
0 .sup.a 100% Activity of PSOMT1 and PSOMT2 is 1.5 and 2.0
pmoles/sec/mg total protein, respectively. Assay conditions are
given in the experimental.
TABLE-US-00010 TABLE V Kinetic parameters of PSOMT1 and PSOMT2 for
various substrates and co-substrate (AdoMet) k.sub.cat/K.sub.m
K.sub.m K.sub.m V.sub.max k.sub.cat Substrate AdoMet Substrate
Substrate Substrate (s.sup.-1 Enzyme Substrate (.mu.M) (.mu.M)
(pmol/s) (s.sup.-1) mM.sup.-1) PSOMT1 Guaiacol 310 17 6 0.1 5.9
(S)-Reticuline 360 16 4 0.07 4.5 (R)-Reticuline 310 17 4 0.07 4.2
(R)-Protosinomenine 320 16 2 0.03 1.7 (R,S)-Isoorientaline 260 17 1
0.02 1.4 Isovanillic acid 150 14 1 0.02 1.2 PSOMT2
(R,S)-Norcoclaurine 100 10 5 0.08 7.4 (R)- 200 5 1 0.02 4.1
Norprotosinomenine (S)- 260 5 1 0.02 4.0 Norprotosinomenine
(R,S)-Isoorientaline 280 29 2 0.03 1.0 Assay conditions are given
in the experimental.
[0163] Structure Elucidation of Enzymic Products--Initial enzyme
activity measurements were carried out using a radioassay. Many of
the substrates tested contained more than one site of potential
methylation. Since the radioassay is only a facile measure of
whether methylation had likely occurred, but does not indicate the
position of methyl transfer, each positive assay was repeated with
unlabeled substrate and the enzymic product was subjected to
HPLC-MS analysis. Tetrahydrobenzylisoquinolines readily cleave at
low ionization energies into the corresponding isoquinoline- and
benzyl ions. This enables identification of methylation at either
moiety. The structures of the ten substrates that were methylated
by either PSOMT1 or PSOMT2 are shown in FIG. 6. Each alkaloidal
substrate was monitored for purity by HLPC-MS and the fragmentation
pattern was determined. Enzymic product fragmentation patterns were
then compared to those of substrate. All substrates were methylated
by either PSOMT1 or PSOMT2 on the isoquinoline moiety. For example,
(R)- or (S)-reticuline ([M+H].sup.+ m/z 330) has the major fragment
ions m/z 192 (isoquinoline) and m/z 137 (benzyl). The methylation
of (R)- or (S)-reticuline by PSOMT1 results in a product of
[M+H].sup.+ m/z 344 (methylated (R)- or (S)-reticuline) with
fragment ions at m/z 206 (isoquinoline+CH.sub.2) and m/z 137
(unmodified benzyl). Likewise, (R,S)-norcoclaurine ([M+H].sup.+ m/z
272) has the major fragment ions m/z 161 (isoquinoline) and m/z 107
(benzyl). The methylation of (R,S)-norcoclaurine by PSOMT2 results
in a product of [M+H].sup.+ m/z 286 (methylated
(R,S)-norcoclaurine) with fragment ions at m/z 175
(isoquinoline+CH.sub.2) and m/z 107 (unmodified benzyl).
[0164] Surprising results were obtained when the PSOMT1 methylation
products of (R,S)-orientaline and (R,S)-isoorientaline were
analyzed by HPLC-MS. The fragment ions obtained for the methylation
products of orientaline are shown in FIG. 7. Methylation of the
7-hydroxyl group resulted in the main enzymic product
7-O-methylorientaline. Approximately 1% of the product produced is
the double methylated 7,4'-O-dimethylorientaline (laudanosine) and
the monomethylated 4'-O-methylorientaline.
[0165] The identification of new O-methyltransferases presented
herein follows on from a first attempt to use proteome analysis to
identify proteins in latex of P. somniferum (24, 30). Latex
collected from capsules was resolved into a cytosolic and a
vesicular fraction by centrifugation and the cytosolic proteins
were then resolved by two-dimensional polyacrylamide gel
electrophoresis. From internal amino acid sequence determination of
these proteins, one with homology to plant O-methyltransferases was
identified. Using RT-PCR followed by RACE-PCR, two cDNAs PSOMT1 and
PSOMT2 encoding complete open reading frames were isolated.
[0166] A sequence comparison of the translations of PSOMT1 and
PSOMT2 with those sequences available in the GenBank/EMBL databases
revealed that PSOMT1 grouped with proteins from P. radiata of
unknown function and that PSOMT2 was likely functionally equivalent
to (R,S)-norcoclaurine 6-O-methyltransferase from C. japonica (5).
Using amino acid sequence comparison to predict the in vivo
function of plant O-methyltransferases is not trivial due to the
broad substrate specificities that can be found for closely related
enzymes (4). To overcome the uncertainties associated with
phylogenetic comparison, PSOMT1 and PSOMT2 were each introduced
into a baculovirus expression vector and the corresponding proteins
PSOMT1 and PSOMT2 were produced in S. frugiperda Sf9 cell culture.
Forty compounds were tested as potential substrates for the two
enzymes. Most of these substances were tetrahydrobenzylisoquinoline
alkaloids, but simple catechols and a few common
phenylpropanoid-derived compounds were also included. PSOMT1
O-methylated guaiacol, isovanillic acid, (R)-reticuline,
(S)-reticuline, (R,S)-orientaline, (R)-protosinomenine and
(R,S)-isoorientaline. PSOMT2 O-methylated (R,S)-norcoclaurine,
(R)-norprotosinomenine, (S)-norprotosinomenine and
(R,S)-isoorientaline.
[0167] The broad substrate specificities of plant
O-methyltransferases can make the assignment of an in vivo role to
these enzymes quite challenging. A comparison of the
k.sub.cat/K.sub.m ratio for the various substrates suggested that
the in vivo substrates for PSOMT1 are likely (R)-reticuline and
(S)-reticuline. Guaiacol demonstrated the highest k.sub.cat/K.sub.m
ratio, but this catechol has not been reported to accumulate in P.
somniferum and could simply represent a fortuitous methylation in
vitro. PSOMT2, on the other hand, clearly methylated
(R,S)-norcoclaurine most efficiently. The k.sub.cat/K.sub.m ratios
for (R)-norprotosinomenine and (S)-norprotosinomenine were 55% of
that for (R,S)-norcoclaurine, but norprotosinomenine has been
reported to occur in the legume Erythrina lithosperma, not in P.
somniferum (25). The O-methylation of norprotosinomenine,
therefore, also appears to be a fortuitous in vitro reaction
catalyzed by PSOMT2.
[0168] Elucidation of the structures of the enzymic products was
done by HPLC-MS. Mass spectroscopic analysis of
tetrahydrobenzylisoquinoline alkaloids exploits the ready
fragmentation of these types of molecules into two halves, an
isoquinoline moiety and a benzyl moiety. Methylation of either
portion of the molecule can be readily identified. PSOMT2
O-methylated (R,S)-norcoclaurine, (R)-norprotosinomenine,
(S)-norprotosinomenine and (R,S)-isoorientaline on the isoquinoline
moiety. In the case of (R,S)-norcoclaurine, both C-6 and C-7 are
hydroxylated. (R)-norprotosinomenine, (S)-norprotosinomenine and
(R,S)-isoorientaline all have a free hydroxyl group at C-6, but C-7
is methoxylated. This indicates that the position of O-methylation
of these molecules is at C-6. Based upon the phylogenetic analysis
and the structures of the methylated alkaloidal products, it can be
concluded that PSOMT2 encodes the tetrahydroisoquinoline
biosynthetic enzyme (R,S)-norcoclaurine 6-O-methyltransferase. In
P. somniferum, this enzyme participates in the early steps of
(S)-reticuline biosynthesis, which intermediate leads to numerous
alkaloids of the morphinan, benzo[c]phenanthridine, papaverine and
phthalideisoquinoline types that are accumulated in this plant. The
distribution of PSOMT2 transcript in bud, stem, leaf, root, and
capsule is consistent with this role since these are all major
sites of accumulation of one or the other of these alkaloid classes
(i.e. morphinans in latex and benzo[c]phenanthridines in root).
[0169] The methylating capacity of PSOMT1 was more promiscuous than
that of PSOMT2. PSOMT1 O-methylation of guaiacol, isovanillic acid,
(R)-reticuline, (S)-reticuline, (R,S)-orientaline,
(R)-protosinomenine and (R,S)-isoorientaline resulted in a more
complicated product profile. HPLC-MS analysis indicated that
(R)-reticuline, (S)-reticuline, (R,S)-orientaline, each of which
has a C-6 methoxy group and a C-7 hydroxy moiety, were O-methylated
at C-7. In contrast, (R)-protosinomenine and (R,S)-isoorientaline
each has a free hydroxyl group at C-6 and is methoxylated at C-7.
These molecules were O-methylated by PSOMT1 at C-6. The ratio of
k.sub.cat/K.sub.m for C-7 O-methylation compared to C-6
O-methylation was 3.8:1, suggesting that C-7 O-methylation is
preferred. Multiple products were detected when either
(R,S)-orientaline or (R,S)-isoorientaline were used as substrate.
In addition to methylation of the isoquinoline half of the
tetrahydrobenzylisoquinolines, the benzyl moiety was also
methylated. (R,S)-orientaline and (R,S)-isoorientaline differ from
the other tetrahydrobenzylisoquinoline substrates in that the
benzyl ring is 3'-methoxylated and 4'-hydroxylated. Reticuline and
the protosinomenines are 4'-methoxylated and 3'-hydroxylated. The
free 4'-hydroxy group of (R,S)-orientaline and (R,S)-isoorientaline
is methylated by PSOMT1. 4'-O-methylation appears to occur
independent of both hydroxyl groups of the isoquinoline nucleus
being methylated, since three products can be identified by
HPLC-MS, representing monomethylation at the isoquinoline moiety,
monomethylation at the benzyl moiety and double methylation. A
heterologously expressed O-methyltransferase from Catharanthus
roseus cell suspension cultures that methylates the flavonol
myricetin at both the 3'- and 5'-hydroxyl groups has recently been
reported (26). Given free rotation around the bond between the B
and C rings, these two hydroxyl moieties can be seen as chemically
equivalent, whereas the two hydroxyl groups methylated by PSOMT1
can be viewed as chemically unique.
[0170] The main enzymic reaction product formed by PSOMT1
(approximately 99%) results from monomethylation of the
isoquinoline group. Based upon these combined kinetic and mass
spectroscopic results, it is concluded that PSOMT1 encodes
(R,S)-reticuline 7-O-methyltransferase, a new enzyme of
tetrahydrobenzylisoquinoline alkaloid biosynthesis in P.
somniferum. The product of this reaction,
7-O-methylreticuline(laudanine) is a natural product that has been
reported to occur in opium (27) and this occurrence has been
confirmed for the variety of P. somniferum used herein (A. J. Fist,
personal communication). The distribution of PSOMT1 transcript
predominantly in bud and stem correlates with latex as the site of
laudanine accumulation.
[0171] Enzymic O-methylation of tetrahydrobenzylisoquinolines has
been reported to be catalyzed by catechol O-methyltrasferase (COMT)
isolated from rat liver as part of a program investigating the
nature and biosynthetic origin of mammalian alkaloids (28). In that
particular report, COMT O-methylated norcoclaurine at the
6-hydroxy- and 7-hydroxy positions in a ratio of 8:2. This low
specificity compares to that of norcoclaurine 6-O-methyltransferase
characterized from T. tuberosum, which methylated
tetrahydrobenzylisoquinolines that contained a catechol- and, to a
lesser degree, a guaiacol moiety (4). The P. somniferum 7-O- and
6-O-methyltransferases characterized herein appear to methylate
with higher regiospecificity.
REFERENCES
[0172] 1. Ibrahim, R. K., Bruneau, A., and Bantignies, B. (1998)
Plant Mol. Biol. 36, 1-10 [0173] 2. Joshi, C. P., and Chiang, V. L.
(1998) Plant Mol. Biol. 37, 663-674 [0174] 3. Schroder, G.,
Wehinger, E., and Schroder, J. (2002) Phytochemistry 59, 1-8 [0175]
4. Frick, S., and Kutchan, T. M. (1999) Plant J. 17, 329-339 [0176]
5. Morishige, T., Tsujita, T., Yamada, Y., and Sato, F. (2000) J.
Biol. Chem. 275, 23398-23405 [0177] 6. Maury, S., Geoffroy, P., and
Legrand, M. (1999) Plant Physiol. 121, 215-223 [0178] 7. Kutchan,
T. M. (1998) in The Alkaloids Vol. 50, (ed. G. Cordell) Academic
Press, San Diego, 257-316 [0179] 8. Pauli, H. H., and Kutchan, T.
M. (1998) Plant J. 13, 793-801 [0180] 9. Huang, F.-C., and Kutchan,
T. M. (2000) Phytochemistry 53, 555-564 [0181] 10. Rosco, A.,
Pauli, H. H., Priesner, W., and Kutchan, T. M. (1997) Arch.
Biochem. Biophys. 348, 369-377 [0182] 11. Dittrich, H. and Kutchan,
T. M. (1991) Proc. Natl. Acad. Sci. USA 88, 9969-9973 [0183] 12.
Facchini, P. J., Penzes, C., Johnson A. G., and Bull, D. (1996)
Plant Physiol. 112, 1669-1677 [0184] 13. Grothe, T., Lenz, R., and
Kutchan, T. M. (2001) J. Biol. Chem. 276, 30717-30723 [0185] 14.
Unterlinner, B., Lenz, R., and Kutchan, T. M. (1999) Plant J. 18,
465-475 [0186] 15. Gamborg, O. L., Miller, R. A., and Ojina, K.
(1968) Exp. Cell. Res. 50, 151-158 [0187] 16. Kutchan, T. M., Bock,
A., and Dittrich, H. (1994) Phytochemistry 35, 353-360 [0188] 17.
Pauli, H., and Kutchan, T. M. (1998) Plant J. 13, 793-801 [0189]
18. Ruffer, M., Nagakura, N., and Zenk, M. H. (1983) Planta Med.
49, 131-137 [0190] 19. Ruffer, M., Nagakura, N., and Zenk, M. H.
(1983) Planta Med. 49, 196-198 [0191] 20. Pauli, H., and Kutchan,
T. M. (1998) Plant J. 13, 793-801 [0192] 21. Bracher, D., and
Kutchan, T. M. (1992) Arch. Biochem. Biophys. 294, 717-723 [0193]
22. Roberts, M. F., McCarthy, D., Kutchan, T. M., and Coscia, C. J.
(1983) Arch. Biochem. Biophys. 222, 599-609 [0194] 23. Antoun, M.
D., and Roberts, M. F. (1975) Phytochemistry 14, 909-914 [0195] 24.
Decker, G., Wanner, G., Zenk, M. H., and Lottspeich, F. (2000)
Electrophoresis 21, 3500-3516 [0196] 25. Ghosal, S., Majumdar, S.
K., and Chakraborti, A. (1971) Austral. J. Chem. 24, 2733-2735
[0197] 26. Cacace, S., Schroder, G., Wehinger, E., Strack, D.,
Schmidt, J., and Schroder, J. (2003) Phytochemistry 62, 127-137
[0198] 27. Small, L. F., and Lutz, R. E. (1932) Chemistry of the
Opium Alkaloids, Supplement No. 103, Public Health Reports,
Washington, p. 34 [0199] 28. Sekine, Y., Creveling, C., Bell, M.,
and Brossi, A. (1990) Helv. Chim. Acta 73, 426-432 [0200] 29.
Fisinger, U. Dissertation zur Erlangung des Doktorgrades der
Fakultat fur Chemie und Pharmazie der
Ludwig-Maximiliens-Universitat zu Munchen: "Untersuchungen zur
Morphinbiosynthese in der Ratte Rattus rattus L. und im Schlafmohn
Papaver somniferum L." 1998. [0201] 30. Decker, G. T. Dissertation
zur Erlangung des Doktorgrades der Fakultat fur Chemie und
Pharmazie der Ludwig-Maximiliens-Universitat zu Munchen: "Der
Milchsaft von Papaver somniferum. Die Proteinanalyse als Ansatz zur
Funktionsanalyse" 2001. [0202] 31. Facchini, P. J. and Park, S. U.
GenBank accession n.sup.o AY217335. [0203] 32. Li et al., (1997),
PNAS 94, 5461-5466.
Sequence CWU 1
1
3311437DNAPapaver somniferumCDS(41)..(1108) 1gaaaacaaaa cataaacaca
atttattcag agatatctgg atg gat act gca gaa 55Met Asp Thr Ala Glu1
5gaa agg ttg aaa ggg caa gct gaa ata tgg gag cat atg ttc gca ttc
103Glu Arg Leu Lys Gly Gln Ala Glu Ile Trp Glu His Met Phe Ala Phe
10 15 20gtg gat tca atg gca ttg aaa tgt gca gtt gag ctt ggc ata cca
gac 151Val Asp Ser Met Ala Leu Lys Cys Ala Val Glu Leu Gly Ile Pro
Asp 25 30 35ata ata aac tct cat ggt cgt ccg gtc aca ata tct gag atc
gtc gac 199Ile Ile Asn Ser His Gly Arg Pro Val Thr Ile Ser Glu Ile
Val Asp 40 45 50agt ttg aaa aca aac aca cca tca tca tct ccc aac atc
gat tat ctt 247Ser Leu Lys Thr Asn Thr Pro Ser Ser Ser Pro Asn Ile
Asp Tyr Leu 55 60 65aca cgt ata atg aga cta ctg gtt cac aag agg cta
ttt act tct gaa 295Thr Arg Ile Met Arg Leu Leu Val His Lys Arg Leu
Phe Thr Ser Glu70 75 80 85ctt cat caa gaa agt aac caa ctt ctc tat
aat tta act cga tca tca 343Leu His Gln Glu Ser Asn Gln Leu Leu Tyr
Asn Leu Thr Arg Ser Ser 90 95 100aaa tgg cta cta aaa gat tcc aag
ttt aat ctg tca cca ctg gtt tta 391Lys Trp Leu Leu Lys Asp Ser Lys
Phe Asn Leu Ser Pro Leu Val Leu 105 110 115tgg gaa act aat ccg ata
tta cta aaa cca tgg caa tat ttg ggc aag 439Trp Glu Thr Asn Pro Ile
Leu Leu Lys Pro Trp Gln Tyr Leu Gly Lys 120 125 130tgt gct caa gaa
aaa agt tct cca ttt gag aga gct cat gga tgt gag 487Cys Ala Gln Glu
Lys Ser Ser Pro Phe Glu Arg Ala His Gly Cys Glu 135 140 145att tgg
gat ctt gct tta gct gat cct aag ttt aat aat ttc ctt aac 535Ile Trp
Asp Leu Ala Leu Ala Asp Pro Lys Phe Asn Asn Phe Leu Asn150 155 160
165ggt gca atg caa tgt tcg act aca aca ata atc aac gag atg ctg ctt
583Gly Ala Met Gln Cys Ser Thr Thr Thr Ile Ile Asn Glu Met Leu Leu
170 175 180gaa tat aaa gat gga ttt agt ggt ata gca gga tcg ctt gtt
gat gtc 631Glu Tyr Lys Asp Gly Phe Ser Gly Ile Ala Gly Ser Leu Val
Asp Val 185 190 195ggg ggt ggg acc ggg tcg ata atc gct gaa ata gtt
aag gct cat cca 679Gly Gly Gly Thr Gly Ser Ile Ile Ala Glu Ile Val
Lys Ala His Pro 200 205 210cac ata caa ggc atc aat ttt gat cta cca
cat gta gtg gct aca gcg 727His Ile Gln Gly Ile Asn Phe Asp Leu Pro
His Val Val Ala Thr Ala 215 220 225gct gaa ttt cca ggg gtg aag cat
gtc ggt ggt gat atg ttt gtc gat 775Ala Glu Phe Pro Gly Val Lys His
Val Gly Gly Asp Met Phe Val Asp230 235 240 245att ccg gaa gct gat
gct gtc atc atg aag tgg ata ttg cac gac tgg 823Ile Pro Glu Ala Asp
Ala Val Ile Met Lys Trp Ile Leu His Asp Trp 250 255 260agt gac gaa
gac tgt aca att ata ctg aag aat tgt tac cga gca ata 871Ser Asp Glu
Asp Cys Thr Ile Ile Leu Lys Asn Cys Tyr Arg Ala Ile 265 270 275aga
aag aag aaa aac gga aaa gtc ata att gtt gat tgt gtg ttg cga 919Arg
Lys Lys Lys Asn Gly Lys Val Ile Ile Val Asp Cys Val Leu Arg 280 285
290cca gat gga aat gac tta ttc gat aaa atg gga ttg ata ttt gat gtg
967Pro Asp Gly Asn Asp Leu Phe Asp Lys Met Gly Leu Ile Phe Asp Val
295 300 305ctg atg atg gca cat act aca gct gga aaa gaa aga aca gaa
gcg gaa 1015Leu Met Met Ala His Thr Thr Ala Gly Lys Glu Arg Thr Glu
Ala Glu310 315 320 325tgg aag atc tta tta aat aat gca ggt ttt cct
cgt tac aat gtc att 1063Trp Lys Ile Leu Leu Asn Asn Ala Gly Phe Pro
Arg Tyr Asn Val Ile 330 335 340cga act ccg gca ttt cct tgc atc atc
gag gcc ttt cca gaa taa 1108Arg Thr Pro Ala Phe Pro Cys Ile Ile Glu
Ala Phe Pro Glu 345 350 355tgatcaaggt gcagctatgg tagcccaacg
atactctcaa gctatatata tgatatttcc 1168aaaagaatgt gttctctttg
ttgtgcatgt tttgtagagt gtggtaactt tggaaagacc 1228atttacaaat
agctatgcta tttgttggct agctaagggt caggttccta caaaataatt
1288cagaacttta tgtttttgag tggtaataaa acaattctcc tgtgagagag
ctgttacctt 1348gtctgttatc tgtattgcta tccttagaca tctggggggg
tggaatgtat tctgattttg 1408cgtttttacg taaaaaaaaa aaaaaaaaa
14372355PRTPapaver somniferum 2Met Asp Thr Ala Glu Glu Arg Leu Lys
Gly Gln Ala Glu Ile Trp Glu1 5 10 15His Met Phe Ala Phe Val Asp Ser
Met Ala Leu Lys Cys Ala Val Glu 20 25 30Leu Gly Ile Pro Asp Ile Ile
Asn Ser His Gly Arg Pro Val Thr Ile 35 40 45Ser Glu Ile Val Asp Ser
Leu Lys Thr Asn Thr Pro Ser Ser Ser Pro 50 55 60Asn Ile Asp Tyr Leu
Thr Arg Ile Met Arg Leu Leu Val His Lys Arg65 70 75 80Leu Phe Thr
Ser Glu Leu His Gln Glu Ser Asn Gln Leu Leu Tyr Asn 85 90 95 Leu
Thr Arg Ser Ser Lys Trp Leu Leu Lys Asp Ser Lys Phe Asn Leu 100 105
110Ser Pro Leu Val Leu Trp Glu Thr Asn Pro Ile Leu Leu Lys Pro Trp
115 120 125Gln Tyr Leu Gly Lys Cys Ala Gln Glu Lys Ser Ser Pro Phe
Glu Arg 130 135 140Ala His Gly Cys Glu Ile Trp Asp Leu Ala Leu Ala
Asp Pro Lys Phe145 150 155 160 Asn Asn Phe Leu Asn Gly Ala Met Gln
Cys Ser Thr Thr Thr Ile Ile 165 170 175Asn Glu Met Leu Leu Glu Tyr
Lys Asp Gly Phe Ser Gly Ile Ala Gly 180 185 190Ser Leu Val Asp Val
Gly Gly Gly Thr Gly Ser Ile Ile Ala Glu Ile 195 200 205Val Lys Ala
His Pro His Ile Gln Gly Ile Asn Phe Asp Leu Pro His 210 215 220 Val
Val Ala Thr Ala Ala Glu Phe Pro Gly Val Lys His Val Gly Gly225 230
235 240Asp Met Phe Val Asp Ile Pro Glu Ala Asp Ala Val Ile Met Lys
Trp 245 250 255Ile Leu His Asp Trp Ser Asp Glu Asp Cys Thr Ile Ile
Leu Lys Asn 260 265 270Cys Tyr Arg Ala Ile Arg Lys Lys Lys Asn Gly
Lys Val Ile Ile Val 275 280 285Asp Cys Val Leu Arg Pro Asp Gly Asn
Asp Leu Phe Asp Lys Met Gly 290 295 300Leu Ile Phe Asp Val Leu Met
Met Ala His Thr Thr Ala Gly Lys Glu305 310 315 320Arg Thr Glu Ala
Glu Trp Lys Ile Leu Leu Asn Asn Ala Gly Phe Pro 325 330 335Arg Tyr
Asn Val Ile Arg Thr Pro Ala Phe Pro Cys Ile Ile Glu Ala 340 345
350Phe Pro Glu 3553346PRTPapaver somniferum 3Met Glu Thr Val Ser
Lys Ile Asp Gln Gln Asn Gln Ala Lys Ile Trp1 5 10 15Lys Gln Ile Tyr
Gly Phe Ala Glu Ser Leu Val Leu Lys Cys Ala Val 20 25 30Gln Leu Glu
Ile Ala Glu Thr Leu His Asn Asn Val Lys Pro Met Ser 35 40 45Leu Ser
Glu Leu Ala Ser Lys Leu Pro Val Ala Gln Pro Val Asn Glu 50 55 60Asp
Arg Leu Phe Arg Ile Met Arg Tyr Leu Val His Met Glu Leu Phe65 70 75
80Lys Ile Asp Ala Thr Thr Gln Lys Tyr Ser Leu Ala Pro Pro Ala Lys
85 90 95Tyr Leu Leu Arg Gly Trp Glu Lys Ser Met Val Asp Ser Ile Leu
Cys 100 105 110Ile Asn Asp Lys Asp Phe Leu Ala Pro Trp His His Leu
Gly Asp Gly 115 120 125Leu Thr Gly Asn Cys Asp Ala Phe Glu Lys Ala
Leu Gly Lys Ser Ile 130 135 140Trp Val Tyr Met Ser Val Asn Pro Glu
Lys Asn Gln Leu Phe Asn Ala145 150 155 160Ala Met Ala Cys Asp Thr
Arg Leu Val Thr Ser Ala Leu Ala Asn Glu 165 170 175Cys Lys Ser Ile
Phe Ser Asp Gly Ile Ser Thr Leu Val Asp Val Gly 180 185 190Gly Gly
Thr Gly Thr Ala Val Lys Ala Ile Ser Lys Ala Phe Pro Asp 195 200
205Ile Lys Cys Thr Ile Tyr Asp Leu Pro His Val Ile Ala Asp Ser Pro
210 215 220Glu Ile Pro Asn Ile Thr Lys Ile Ser Gly Asp Met Phe Lys
Ser Ile225 230 235 240Pro Ser Ala Asp Ala Ile Phe Met Lys Cys Ile
Leu His Asp Trp Asn 245 250 255Asp Asp Glu Cys Ile Gln Ile Leu Lys
Arg Cys Lys Glu Ala Leu Pro 260 265 270Lys Gly Gly Lys Val Ile Ile
Val Asp Val Val Ile Asp Met Asp Ser 275 280 285Thr His Pro Tyr Ala
Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met 290 295 300Leu Asn Thr
Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys Thr Leu305 310 315
320Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val Thr Gln Ile Ser Ala
325 330 335Val Gln Ser Val Ile Glu Ala Tyr Pro Tyr 340
345424DNAArtificialprimer 4agtcatttcc atctggtcgc aaca
24524DNAArtificialprimer 5atggatactg cagaagaaag gttg
24627DNAArtificialprimer 6ataagggtaa gcctcaatta cagattg
27722DNAArtificialprimer 7gctgcagtga aagccataat ct
22825DNAArtificialprimer 8tatcggatcc atggatactg cagaa
25927DNAArtificialprimer 9ttaggcggcc gcttattctg gaaaggc
271025DNAArtificialprimer 10tatcggatcc atggaaacag taagc
251127DNAArtificialprimer 11ttaggcggcc gcttaataag ggtaagc
271215PRTPapaver somniferum 12Val Ile Ile Val Asp Cys Val Leu Arg
Pro Asp Gly Asn Asp Leu1 5 10 151315PRTPapaver somniferum 13Val Gly
Gly Asp Met Phe Val Asp Ile Pro Glu Ala Asp Ala Val1 5 10
151425PRTPapaver somniferum 14Ile Leu Leu Asn Asn Ala Gly Phe Pro
Arg Tyr Asn Val Ile Arg Thr1 5 10 15Pro Ala Phe Pro Cys Ile Ile Glu
Ala 20 251514PRTPapaver somniferum 15Asp Gly Phe Ser Gly Ile Ala
Gly Ser Leu Val Asp Gly Gly1 5 101626DNAArtificialprimer
16gcnggnwsny tngtngaygt nggngg 261721DNAArtificialprimer
17ytcngcytcn gtnckytcct t 21181346DNAPapaver
somniferumCDS(60)..(1100)misc_feature(731)..(731)a, g, c ou t
18gagctcaaat cattcaatca ttcttctcat caacagctaa agtgtctaaa cagagagaa
59atg gaa aca gta agc aag att gat caa caa aac caa gca aaa atc tgg
107Met Glu Thr Val Ser Lys Ile Asp Gln Gln Asn Gln Ala Lys Ile Trp1
5 10 15aaa caa att tac ggt ttc gca gaa tca cta gtt ctg aaa tgt gca
gtc 155Lys Gln Ile Tyr Gly Phe Ala Glu Ser Leu Val Leu Lys Cys Ala
Val 20 25 30caa cta gag att gct gaa aca ctt cac aac aat gtc aaa ccc
atg tct 203Gln Leu Glu Ile Ala Glu Thr Leu His Asn Asn Val Lys Pro
Met Ser 35 40 45tta tcc gaa ttg gca tcg aaa ctt ccc gtt gct caa ccc
gtt aac gaa 251Leu Ser Glu Leu Ala Ser Lys Leu Pro Val Ala Gln Pro
Val Asn Glu 50 55 60gac cgt ctg ttc cga att atg cgt tac ttg gtt cac
atg gag ctc ttc 299Asp Arg Leu Phe Arg Ile Met Arg Tyr Leu Val His
Met Glu Leu Phe65 70 75 80aaa ata gat gct acc acg cag aaa tac tca
tta gct cca cca gct aag 347Lys Ile Asp Ala Thr Thr Gln Lys Tyr Ser
Leu Ala Pro Pro Ala Lys 85 90 95tat ttg ttg aga ggc tgg gag aaa tca
atg gtt gat tca att tta tgc 395Tyr Leu Leu Arg Gly Trp Glu Lys Ser
Met Val Asp Ser Ile Leu Cys 100 105 110ata aat gat aag gat ttc tta
gct cca tgg cac cat tta ggc gac ggt 443Ile Asn Asp Lys Asp Phe Leu
Ala Pro Trp His His Leu Gly Asp Gly 115 120 125ttg acc ggt aac tgt
gac gct ttt gag aaa gcg ttg ggg aag agt att 491Leu Thr Gly Asn Cys
Asp Ala Phe Glu Lys Ala Leu Gly Lys Ser Ile 130 135 140tgg gtg tat
atg agt gta aat cct gaa aag aat caa ttg ttt aat gca 539Trp Val Tyr
Met Ser Val Asn Pro Glu Lys Asn Gln Leu Phe Asn Ala145 150 155
160gca atg gct tgt gat act aga ttg gtt act tct gca ttg gct aat gag
587Ala Met Ala Cys Asp Thr Arg Leu Val Thr Ser Ala Leu Ala Asn Glu
165 170 175tgc aaa agt att ttc agt gat gga atc agt aca ctg gtt gat
gtc ggc 635Cys Lys Ser Ile Phe Ser Asp Gly Ile Ser Thr Leu Val Asp
Val Gly 180 185 190ggt ggt acg ggt act gca gtg aaa gcc ata tct aaa
gct ttt ccg gat 683Gly Gly Thr Gly Thr Ala Val Lys Ala Ile Ser Lys
Ala Phe Pro Asp 195 200 205att aag tgc act atc tat gat ctt cct cat
gtc ata gct gat tct ccn 731Ile Lys Cys Thr Ile Tyr Asp Leu Pro His
Val Ile Ala Asp Ser Pro 210 215 220gaa atc ccc aat atc act aaa att
tct gga gat atg ttc aag tct att 779Glu Ile Pro Asn Ile Thr Lys Ile
Ser Gly Asp Met Phe Lys Ser Ile225 230 235 240cct agt gct gat gcc
atc ttc atg aag tgc ata ctt cac gac tgg aac 827Pro Ser Ala Asp Ala
Ile Phe Met Lys Cys Ile Leu His Asp Trp Asn 245 250 255gat gac gaa
tgc att caa atc ttg aag aga tgc aaa gaa gca tta cca 875Asp Asp Glu
Cys Ile Gln Ile Leu Lys Arg Cys Lys Glu Ala Leu Pro 260 265 270aaa
ggt ggc aaa gtt att atc gtg gat gtc gtg ata gac atg gat tcg 923Lys
Gly Gly Lys Val Ile Ile Val Asp Val Val Ile Asp Met Asp Ser 275 280
285act cat cca tat gca aaa att aga ctc aca ctg gat ttg gat atg atg
971Thr His Pro Tyr Ala Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met
290 295 300ctt aac act ggt gga aaa gag aga acc aaa gaa gaa tgg aag
aca ctt 1019Leu Asn Thr Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys
Thr Leu305 310 315 320ttt gat gcc gct ggt ttt gct agc cac aaa gtc
act cag ata tct gct 1067Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val
Thr Gln Ile Ser Ala 325 330 335gtc caa tct gta att gag gct tac cct
tat taa ggaacatttt aaccggtttt 1120Val Gln Ser Val Ile Glu Ala Tyr
Pro Tyr 340 345ccctttgatt aattgttgct ttctctttgg attatgttta
tgtttataac aattgcaaga 1180tgaatgactt ccaactcgca ttggattaat
gtttttcgtt tactttactt tctagatatt 1240ttgaggggct ttgtttaaat
ttgatatccc acgtttgtaa ctgtaaagag tagagtggat 1300gaatgatact
ccctccgttt ccaaaaaaaa aaaaaaaaaa aaaaaa 134619346PRTPapaver
somniferum 19Met Glu Thr Val Ser Lys Ile Asp Gln Gln Asn Gln Ala
Lys Ile Trp1 5 10 15Lys Gln Ile Tyr Gly Phe Ala Glu Ser Leu Val Leu
Lys Cys Ala Val 20 25 30Gln Leu Glu Ile Ala Glu Thr Leu His Asn Asn
Val Lys Pro Met Ser 35 40 45Leu Ser Glu Leu Ala Ser Lys Leu Pro Val
Ala Gln Pro Val Asn Glu 50 55 60Asp Arg Leu Phe Arg Ile Met Arg Tyr
Leu Val His Met Glu Leu Phe65 70 75 80Lys Ile Asp Ala Thr Thr Gln
Lys Tyr Ser Leu Ala Pro Pro Ala Lys 85 90 95Tyr Leu Leu Arg Gly Trp
Glu Lys Ser Met Val Asp Ser Ile Leu Cys 100 105 110 Ile Asn Asp Lys
Asp Phe Leu Ala Pro Trp His His Leu Gly Asp Gly 115 120 125Leu Thr
Gly Asn Cys Asp Ala Phe Glu Lys Ala Leu Gly Lys Ser Ile 130 135
140Trp Val Tyr Met Ser Val Asn Pro Glu Lys Asn Gln Leu Phe Asn
Ala145 150 155 160Ala Met Ala Cys Asp Thr Arg Leu Val Thr Ser Ala
Leu Ala Asn Glu 165 170 175Cys Lys Ser Ile Phe Ser Asp Gly Ile Ser
Thr Leu Val Asp Val Gly 180 185 190 Gly Gly Thr Gly Thr Ala Val Lys
Ala Ile Ser Lys Ala Phe Pro Asp 195 200 205Ile Lys Cys Thr Ile Tyr
Asp Leu Pro His Val Ile Ala Asp Ser Pro 210 215 220Glu Ile Pro Asn
Ile Thr Lys Ile Ser Gly Asp Met Phe Lys Ser Ile225 230 235 240Pro
Ser Ala Asp Ala Ile Phe Met Lys Cys Ile Leu His Asp Trp Asn 245 250
255Asp Asp Glu Cys Ile Gln Ile
Leu Lys Arg Cys Lys Glu Ala Leu Pro 260 265 270 Lys Gly Gly Lys Val
Ile Ile Val Asp Val Val Ile Asp Met Asp Ser 275 280 285Thr His Pro
Tyr Ala Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met 290 295 300Leu
Asn Thr Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys Thr Leu305 310
315 320Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val Thr Gln Ile Ser
Ala 325 330 335Val Gln Ser Val Ile Glu Ala Tyr Pro Tyr 340 345
201041DNAPapaver somniferumCDS(1)..(1041) 20atg gaa aca gta agc aag
att gat caa caa aac caa gca aaa atc tgg 48Met Glu Thr Val Ser Lys
Ile Asp Gln Gln Asn Gln Ala Lys Ile Trp1 5 10 15aaa caa att tac ggt
ttc gca gaa tca cta gtt ctg aaa tgt gca gtc 96Lys Gln Ile Tyr Gly
Phe Ala Glu Ser Leu Val Leu Lys Cys Ala Val 20 25 30caa cta gag att
gct gaa aca ctt cac aac aat gtc aaa ccc atg tct 144Gln Leu Glu Ile
Ala Glu Thr Leu His Asn Asn Val Lys Pro Met Ser 35 40 45tta tcc gaa
ttg gca tcg aaa ctt ccc gtt gct caa ccc gtt aac gaa 192Leu Ser Glu
Leu Ala Ser Lys Leu Pro Val Ala Gln Pro Val Asn Glu 50 55 60gac cgt
ctg ttc cga att atg cgt tac ttg gtt cac atg gag ctc ttc 240Asp Arg
Leu Phe Arg Ile Met Arg Tyr Leu Val His Met Glu Leu Phe65 70 75
80aaa ata gat gct acc acg cag aaa tac tca tta gtt cca cca gct aag
288Lys Ile Asp Ala Thr Thr Gln Lys Tyr Ser Leu Val Pro Pro Ala Lys
85 90 95tat ttg ttg aga ggc tgg gag aaa tca atg gtt gat tca att tta
tgc 336Tyr Leu Leu Arg Gly Trp Glu Lys Ser Met Val Asp Ser Ile Leu
Cys 100 105 110ata aat gat aag gat ttc tta gct cca tgg cac cat tta
ggc gac ggt 384Ile Asn Asp Lys Asp Phe Leu Ala Pro Trp His His Leu
Gly Asp Gly 115 120 125ttg acc ggt aac tgt gac gct ttt gag aaa gcg
ttg ggg aag agt att 432Leu Thr Gly Asn Cys Asp Ala Phe Glu Lys Ala
Leu Gly Lys Ser Ile 130 135 140tgg gtg tat atg agt gaa aat cct gaa
aag aat caa ttg ttt aat gca 480Trp Val Tyr Met Ser Glu Asn Pro Glu
Lys Asn Gln Leu Phe Asn Ala145 150 155 160gca atg gct tgt gat act
aga ttg gtt act tct gca ttg gct aat gag 528Ala Met Ala Cys Asp Thr
Arg Leu Val Thr Ser Ala Leu Ala Asn Glu 165 170 175tgc aaa agt att
ttc agt gat gga atc agt aca ctg gtt gat gtc ggc 576Cys Lys Ser Ile
Phe Ser Asp Gly Ile Ser Thr Leu Val Asp Val Gly 180 185 190ggt ggt
acg ggt act gca gtg aaa gcc ata tct aaa gct ttt ccg gat 624Gly Gly
Thr Gly Thr Ala Val Lys Ala Ile Ser Lys Ala Phe Pro Asp 195 200
205att aag tgc act atc tat gat ctt cct cat gtc ata gct gat tct cct
672Ile Lys Cys Thr Ile Tyr Asp Leu Pro His Val Ile Ala Asp Ser Pro
210 215 220gaa atc ccc aat atc act aaa att cct gga gat atg ttc aag
tct att 720Glu Ile Pro Asn Ile Thr Lys Ile Pro Gly Asp Met Phe Lys
Ser Ile225 230 235 240cct agt gct gat ggc atc ttc atg aag tgc ata
ctt cac gac tgg aac 768Pro Ser Ala Asp Gly Ile Phe Met Lys Cys Ile
Leu His Asp Trp Asn 245 250 255gat gac gaa tgc att caa atc ttg aag
aga tgc aaa gaa gca tta cca 816Asp Asp Glu Cys Ile Gln Ile Leu Lys
Arg Cys Lys Glu Ala Leu Pro 260 265 270aaa gtt ggc aaa gtt att atc
gtg gat gtc gtg ata gac atg gat tcg 864Lys Val Gly Lys Val Ile Ile
Val Asp Val Val Ile Asp Met Asp Ser 275 280 285act cat cca tat gca
aaa att aga ctc aca ctg gat ttg gat atg atg 912Thr His Pro Tyr Ala
Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met 290 295 300ctt aac act
ggt gga aaa gag aga acc aaa gaa gaa tgg aag aca ctt 960Leu Asn Thr
Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys Thr Leu305 310 315
320ttt gat gcc gct ggt ttt gct agc cac aaa gtc act cag ata tct gct
1008Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val Thr Gln Ile Ser Ala
325 330 335gtc caa tct gta att gag gct tac cct tat taa 1041Val Gln
Ser Val Ile Glu Ala Tyr Pro Tyr 340 34521346PRTPapaver somniferum
21Met Glu Thr Val Ser Lys Ile Asp Gln Gln Asn Gln Ala Lys Ile Trp1
5 10 15Lys Gln Ile Tyr Gly Phe Ala Glu Ser Leu Val Leu Lys Cys Ala
Val 20 25 30Gln Leu Glu Ile Ala Glu Thr Leu His Asn Asn Val Lys Pro
Met Ser 35 40 45Leu Ser Glu Leu Ala Ser Lys Leu Pro Val Ala Gln Pro
Val Asn Glu 50 55 60Asp Arg Leu Phe Arg Ile Met Arg Tyr Leu Val His
Met Glu Leu Phe65 70 75 80Lys Ile Asp Ala Thr Thr Gln Lys Tyr Ser
Leu Val Pro Pro Ala Lys 85 90 95Tyr Leu Leu Arg Gly Trp Glu Lys Ser
Met Val Asp Ser Ile Leu Cys 100 105 110Ile Asn Asp Lys Asp Phe Leu
Ala Pro Trp His His Leu Gly Asp Gly 115 120 125Leu Thr Gly Asn Cys
Asp Ala Phe Glu Lys Ala Leu Gly Lys Ser Ile 130 135 140Trp Val Tyr
Met Ser Glu Asn Pro Glu Lys Asn Gln Leu Phe Asn Ala145 150 155
160Ala Met Ala Cys Asp Thr Arg Leu Val Thr Ser Ala Leu Ala Asn Glu
165 170 175Cys Lys Ser Ile Phe Ser Asp Gly Ile Ser Thr Leu Val Asp
Val Gly 180 185 190Gly Gly Thr Gly Thr Ala Val Lys Ala Ile Ser Lys
Ala Phe Pro Asp 195 200 205Ile Lys Cys Thr Ile Tyr Asp Leu Pro His
Val Ile Ala Asp Ser Pro 210 215 220Glu Ile Pro Asn Ile Thr Lys Ile
Pro Gly Asp Met Phe Lys Ser Ile225 230 235 240Pro Ser Ala Asp Gly
Ile Phe Met Lys Cys Ile Leu His Asp Trp Asn 245 250 255Asp Asp Glu
Cys Ile Gln Ile Leu Lys Arg Cys Lys Glu Ala Leu Pro 260 265 270Lys
Val Gly Lys Val Ile Ile Val Asp Val Val Ile Asp Met Asp Ser 275 280
285Thr His Pro Tyr Ala Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met
290 295 300Leu Asn Thr Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys
Thr Leu305 310 315 320Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val
Thr Gln Ile Ser Ala 325 330 335Val Gln Ser Val Ile Glu Ala Tyr Pro
Tyr 340 345221194DNAPapaver somniferumCDS(28)..(1068) 22gcagctaaag
tgtctaaaca gagagaa atg gaa aca gta agc aag att gat caa 54Met Glu
Thr Val Ser Lys Ile Asp Gln1 5caa aac caa gca aaa atc tgg aaa caa
att tac ggt ttc gca gaa tca 102Gln Asn Gln Ala Lys Ile Trp Lys Gln
Ile Tyr Gly Phe Ala Glu Ser10 15 20 25cta gtt ctg aaa tgt gca gtc
caa cta gag att gct gaa aca ctt cac 150Leu Val Leu Lys Cys Ala Val
Gln Leu Glu Ile Ala Glu Thr Leu His 30 35 40aac aat gtc aaa ccc atg
tct tta tcc gaa ttg gca tcg aaa ctt ccc 198Asn Asn Val Lys Pro Met
Ser Leu Ser Glu Leu Ala Ser Lys Leu Pro 45 50 55gtt gct caa ccc gtt
aac gaa gac cgt ctg ttc cga att atg cgt tac 246Val Ala Gln Pro Val
Asn Glu Asp Arg Leu Phe Arg Ile Met Arg Tyr 60 65 70ttg gtt cac atg
gag ctc ttc aaa ata gat gct acc acg cag aaa tac 294Leu Val His Met
Glu Leu Phe Lys Ile Asp Ala Thr Thr Gln Lys Tyr 75 80 85tca tta gct
cca cca gct aag tat ttg ttg aga ggc tgg gag aaa tca 342Ser Leu Ala
Pro Pro Ala Lys Tyr Leu Leu Arg Gly Trp Glu Lys Ser90 95 100 105atg
gtt gat tca att tta tgc ata aat gat aag gat ttc tta gct cca 390Met
Val Asp Ser Ile Leu Cys Ile Asn Asp Lys Asp Phe Leu Ala Pro 110 115
120tgg cac cat tta ggc gac ggt ttg acc ggt aac tgt gac gct ttt gag
438Trp His His Leu Gly Asp Gly Leu Thr Gly Asn Cys Asp Ala Phe Glu
125 130 135aaa gcg ttg ggg aag agt att tgg gtg tat atg agt gaa aat
cct gaa 486Lys Ala Leu Gly Lys Ser Ile Trp Val Tyr Met Ser Glu Asn
Pro Glu 140 145 150aag aat caa ttg ttt aat gca gca atg gct tgt gat
act aga ttg gtt 534Lys Asn Gln Leu Phe Asn Ala Ala Met Ala Cys Asp
Thr Arg Leu Val 155 160 165act tct gca ttg gct aat gag tgc aaa agt
att ttc agt gat gga atc 582Thr Ser Ala Leu Ala Asn Glu Cys Lys Ser
Ile Phe Ser Asp Gly Ile170 175 180 185agt aca ctg gtt gat gtc ggc
ggt ggt acg ggt act gca gtg aaa gcc 630Ser Thr Leu Val Asp Val Gly
Gly Gly Thr Gly Thr Ala Val Lys Ala 190 195 200ata tct aaa gct ttt
ccg gat att aag tgc act atc tat gat ctt cct 678Ile Ser Lys Ala Phe
Pro Asp Ile Lys Cys Thr Ile Tyr Asp Leu Pro 205 210 215cat gtc ata
gct gat tct cct gaa atc ccc aat atc act aaa att tct 726His Val Ile
Ala Asp Ser Pro Glu Ile Pro Asn Ile Thr Lys Ile Ser 220 225 230gga
gat atg ttc aag tct att cct agt gct gat gcc atc ttc atg aag 774Gly
Asp Met Phe Lys Ser Ile Pro Ser Ala Asp Ala Ile Phe Met Lys 235 240
245tgc ata ctt cac gac tgg aac gat gat gaa tgc att caa atc ttg aag
822Cys Ile Leu His Asp Trp Asn Asp Asp Glu Cys Ile Gln Ile Leu
Lys250 255 260 265aga tgc aaa gaa gca tta cca aaa gtt ggc aaa gtt
att atc gtg gat 870Arg Cys Lys Glu Ala Leu Pro Lys Val Gly Lys Val
Ile Ile Val Asp 270 275 280gtc gtg ata gac atg gat tcg act cat cca
tat gca aaa att aga ctc 918Val Val Ile Asp Met Asp Ser Thr His Pro
Tyr Ala Lys Ile Arg Leu 285 290 295aca ctg gat ttg gat atg atg ctt
aac act ggt gga aaa gag aga acc 966Thr Leu Asp Leu Asp Met Met Leu
Asn Thr Gly Gly Lys Glu Arg Thr 300 305 310aaa gaa gaa tgg aag aca
ctt ttt gat gcc gct ggt ttt gct agc cac 1014Lys Glu Glu Trp Lys Thr
Leu Phe Asp Ala Ala Gly Phe Ala Ser His 315 320 325aaa gtc act cag
ata tct gct gtc caa tct gta att gag gct tac cct 1062Lys Val Thr Gln
Ile Ser Ala Val Gln Ser Val Ile Glu Ala Tyr Pro330 335 340 345tat
taa ggaacatttt aaccggtttt ccctttgatt aattgttgct ttctctttgg
1118Tyrattatgttta tgtttataac aattgcaaga tgaatgaatt tccaacttgc
attggattaa 1178aaaaaaaaaa aaaaaa 119423346PRTPapaver somniferum
23Met Glu Thr Val Ser Lys Ile Asp Gln Gln Asn Gln Ala Lys Ile Trp1
5 10 15Lys Gln Ile Tyr Gly Phe Ala Glu Ser Leu Val Leu Lys Cys Ala
Val 20 25 30Gln Leu Glu Ile Ala Glu Thr Leu His Asn Asn Val Lys Pro
Met Ser 35 40 45Leu Ser Glu Leu Ala Ser Lys Leu Pro Val Ala Gln Pro
Val Asn Glu 50 55 60Asp Arg Leu Phe Arg Ile Met Arg Tyr Leu Val His
Met Glu Leu Phe65 70 75 80Lys Ile Asp Ala Thr Thr Gln Lys Tyr Ser
Leu Ala Pro Pro Ala Lys 85 90 95Tyr Leu Leu Arg Gly Trp Glu Lys Ser
Met Val Asp Ser Ile Leu Cys 100 105 110Ile Asn Asp Lys Asp Phe Leu
Ala Pro Trp His His Leu Gly Asp Gly 115 120 125Leu Thr Gly Asn Cys
Asp Ala Phe Glu Lys Ala Leu Gly Lys Ser Ile 130 135 140Trp Val Tyr
Met Ser Glu Asn Pro Glu Lys Asn Gln Leu Phe Asn Ala145 150 155
160Ala Met Ala Cys Asp Thr Arg Leu Val Thr Ser Ala Leu Ala Asn Glu
165 170 175Cys Lys Ser Ile Phe Ser Asp Gly Ile Ser Thr Leu Val Asp
Val Gly 180 185 190Gly Gly Thr Gly Thr Ala Val Lys Ala Ile Ser Lys
Ala Phe Pro Asp 195 200 205Ile Lys Cys Thr Ile Tyr Asp Leu Pro His
Val Ile Ala Asp Ser Pro 210 215 220Glu Ile Pro Asn Ile Thr Lys Ile
Ser Gly Asp Met Phe Lys Ser Ile225 230 235 240Pro Ser Ala Asp Ala
Ile Phe Met Lys Cys Ile Leu His Asp Trp Asn 245 250 255Asp Asp Glu
Cys Ile Gln Ile Leu Lys Arg Cys Lys Glu Ala Leu Pro 260 265 270Lys
Val Gly Lys Val Ile Ile Val Asp Val Val Ile Asp Met Asp Ser 275 280
285Thr His Pro Tyr Ala Lys Ile Arg Leu Thr Leu Asp Leu Asp Met Met
290 295 300Leu Asn Thr Gly Gly Lys Glu Arg Thr Lys Glu Glu Trp Lys
Thr Leu305 310 315 320Phe Asp Ala Ala Gly Phe Ala Ser His Lys Val
Thr Gln Ile Ser Ala 325 330 335Val Gln Ser Val Ile Glu Ala Tyr Pro
Tyr 340 345245PRTPapaver somniferum 24Arg Thr Glu Ala Glu1
525346PRTPapaver somniferummisc_feature(93)..(93)Xaa can be any
naturally occurring amino acid 25Met Glu Thr Val Ser Lys Ile Asp
Gln Gln Asn Gln Ala Lys Ile Trp1 5 10 15Lys Gln Ile Tyr Gly Phe Ala
Glu Ser Leu Val Leu Lys Cys Ala Val 20 25 30Gln Leu Glu Ile Ala Glu
Thr Leu His Asn Asn Val Lys Pro Met Ser 35 40 45Leu Ser Glu Leu Ala
Ser Lys Leu Pro Val Ala Gln Pro Val Asn Glu 50 55 60Asp Arg Leu Phe
Arg Ile Met Arg Tyr Leu Val His Met Glu Leu Phe65 70 75 80Lys Ile
Asp Ala Thr Thr Gln Lys Tyr Ser Leu Ala Xaa Pro Ala Lys 85 90 95Tyr
Leu Leu Arg Gly Trp Glu Lys Ser Met Val Asp Ser Ile Leu Cys 100 105
110Ile Asn Asp Lys Asp Phe Leu Ala Pro Trp His His Leu Gly Asp Gly
115 120 125Leu Thr Gly Asn Cys Asp Ala Phe Glu Lys Ala Leu Gly Lys
Ser Ile 130 135 140Trp Val Tyr Met Ser Xaa Asn Pro Glu Lys Asn Gln
Leu Phe Asn Ala145 150 155 160Ala Met Ala Cys Asp Thr Arg Leu Val
Thr Ser Ala Leu Ala Asn Glu 165 170 175Cys Lys Ser Ile Phe Ser Asp
Gly Ile Ser Thr Leu Val Asp Val Gly 180 185 190Gly Gly Thr Gly Thr
Ala Val Lys Ala Ile Ser Lys Ala Phe Pro Asp 195 200 205Ile Lys Cys
Thr Ile Tyr Asp Leu Pro His Val Ile Ala Asp Ser Pro 210 215 220Glu
Ile Pro Asn Ile Thr Lys Ile Xaa Gly Asp Met Phe Lys Ser Ile225 230
235 240Pro Ser Ala Asp Xaa Ile Phe Met Lys Cys Ile Leu His Asp Trp
Asn 245 250 255Asp Asp Glu Cys Ile Gln Ile Leu Lys Arg Cys Lys Glu
Ala Leu Pro 260 265 270Lys Xaa Gly Lys Val Ile Ile Val Asp Val Val
Ile Asp Met Asp Ser 275 280 285Thr His Pro Tyr Ala Lys Ile Arg Leu
Thr Leu Asp Leu Asp Met Met 290 295 300Leu Asn Thr Gly Gly Lys Glu
Arg Thr Lys Glu Glu Trp Lys Thr Leu305 310 315 320Phe Asp Ala Ala
Gly Phe Ala Ser His Lys Val Thr Gln Ile Ser Ala 325 330 335Val Gln
Ser Val Ile Glu Ala Tyr Pro Tyr 340 345267PRTPapaver somniferum
26Leu Val Asp Val Gly Gly Gly1 5279PRTPapaver
somniferummisc_feature(2)..(3)Xaa can be any naturally occurring
amino acid 27Pro Xaa Xaa Asp Ala Xaa Xaa Met Lys1 5285PRTPapaver
somniferummisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 28Xaa Gly Lys Val Ile1 5295PRTPapaver somniferum 29Asp
Leu Pro His Val1 5307PRTPapaver somniferum 30His Val Gly Gly Asp
Met Phe1 5315PRTPapaver somniferum 31Gly Lys Glu Arg Thr1
5329PRTPapaver somniferum 32Leu Val Asp Val Gly Gly Gly Thr Gly1
5339PRTPapaver somniferum 33Ala Gly Lys Glu Arg Thr Glu Ala Glu1
5
* * * * *