U.S. patent application number 15/034773 was filed with the patent office on 2017-08-24 for s-adenosylmethionine (sam) synthase variants for the synthesis of artificial cofactors.
The applicant listed for this patent is LEIBNIZ-INSTITUT FUR PFLANZEN-BIOCHEMIE STIFTUNG DES OFFENTLICHEN RECHTS. Invention is credited to Wolfgang Brandt, Martin Dippe, Andrea Porzel, Hannes Rost, Ludger A. Wessjohann.
Application Number | 20170240870 15/034773 |
Document ID | / |
Family ID | 49551498 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240870 |
Kind Code |
A9 |
Dippe; Martin ; et
al. |
August 24, 2017 |
S-ADENOSYLMETHIONINE (SAM) SYNTHASE VARIANTS FOR THE SYNTHESIS OF
ARTIFICIAL COFACTORS
Abstract
The present invention relates to isolated polypeptides that are
derived from wildtype Bacillus subtilis S-Adenosylmethionine (SAM)
synthase or from a biologically active fragment thereof, wherein
said isolated polypeptides comprise an amino acid sequence that, in
relation to the amino acid sequence of said wildtype Bacillus
subtilis SAM synthase or of the biologically active fragment
thereof, comprises at least one amino acid substitution, selected
from the group consisting of amino acid substitutions at positions
I317 and I105. The present invention further relates to respective
isolated nucleic acids, vectors, host cells, uses and methods for
the production of SAM derivatives.
Inventors: |
Dippe; Martin; (Halle
(Saale), DE) ; Brandt; Wolfgang; (Halle (Saale),
DE) ; Wessjohann; Ludger A.; (Halle (Saale), DE)
; Rost; Hannes; (Halle (Saale), DE) ; Porzel;
Andrea; (Halle (Saale), DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEIBNIZ-INSTITUT FUR PFLANZEN-BIOCHEMIE STIFTUNG DES OFFENTLICHEN
RECHTS |
Halle (Saale) |
|
DE |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160264946 A1 |
September 15, 2016 |
|
|
Family ID: |
49551498 |
Appl. No.: |
15/034773 |
Filed: |
September 4, 2014 |
PCT Filed: |
September 4, 2014 |
PCT NO: |
PCT/EP2014/002398 PCKC 00 |
371 Date: |
May 5, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1085 20130101;
C12Y 205/01006 20130101; C12P 19/40 20130101 |
International
Class: |
C12N 9/10 20060101
C12N009/10; C12P 19/40 20060101 C12P019/40 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 6, 2013 |
EP |
13 005 228.5 |
Claims
1. An isolated polypeptide derived from wildtype Bacillus subtilis
S-Adenosylmethionine (SAM) synthase or from a biologically active
fragment thereof, comprising the amino acid sequence of SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6 or SEQ ID
NO:7.
2-8. (canceled)
9. An isolated nucleic acid encoding a polypeptide according to
claim 1.
10. A vector comprising the nucleic acid of claim 9.
11. A host cell comprising the nucleic acid of claim 9.
12. A method for the biocatalytic generation of
S-Adenosylmethionine (SAM) and/or SAM analogues having artificial
alkyl chains or allyl chains, or chains of the type
--(CH.sub.2).sub.n--OR, --(CH.sub.2).sub.n--SR, or
--(CH.sub.2).sub.n-Hal, wherein n is 1 to 3, R is an alkyl, and Hal
is a halogen, comprising the step of reacting a suitable S-alkyl
homocysteine, S-methylvinyl homocysteine, or other homocysteine
derivative with a polypeptide of claim 1.
13. (canceled)
14. A host cell comprising the vector of claim 10.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. National Phase patent application
of International Application Serial Number PCT/EP2014/002398, filed
on Sep. 4, 2014, which is hereby incorporated by reference in its
entirety, and which claims the benefit of European Patent
Application Number 13005228.5, filed on Nov. 6, 2013, which is
hereby incorporated by reference in its entirety.
STATEMENT IN SUPPORT OF FILING A SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing. A paper
copy of the Sequence Listing and a computer readable copy of the
Sequence Listing in ASCII format are provided herein and are herein
incorporated by reference in their entirety. Said computer readable
copy, created on Apr. 21, 2016, is named "23311777_1.txt" and is
24,918 bytes in size (as measured in MICROSOFT WINDOWS.RTM.
EXPLORER). This Sequence Listing consists of SEQ ID NOs: 1-7.
BACKGROUND OF THE DISCLOSURE
[0003] The present invention relates to isolated polypeptides that
are derived from wildtype Bacillus subtilis S-Adenosylmethionine
(SAM) synthase or from a biologically active fragment thereof,
wherein said isolated polypeptides comprise an amino acid sequence
that, in relation to the amino acid sequence of said wildtype
Bacillus subtilis SAM synthase or of the biologically active
fragment thereof, comprises at least one amino acid substitution,
selected from the group consisting of amino acid substitutions at
positions I317 and I105. The present invention further relates to
respective isolated nucleic acids, vectors, host cells, uses and
methods for the production of SAM derivatives.
[0004] Almost all enzyme-catalyzed methyl transfer reactions depend
on the cofactor S-adenosylmethionine (SAM). Thus, availability of
this compound is crucial for application of the corresponding
methyltransferases in biotechnology, e.g. in the production of
valuable fine chemicals and pharmaceuticals. In cellular
metabolism, this universal methyl donor is formed by adenosylation
of L-methionine by SAM synthases (SAMS). However, enzymatic
synthesis of SAM is not feasible for industrial application due to
inhibition of most SAMS by their product SAM which results in
stagnating conversion, low yield or--in life whole-cell
biocatalysts--in insufficient availability of the cofactor. In
addition, most SAMS enzymes does not tolerate changes in the
structure of their amino acid substrate. Thus, these enzymes lack
the ability to synthesize cofactor analogues from artificial
methionine derivatives. From the large number of SAMS enzymes which
have been characterized, only two proteins tolerably convert
S-ethyl-L-homocysteine (ethionine) to S-adenosylethionine. However,
these proteins are not feasible for the synthesis of SAM
derivatives which enable transfer of non-natural long-chain or
functionalized alkyl groups.
BRIEF SUMMARY OF THE DISCLOSURE
[0005] Accordingly, the technical problem underlying the present
invention is to provide variants of SAM synthase that can be used
for the biocatalytic production of SAM and SAM analogues having
artificial alkyl chains or allyl chains, or chains of the type
--(CH.sub.2).sub.n--OR, --(CH.sub.2).sub.n--SR, or
--(CH.sub.2).sub.n-Hal, wherein n is 1 to 3, R is an alkyl,
preferably a C.sub.1 to C.sub.4 alkyl, and Hal is a halogen,
preferably on an industrial scale, wherein said variants should
display a broad substrate specificity, high catalytic efficiency
and reduced product inhibition as compared to conventional SAM
synthases.
[0006] The solution to the above technical problem is achieved by
the embodiments characterized in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1: SAM Synthase Enzymes
[0008] FIG. 1 shows a SDS-PAGE of purified SAM synthase from
Bacillus subtilis (lane 1) and its variants I317V (lane 2), I317A
(lane 3), I105V/I317A (lane 4), I105A/I317A (lane 5), I105V/I3175
(lane 6) and I105S/I317S (lane 7).
[0009] FIGS. 2A-2C: Model of the SAM Synthase from Bacillus
subtilis
[0010] FIG. 2A shows the tertiary structure of the tetramer with
SAM (space fill representations) bound between two monomer units.
FIG. 2B shows the active site with the bound product SAM. The space
for docking of SAM derivatives with larger functional groups R is
restricted by the two isoleucines I105 and I317 which were
therefore subject of site-directed mutagenesis. FIG. 2C shows the
arrangement of the substrate analogues S-n-butyl-L-homocysteine and
adenylyl imidodiphosphate in the active center. The arrows indicate
the nucleophilic attack (short arrow) during catalysis. The
reaction is accompanied with conformational change of the protein
which will lead to steric hindrance (long arrow) with the amino
acid side chain.
[0011] FIGS. 3A-3E: Conversion of Methionine and Derivatives
[0012] FIGS. 3A-3E show the rates of conversion of amino acid
substrates catalyzed by the SAMS enzyme from Bacillus subtilis
(FIG. 3A) and its variants I317V (FIG. 3B), I317A (FIG. 3C),
I105V/I317A (FIG. 3D) or I105A/I317A (FIG. 3E) as a function of the
substrate concentration. The reaction with L-methionine (open
circles), D,L-methionine (closed circles),
D,L-methionine-(methyl-D3) (.times.), D,L-ethionine (hatched
circles), S-n-propyl-D,L-homocysteine (open triangles),
S-n-butyl-D,L-homocysteine (closed triangles) or
S-(2-methylvinyl)-D,L-homocysteine (diamonds) was assessed by
spectrophotometric determination of phosphate which is released
from the co-substrate ATP during the reaction.
[0013] FIGS. 4A-4C: Formation of SAM and SAM Derivatives
[0014] FIGS. 4A-4C show the formation of SAM and SAM derivatives by
the SAM synthase enzyme from Bacillus subtilis (FIG. 4A) or its
variants I317V (FIG. 4B) and I317A (FIG. 4C). The conversion of the
L-enantiomer of methionine (closed circles), ethionine (hatched
circles), S-n-propylhomocysteine (open triangles) and
S-n-butylhomocysteine (closed triangles) from the corresponding
racemic amino acid (10 mM) was performed in the presence of equal
amounts of enzyme (10 mU ml.sup.-1). The reactions were analyzed by
HPTLC.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0015] In particular, in a first aspect, the present invention
relates to an isolated polypeptide derived from wildtype Bacillus
subtilis S-Adenosylmethionine (SAM) synthase or from a biologically
active fragment thereof, wherein said isolated polypeptide
comprises an amino acid sequence that, in relation to the amino
acid sequence of said wildtype Bacillus subtilis SAM synthase (SEQ
ID NO: 1) or of the biologically active fragment thereof, comprises
at least one amino acid substitution, selected from the group
consisting of amino acid substitutions at positions I317 and I105.
In particular, the isolated polypeptide of the present invention
can have any amino acid substitution at position I317, at position
I105, or at both positions I317 and I105, wherein an amino acid
substitution at position I317, or at both positions I317 and I105
is particularly preferred. The substitute amino acid is not
particularly limited and can be any proteinogenic amino acid with
the obvious exception of isoleucine (I). However, preferably the
substitute amino acid is an amino acid that is less spacious, i.e.
less voluminous than isoleucine. Particularly preferred amino acids
in this respect are glycine (G), leucine (L), proline (P),
threonine (T), cysteine (C), serine (S), aspartic acid (D),
glutamic acid (E), asparagine (N), valine (V), and alanine (A),
wherein valine (V), and alanine (A) are most preferred.
[0016] Thus, in a particularly preferred embodiment, the present
invention relates to an isolated polypeptide derived from wildtype
Bacillus subtilis S-Adenosylmethionine (SAM) synthase or from a
biologically active fragment thereof, wherein said isolated
polypeptide comprises an amino acid sequence that, in relation to
the amino acid sequence of said wildtype Bacillus subtilis SAM
synthase (SEQ ID NO: 1) or of the biologically active fragment
thereof, comprises at least one amino acid substitution, selected
from the group consisting of the amino acid substitutions I317G,
I317L, I317P, I317T, I317C, I317D, I317E, I317N, I317A and
I317V.
[0017] In another particularly preferred embodiment, the present
invention relates to an isolated polypeptide derived from wildtype
Bacillus subtilis S-Adenosylmethionine (SAM) synthase or from a
biologically active fragment thereof, wherein said isolated
polypeptide comprises an amino acid sequence that, in relation to
the amino acid sequence of said wildtype Bacillus subtilis SAM
synthase (SEQ ID NO: 1) or of the biologically active fragment
thereof, comprises at least one amino acid substitution, selected
from the group consisting of the amino acid substitutions I317A and
I317V.
[0018] In this context, the term "polypeptide derived from wildtype
Bacillus subtilis SAM synthase or from a biologically active
fragment thereof" as used herein is intended to relate to
polypeptides that essentially correspond to wildtype Bacillus
subtilis SAM synthase, provided that in relation to said wildtype
Bacillus subtilis SAM synthase they comprise at least one of the
amino acid substitutions defined above. Further, said term is
intended to relate to polypeptides that can comprise any number of
additional amino acid substitutions, additions, or deletions,
provided that the resulting polypeptide retains the biological
activity of a SAM synthase. In this context, the term "retains the
biological activity of a SAM synthase" as used herein, as well as
the term "biologically active fragment thereof" as used herein,
relates to polypeptides that have at least 0.1%, at least 0.5%, at
least 1%, at least 5%, at least 10%, at least 25%, at least 50%,
preferably at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 82%, at least 84%, at least
86%, at least 88%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 97.5%, at least 98%, at least 98.5%, at least 99%, at
least 99.5%, 100%, or more than 100% of the activity of wildtype
Bacillus subtilis SAM synthase, as determined in a standard SAM
synthase activity assay known in the art.
[0019] While the number of additional amino acid substitutions,
additions, or deletions is generally only limited by the above
proviso concerning the biological activity of the resulting
polypeptide, it is preferable that the resulting polypeptide has at
least 50%, at least 52.5%, at least 55%, at least 57.5%, at least
60%, at least 62.5%, at least 65%, at least 67.5%, at least 70%, at
least 72.5%, at least 75%, at least 76.25%, at least 77.5%, at
least 78.75%, at least 80%, at least 81.25%, at least 83.75%, at
least 85%, at least 86.25%, at least 87.5%, at least 88%, at least
88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%,
at least 91%, at least 91.5%, at least 92%, at least 92.5%, at
least 93%, at least 93.5%, at least 94%, at least 94.5%, at least
95%, at least 95.25%, at least 95.5%, at least 95.75%, at least
96%, at least 96.25%, at least 96.5%, at least 96.75%, at least
97%, at least 97.25%, at least 97.5%, at least 97.75%, at least
98%, at least 98.25%, at least 98.5%, at least 98.75%, at least
99%, at least 99.25%, at least 99.5%, or 99.75% identity to
wildtype Bacillus subtilis SAM synthase.
[0020] Further, the terms "an amino acid sequence that, in relation
to the amino acid sequence of said wildtype Bacillus subtilis SAM
synthase (SEQ ID NO: 1) or of the biologically active fragment
thereof, comprises at least one amino acid substitution, selected
from the group consisting of amino acid substitutions at positions
I317 and I105" and "an amino acid sequence that, in relation to the
amino acid sequence of said wildtype Bacillus subtilis SAM synthase
(SEQ ID NO: 1) or of the biologically active fragment thereof,
comprises at least one amino acid substitution, selected from the
group consisting of the amino acid substitutions I317A and I317V"
as used herein are intended to relate to a respective amino acid
sequence, i.e. an amino acid sequence that (i) essentially
corresponds to the amino acid sequence of said wildtype Bacillus
subtilis SAM synthase (SEQ ID NO: 1), (ii) in relation to said
sequence comprises at least one of the above amino acid
substitutions, and (iii) can comprise any further amino acid
substitutions, additions or deletions as defined above, wherein the
number of said further amino acid substitutions, additions or
deletions is also as defined above with respect to the biological
activity of the resulting polypeptide, preferably with respect to
the identity of the resulting polypeptide to wildtype Bacillus
subtilis SAM synthase.
[0021] In a further embodiment, the isolated polypeptide of the
present invention further comprises an amino acid substitution,
selected from the group consisting of the amino acid substitutions
I105G, I105L, I105P, I105T, I105C, I105S, I105A and I105V.
[0022] Preferably, in a further embodiment, the isolated
polypeptide of the present invention further comprises an amino
acid substitution, selected from the group consisting of the amino
acid substitutions I105A and I105V.
[0023] In a preferred embodiment, the isolated polypeptide of the
present invention comprises the amino acid sequence of SEQ ID NO:
2, said amino acid sequence containing the amino acid substitution
I317A in relation to the amino acid sequence of wildtype Bacillus
subtilis SAM synthase (SEQ ID NO: 1). Even more preferably, the
isolated polypeptide of the present invention consists of the amino
acid sequence of SEQ ID NO: 2.
[0024] In a further preferred embodiment, the isolated polypeptide
of the present invention comprises the amino acid sequence of SEQ
ID NO: 3, said amino acid sequence containing the amino acid
substitution I317V in relation to the amino acid sequence of
wildtype Bacillus subtilis SAM synthase (SEQ ID NO: 1). Even more
preferably, the isolated polypeptide of the present invention
consists of the amino acid sequence of SEQ ID NO: 3.
[0025] In a further preferred embodiment, the isolated polypeptide
of the present invention comprises the amino acid sequence of SEQ
ID NO: 4, said amino acid sequence containing the amino acid
substitutions I317A and I105A in relation to the amino acid
sequence of wildtype Bacillus subtilis SAM synthase (SEQ ID NO: 1).
Even more preferably, the isolated polypeptide of the present
invention consists of the amino acid sequence of SEQ ID NO: 4.
[0026] In a further preferred embodiment, the isolated polypeptide
of the present invention comprises the amino acid sequence of SEQ
ID NO: 5, said amino acid sequence containing the amino acid
substitutions I317V and I105A in relation to the amino acid
sequence of wildtype Bacillus subtilis SAM synthase (SEQ ID NO: 1).
Even more preferably, the isolated polypeptide of the present
invention consists of the amino acid sequence of SEQ ID NO: 5.
[0027] In a further preferred embodiment, the isolated polypeptide
of the present invention comprises the amino acid sequence of SEQ
ID NO: 6, said amino acid sequence containing the amino acid
substitutions I317A and I105V in relation to the amino acid
sequence of wildtype Bacillus subtilis SAM synthase (SEQ ID NO: 1).
Even more preferably, the isolated polypeptide of the present
invention consists of the amino acid sequence of SEQ ID NO: 6.
[0028] In a further preferred embodiment, the isolated polypeptide
of the present invention comprises the amino acid sequence of SEQ
ID NO: 7, said amino acid sequence containing the amino acid
substitutions I317V and I105V in relation to the amino acid
sequence of wildtype Bacillus subtilis SAM synthase (SEQ ID NO: 1).
Even more preferably, the isolated polypeptide of the present
invention consists of the amino acid sequence of SEQ ID NO: 7.
[0029] In a further aspect, the present invention relates to an
isolated nucleic acid encoding a polypeptide according to the
present invention. Such nucleic acids are not particularly limited
and can be single-stranded or double-stranded DNA molecules, RNA
molecules, DNA/RNA hybrid molecules or artificial and/or modified
nucleic acid molecules. The term "nucleic acid encoding a
polypeptide according to the present invention" as used herein is
intended to relate to nucleic acid molecules comprising the coding
sequence of the respective polypeptide, as well as nucleic acid
molecules comprising a sequence that is fully complementary to the
coding sequence of the respective polypeptide.
[0030] In a further aspect, the present invention relates to a
vector comprising the nucleic acid according to the present
invention. Suitable vectors are not particularly limited and are
known in the art. They include for example plasmid vectors, e.g.
well known expression vectors, in particular bacterial expression
vectors such as pET28a(+), viral vectors, cosmid vectors and
artificial chromosomes.
[0031] In yet another aspect, the present invention relates to a
host cell comprising the nucleic acid according to the present
invention or the vector according to the present invention.
Suitable host cells are not particularly limited and are known in
the art. They include for example bacterial cells, e.g. well known
bacterial cells for recombinant protein expression such as cells
based on the E. coli strain K12, e.g. E. coli BL21 (DE3),
gram-positive bacterial cells such as Bacillus subtilis, but also
yeast cells, insect cells, plant cells, and mammalian cells.
[0032] In a further aspect, the present invention relates to a
method for the biocatalytic generation of S-Adenosylmethionine
(SAM) and/or SAM analogues having artificial alkyl chains or allyl
chains, or chains of the type --(CH.sub.2).sub.n--OR,
--(CH.sub.2).sub.n--SR, or --(CH.sub.2).sub.n-Hal, wherein n is 1
to 3, R is an alkyl, preferably a C.sub.1 to C.sub.4 alkyl, and Hal
is a halogen, comprising the step of reacting a suitable S-alkyl
homocysteine, S-methylvinyl homocysteine, or other homocysteine
derivative with a polypeptide of the present invention.
[0033] SAM analogues that can be generated with the method of the
present invention are not particularly limited, depending only on
the substrate specificity of the SAM synthase variant used and the
selected starting product. Examples include S-adenosylethionine
generated from S-ethyl-L-homocysteine, S-adenosylpropionine
generated from S-n-propyl-D,L-homocysteine, S-adenosyl buthionine
generated from S-n-butyl-D, L-homocysteine, and
S-adenosyl-S-methylvinyl homocysteine from
S-methylvinyl-D,L-homocysteine.
[0034] In a final aspect, the present invention relates to the use
of a polypeptide according to the present invention for the
generation of S-Adenosylmethionine (SAM) and/or SAM analogues
having artificial alkyl chains or allyl chains, or chains of the
type --(CH.sub.2).sub.n--OR, --(CH.sub.2).sub.n--SR, or
--(CH.sub.2).sub.n-Hal, wherein n is 1 to 3, R is an alkyl,
preferably a C.sub.1 to C.sub.4 alkyl, and Hal is a halogen. In
this aspect, SAM analogues that can be generated and their
respective starting products are as defined above for the method of
the present invention.
[0035] The present invention provides SAM synthase variants that
can be advantageously used in the generation of SAM and SAM
analogues, in particular on an industrial scale. The SAM synthase
variants according to the present invention can be highly expressed
in suitable host cells and are characterized by a high
productivity, a broad substrate specificity for a wide range of
starting products resulting in a broad range of SAM analogues that
can be generated, and a reduced product inhibition. These
characteristics make the SAM synthase variants according to the
present invention ideal candidates for the large-scale generation
of SAM and SAM analogues in an industrial setting.
[0036] To obtain a SAMS enzyme suitable for biocatalytic production
of SAM and analogues, the protein from Bacillus subtilis was
improved by rational protein design. Substitution of two conserved
isoleucine residues (I105 and I317) located in close proximity to
the active site extended the substrate spectrum of the enzyme to
artificial methionine derivatives. An introduction of a less
spacious valine or alanine residue into position 105 and 317 led to
variants which were able to convert methionine and
S-alkylhomocysteines bearing substituents with 2 to 4 carbon atoms.
In contrast to the wild-type enzyme, the variants I317V and I317A
were much less affected by product inhibition and proved to be
favorable for the preparative synthesis of SAM and its long-chain
analogues. In addition, these variants might be applied to generate
a high-level intracellular concentration of SAM in prokaryotic
hosts upon addition of methionine to the growth medium.
[0037] The present invention discloses the following amino acid
sequences.
TABLE-US-00001 wildtype Bacillus subtilis SAM synthase (SEQ ID NO:
1) msknrrlfts esvteghpdk icdqisdsil deilkkdpna rvacetsvtt
glvlvsgeit tstyvdipkt vrqtikeigy trakygfdae tcavltside qsadiamgvd
qalearegtm sdeeieaiga gdqglmfgya cnetkelmpl pislahklar rlsevrkedi
lpylrpdgkt qvtveydenn kpvridaivi stqhhpeitl eqiqrnikeh vinpvvpeel
ideetkyfin ptgrfviggp qgdagltgrk iivdtyggya rhgggafsgk datkvdrsaa
yaaryvakni vaaeladsce vqlayaigva qpvsisintf gsgkaseekl ievvrnnfdl
rpagiikmld lrrpiykqta ayghfgrhdv dlpwertdka eqlrkealge I317A SAM
synthase variant (SEQ ID NO: 2) msknrrlfts esvteghpdk icdqisdsil
deilkkdpna rvacetsvtt glvlvsgeit tstyvdipkt vrqtikeigy trakygfdae
tcavltside qsadiamgvd qalearegtm sdeeieaiga gdqglmfgya cnetkelmpl
pislahklar rlsevrkedi lpylrpdgkt qvtveydenn kpvridaivi stqhhpeitl
eqiqrnikeh vinpvvpeel ideetkyfin ptgrfviggp qgdagltgrk iivdtyggya
rhgggafsgk datkvdrsaa yaaryvakni vaaeladsce vqlayaagva qpvsisintf
gsgkaseekl ievvrnnfdl rpagiikmld lrrpiykqta ayghfgrhdv dlpwertdka
eqlrkealge I317V SAM synthase variant (SEQ ID NO: 3) msknrrlfts
esvteghpdk icdqisdsil deilkkdpna rvacetsvtt glvlvsgeit tstyvdipkt
vrqtikeigy trakygfdae tcavltside qsadiamgvd qalearegtm sdeeieaiga
gdqglmfgya cnetkelmpl pislahklar rlsevrkedi lkpylrpdgkt qvtveydenn
kpvridaivi stqhhpeitl eqiqrnikeh vinpvvpeel ideetkyfin ptgrfviggp
qgdagltgrk iivdtyggya rhgggafsgk datkvdrsaa yaaryvakni vaaeladsce
vqlayavgva qpvsisintf gsgkaseekl ievvrnnfdl rpagiikmld lrrpiykqta
ayghfgrhdv dlpwertdka eqlrkealge I105A/I317A SAM synthase variant
(SEQ ID NO: 4) msknrrlfts esvteghpdk icdqisdsil deilkkdpna
rvacetsvtt glvlvsgeit tstyvdipkt vrqtikeigy trakygfdae tcavltside
qsadaamgvd qalearegtm sdeeieaiga gdqglmfgya cnetkelmpl pislahklar
rlsevrkedi lpylrpdgkt qvtveydenn kpvridaivi stqhhpeitl eqiqrnikeh
vinpvvpeel ideetkyfin ptgrfviggp qgdagltgrk iivdtyggya rhgggafsgk
datkvdrsaa yaaryvakni vaaeladsce vqlayaagva qpvsisintf gsgkaseekl
ievvrnnfdl rpagiikmld lrrpiykqta ayghfgrhdv dlpwertdka eqlrkealge
I105A/I317V SAM synthase variant (SEQ ID NO: 5) msknrrlfts
esvteghpdk icdqisdsil deilkkdpna rvacetsvtt glvlvsgeit tstyvdipkt
vrqtikeigy trakygfdae tcavltside qsadaamgvd qalearegtm sdeeieaiga
gdqglmfgya cnetkelmpl pislahklar rlsevrkedi lpylrpdgkt qvtveydenn
kpvridaivi stqhhpeitl eqiqrnikeh vinpvvpeel ideetkyfin ptgrfviggp
qgdagltgrk iivdtyggya rhgggafsgk datkvdrsaa yaaryvakni vaaeladsce
vqlayavgva qpvsisintf gsgkaseekl ievvrnnfdl rpagiikmld lrrpiykqta
ayghfgrhdv dlpwertdka eqlrkealge I105V/I317A SAM synthase variant
(SEQ ID NO: 6) msknrrlfts esvteghpdk icdqisdsil deilkkdpna
rvacetsvtt glvlvsgeit tstyvdipkt vrqtikeigy trakygfdae tcavltside
qsadvamgvd qalearegtm sdeeieaiga gdqglmfgya cnetkelmpl pislahklar
rlsevrkedi lpylrpdgkt qvtveydenn kpvridaivi stqhhpeitl eqiqrnikeh
vinpvvpeel ideetkyfin ptgrfviggp qgdagltgrk iivdtyggya rhgggafsgk
datkvdrsaa yaaryvakni vaaeladsce vqlayaagva qpvsisintf gsgkaseekl
ievvrnnfdl rpagiikmld lrrpiykqta ayghfgrhdv dlpwertdka eqlrkealge
I105V/I317V SAM synthase variant (SEQ ID NO: 7) msknrrlfts
esvteghpdk icdqisdsil deilkkdpna rvacetsvtt glvlvsgeit tstyvdipkt
vrqtikeigy trakygfdae tcavltside qsadvamgvd qalearegtm sdeeieaiga
gdqglmfgya cnetkelmpl pislahklar rlsevrkedi lpylrpdgkt qvtveydenn
kpvridaivi stqhhpeitl eqiqrnikeh vinpvvpeel ideetkyfin ptgrfviggp
qgdagltgrk iivdtyggya rhgggafsgk datkvdrsaa yaaryvakni vaaeladsce
vqlayavgva qpvsisintf gsgkaseekl ievvrnnfdl rpagiikmld lrrpiykqta
ayghfgrhdv dlpwertdka eqlrkealge
[0038] The present invention will be further illustrated in the
following examples without being limited thereto.
EXAMPLES
Example 1
Recombinant Expression of Wildtype Bacillus subtilis SAM
Synthase
[0039] To address the problem of availability of the cofactor
S-adenosyl-L-methionine (SAM) in Escherichia coli, an enhancement
of endogenous SAM synthesis was envisioned. Therefore, the SAM
synthase gene from Bacillus subtilis was cloned into the vector
pET28a(+) and introduced in E. coli BL21 (DE3). Purified enzyme
(FIG. 1, lane 1) could be obtained in high yields (59 mg/l of
culture). Since the protein is not inhibited by the D-enantiomer of
methionine (FIG. 3A), even inexpensive racemic mixtures of this
amino acid can be used as substrate.
Example 2
Generation and Characterization of SAM Synthase Variants
[0040] Beside regeneration of the natural cofactor SAM, recombinant
SAM synthase variants (FIG. 1, lanes 2 to 7) were applied for the
synthesis of cofactor analogues having artificial alkyl chains.
[0041] For these studies, a series of S-alkylhomocysteines with
linear (D,L-methionine-(methyl-D3), -ethionine, -propionine and
-buthionine), branched (D,L-isopropionine and -isobuthionines),
unsaturated (D,L-methylvinylhomocysteine) and functionalized
(D,L-hydroxyethionine and -carboxymethionine) alkyl groups were
synthesized from D,L-homocysteine thiolactone and tested for
conversion by the cloned (wildtype) SAM synthase. However, the
enzyme did not accept any of these methionine derivatives.
[0042] As derived from a homology model which is based on the
crystal structure of the protein from E. coli (PDB code 1XRA), a
hydrophobic cleft formed from two conserved isoleucine residues
hinders the generation of cofactor analogues from amino acid
substrates with bulky alkyl residues (FIG. 2). Accordingly, both
residues were exchanged against less voluminous amino acids by
site-directed mutagenesis. Especially I317 proved to be crucial for
substrate selectivity. In addition to methionine, the variant I317V
also converts ethionine (FIG. 3B). If I317 is substituted by an
even smaller alanine residue, the corresponding variant is active
on ethionine, propionine, buthionine and the unsaturated
D,L-methylvinylhomocysteine (FIG. 3C). Finally, an exchange of both
isoleucines to alanine results in acceptance of the artificial
long-chain substrates only (FIG. 3D).
Example 3
Production of SAM Derivatives by SAM Synthase Variants
[0043] Due to their broad substrate spectrum, the variants I317V
and I317A were used for the production of the SAM derivatives
S-adenosylethionine, -propionine, -buthionine and
--S-methylvinylhomocysteine in preparative scale. The compounds
were synthesized from 0.2 mmol of D,L-amino acid and purified by
cation exchange chromatography on SP-Sephadex C-25. Yields ranged
from 10 to 52%, related to the L-form of the amino acid. As tested
by enzymatic conversion of produced SAM by
catechol-O-methyltransferase, the preparations contained the
biological active form of the cofactor (80 to 92% of total
SAM).
Example 4
Characterization of Further SAM Synthase Variants
[0044] The influence of position 317 on catalytic turnover and its
crucial role in determination of substrate spectrum was
additionally confirmed by introduction of other small- and
medium-sized amino acid residues. Enzymes substituted for aliphatic
(I317G, I317P, I317L) or polar (I317E, I317D, I317N) amino acids
showed a strongly reduced activity (.gtoreq.5.4 nmol min.sup.-1
mg.sup.-1). On the other hand, substitution by cysteine resulted in
a tolerably active enzyme which--similar to the variant
I317A--converted methionine and homologs (59.4.+-.0.4, 16.4.+-.0.7,
8.4.+-.0.3 and 6.0.+-.0.3 nmol min.sup.-1 mg.sup.-1 for conversion
of 5 mM D,L-methionine, -ethionine, -propionine and -buthionine,
respectively).
Example 5
Characterization of Double Mutant SAM Synthase Variants
[0045] To further probe the role of the proximate isoleucine
residue I105 in substrate recognition, the variants I105V/I317A and
I105A/I317A were generated. With increasing size of the active
center of the enzymes, conversion of the natural substrate
methionine (FIGS. 3D, E) is progressively reduced. On the other
hand, the variants prefer long-chain substrates. Compared to the
wild-type enzyme (FIG. 3A), substrate specificity of the variant
I105A/I317A (FIG. 3E) is inverted
(S-n-propylhomocysteine>S-n-butylhomocysteine>ethionine>>meth-
ionine). Hence, steric effects play a major role in substrate
conversion by SAMS enzymes, and selectivity for certain substrates
can be engineered by exchange of two amino acid positions only.
Example 6
Synthesis of SAM Derivatives by SAM Synthase Variants
[0046] The suitability of the wild-type protein and of variants
with appropriate substrate specificity (I317V and I317A) for the
synthesis of SAM and analogues was evaluated by high-performance
thin-layer chromatography. Since the enzymes were not inhibited by
the D-enantiomer of the amino acid substrates, methionine and its
derivatives could be advantageously applied as racemic mixture of
its stereoisomeres in these reactions. The time course of SAM
formation by the wild-type enzyme is shown in FIG. 4A. As described
for several other SAMS proteins, the enzyme is inhibited by its
product SAM which results in stagnating conversion and low yield.
In contrast, this product inhibition is completely reduced in the
variant I317V (FIG. 4B) and less pronounced in case of the mutant
enzyme I317A (FIG. 4C). Accordingly, transformation of methionine
and ethionine by SAMS-I317V in preparative synthetic reactions led
to high conversion rates for the amino acid substrates. The
syntheses were performed similar to the kinetic experiments but in
larger scale (200 .mu.mol). After prolonged incubation (18 hours)
to reach maximum product yield, 84 and 89% of the L-amino acid was
converted. In the production of the n-propyl- and n-butyl analogues
of SAM by the variant I317A, 43% and 28% of conversion could be
reached after 8 hours of reaction time. After product purification
by cation exchange chromatography on SP-Sephadex C-25, SAM and its
homologs could be recovered in final yields of 25, 17, 8 and 11%,
respectively. As proven by .sup.1H NMR, the enzymatically produced
SAM contained a high excess (.gtoreq.90%) of the biologically
active (S,S)-epimer. Interestingly, preparations of the
S-adenosyl-L-ethionine, -propionine and -buthionine were racemic
with respect to the chiral sulfonium center, which might be caused
by faster racemization under the strongly acidic conditions in
column purification.
Example 7
Kinetic Parameters for the Conversion of S-alkylhomocysteines
[0047] The kinetic parameters for the conversion of
S-alkylhomocysteines by Bacillus subtilis SAM synthase and its
variants as shown in Table 1 were calculated from the conversion
rates of methionine and analogues as a function of the substrate
concentration (FIGS. 2A-2C) which were determined by the standard
SAM synthase assay known in the art, i.e. by photometric detection
of the cleaved phosphate.
TABLE-US-00002 TABLE 1 V.sub.max (nmol Enzyme Substrate S.sub.0.5
(mM) min.sup.-1 mg.sup.-1) h Wild-type L-methionine 1.04 .+-. 0.14
93.2 .+-. 3.1 1.1 .+-. 0.1 D,L-methionine 1.92 .+-. 0.12 90.8 .+-.
1.6 1.7 .+-. 0.2 D,L-methionine-(methyl-D.sub.3) 1.86 .+-. 0.07
79.8 .+-. 2.3 2.2 .+-. 0.2 I317V L-methionine 0.72 .+-. 0.04 408.0
.+-. 7.7 1.2 .+-. 0.1 D,L-methionine 1.71 .+-. 0.12 446.2 .+-. 13.8
1.3 .+-. 0.1 D,L-ethionine 10.94 .+-. 2.70 51.2 .+-. 9.1 1.3 .+-.
0.1 I317A L-methionine 0.46 .+-. 0.02 34.8 .+-. 0.5 1.3 .+-. 0.1
D,L-methionine 0.92 .+-. 0.05 37.7 .+-. 0.8 1.1 .+-. 0.1
D,L-ethionine 2.99 .+-. 0.23 44.1 .+-. 1.6 1.2 .+-. 0.1
S-n-propyl-D,L-homocysteine 0.90 .+-. 0.05 34.2 .+-. 0.7 1.1 .+-.
0.1 S-n-butyl-D,L-homocysteine 3.56 .+-. 0.45 35.4 .+-. 2.6 1.4
.+-. 0.1 S-(2-methylvinyl)-D,L-homocysteine 0.79 .+-. 0.04 22.4
.+-. 0.4 1.2 .+-. 0.1 I105V/I317A D,L-methionine 3.77 .+-. 1.02
17.0 .+-. 2.0 1.0 .+-. 0.1 D,L-ethionine 4.25 .+-. 0.20 5.7 .+-.
0.3 4.6 .+-. 0.8 S-n-propyl-D,L-homocysteine 1.86 .+-. 0.41 17.7
.+-. 1.5 1.0 .+-. 0.1 S-n-butyl-D,L-homocysteine 4.20 .+-. 0.41
11.9 .+-. 0.9 2.0 .+-. 0.2 I105A/I317A D,L-methionine n.d. 0.8 .+-.
0.2 n.d D,L-ethionine 3.91 .+-. 0.27 3.0 .+-. 1.8 2.9 .+-. 0.4
S-n-propyl-D,L-homocysteine =10.0 =10.8 .+-. 0.1 1.0 .+-. 0.1
S-n-butyl-D,L-homocysteine =10.0 =4.0 .+-. 0.2 0.8 .+-. 0.1 n.d.
not determined
Sequence CWU 1
1
71400PRTBacillus subtilis 1Met Ser Lys Asn Arg Arg Leu Phe Thr Ser
Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp Gln
Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp Pro
Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly Leu
Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val Asp
Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70 75 80
Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu Thr 85
90 95 Ser Ile Asp Glu Gln Ser Ala Asp Ile Ala Met Gly Val Asp Gln
Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu Glu Ile
Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr
Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro Ile Ser
Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu Val Arg
Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly Lys Thr
Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190 Val Arg
Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195 200 205
Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn Pro 210
215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr Phe Ile
Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Gln Gly
Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly Ala Phe Ser Gly Lys
Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala Ala Tyr Ala Ala Arg
Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295 300 Leu Ala Asp Ser
Cys Glu Val Gln Leu Ala Tyr Ala Ile Gly Val Ala 305 310 315 320 Gln
Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly Lys Ala Ser 325 330
335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn Phe Asp Leu Arg Pro
340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu Arg Arg Pro Ile Tyr
Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe Gly Arg His Asp Val
Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys Ala Glu Gln Leu Arg
Lys Glu Ala Leu Gly Glu 385 390 395 400 2400PRTArtificial
Sequencemutant SAM synthase 2Met Ser Lys Asn Arg Arg Leu Phe Thr
Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp
Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp
Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly
Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val
Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70
75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu
Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Ile Ala Met Gly Val
Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu
Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe
Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro
Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu
Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly
Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190
Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195
200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn
Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr
Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro
Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp
Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly Ala Phe Ser
Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala Ala Tyr Ala
Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295 300 Leu Ala
Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Ala Gly Val Ala 305 310 315
320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly Lys Ala Ser
325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn Phe Asp Leu
Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu Arg Arg Pro
Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe Gly Arg His
Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys Ala Glu Gln
Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400 3400PRTArtificial
Sequencemutant SAM synthase 3Met Ser Lys Asn Arg Arg Leu Phe Thr
Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp
Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp
Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly
Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val
Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70
75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu
Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Ile Ala Met Gly Val
Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu
Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe
Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro
Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu
Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly
Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190
Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195
200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn
Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr
Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro
Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp
Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly Ala Phe Ser
Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala Ala Tyr Ala
Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295 300 Leu Ala
Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Val Gly Val Ala 305 310 315
320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly Lys Ala Ser
325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn Phe Asp Leu
Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu Arg Arg Pro
Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe Gly Arg His
Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys Ala Glu Gln
Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400 4400PRTArtificial
Sequencemutant SAM synthase 4Met Ser Lys Asn Arg Arg Leu Phe Thr
Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp
Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp
Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly
Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val
Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70
75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu
Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Ala Ala Met Gly Val
Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu
Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe
Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro
Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu
Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly
Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190
Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195
200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn
Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr
Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro
Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp
Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly Ala Phe Ser
Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala Ala Tyr Ala
Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295 300 Leu Ala
Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Ala Gly Val Ala 305 310 315
320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly Lys Ala Ser
325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn Phe Asp Leu
Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu Arg Arg Pro
Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe Gly Arg His
Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys Ala Glu Gln
Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400 5400PRTArtificial
Sequencemutant SAM synthase 5Met Ser Lys Asn Arg Arg Leu Phe Thr
Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp
Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp
Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly
Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val
Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70
75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu
Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Ala Ala Met Gly Val
Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu
Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe
Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro
Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu
Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly
Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190
Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195
200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn
Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr
Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro
Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp
Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly Ala Phe Ser
Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala Ala Tyr Ala
Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295 300 Leu Ala
Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Val Gly Val Ala 305 310 315
320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly Lys Ala Ser
325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn Phe Asp Leu
Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu Arg Arg Pro
Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe Gly Arg His
Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys Ala Glu Gln
Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400 6400PRTArtificial
Sequencemutant SAM synthase 6Met Ser Lys Asn Arg Arg Leu Phe Thr
Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp Lys Ile Cys Asp
Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile Leu Lys Lys Asp
Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40 45 Thr Thr Gly
Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr 50 55 60 Val
Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile Gly Tyr 65 70
75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys Ala Val Leu
Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Val Ala Met Gly Val
Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met Ser Asp Glu
Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly Leu Met Phe
Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met Pro Leu Pro
Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg Leu Ser Glu
Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170 175 Asp Gly
Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro 180 185 190
Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro Glu Ile 195
200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His Val Ile Asn
Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu Thr Lys Tyr
Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro
Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile Ile Val Asp
Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly
Ala Phe Ser Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala
Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295
300 Leu Ala Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Ala Gly Val Ala
305 310 315 320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly
Lys Ala Ser 325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn
Phe Asp Leu Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu
Arg Arg Pro Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe
Gly Arg His Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys
Ala Glu Gln Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400
7400PRTArtificial Sequencemutant SAM synthase 7Met Ser Lys Asn Arg
Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly 1 5 10 15 His Pro Asp
Lys Ile Cys Asp Gln Ile Ser Asp Ser Ile Leu Asp Glu 20 25 30 Ile
Leu Lys Lys Asp Pro Asn Ala Arg Val Ala Cys Glu Thr Ser Val 35 40
45 Thr Thr Gly Leu Val Leu Val Ser Gly Glu Ile Thr Thr Ser Thr Tyr
50 55 60 Val Asp Ile Pro Lys Thr Val Arg Gln Thr Ile Lys Glu Ile
Gly Tyr 65 70 75 80 Thr Arg Ala Lys Tyr Gly Phe Asp Ala Glu Thr Cys
Ala Val Leu Thr 85 90 95 Ser Ile Asp Glu Gln Ser Ala Asp Val Ala
Met Gly Val Asp Gln Ala 100 105 110 Leu Glu Ala Arg Glu Gly Thr Met
Ser Asp Glu Glu Ile Glu Ala Ile 115 120 125 Gly Ala Gly Asp Gln Gly
Leu Met Phe Gly Tyr Ala Cys Asn Glu Thr 130 135 140 Lys Glu Leu Met
Pro Leu Pro Ile Ser Leu Ala His Lys Leu Ala Arg 145 150 155 160 Arg
Leu Ser Glu Val Arg Lys Glu Asp Ile Leu Pro Tyr Leu Arg Pro 165 170
175 Asp Gly Lys Thr Gln Val Thr Val Glu Tyr Asp Glu Asn Asn Lys Pro
180 185 190 Val Arg Ile Asp Ala Ile Val Ile Ser Thr Gln His His Pro
Glu Ile 195 200 205 Thr Leu Glu Gln Ile Gln Arg Asn Ile Lys Glu His
Val Ile Asn Pro 210 215 220 Val Val Pro Glu Glu Leu Ile Asp Glu Glu
Thr Lys Tyr Phe Ile Asn 225 230 235 240 Pro Thr Gly Arg Phe Val Ile
Gly Gly Pro Gln Gly Asp Ala Gly Leu 245 250 255 Thr Gly Arg Lys Ile
Ile Val Asp Thr Tyr Gly Gly Tyr Ala Arg His 260 265 270 Gly Gly Gly
Ala Phe Ser Gly Lys Asp Ala Thr Lys Val Asp Arg Ser 275 280 285 Ala
Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Glu 290 295
300 Leu Ala Asp Ser Cys Glu Val Gln Leu Ala Tyr Ala Val Gly Val Ala
305 310 315 320 Gln Pro Val Ser Ile Ser Ile Asn Thr Phe Gly Ser Gly
Lys Ala Ser 325 330 335 Glu Glu Lys Leu Ile Glu Val Val Arg Asn Asn
Phe Asp Leu Arg Pro 340 345 350 Ala Gly Ile Ile Lys Met Leu Asp Leu
Arg Arg Pro Ile Tyr Lys Gln 355 360 365 Thr Ala Ala Tyr Gly His Phe
Gly Arg His Asp Val Asp Leu Pro Trp 370 375 380 Glu Arg Thr Asp Lys
Ala Glu Gln Leu Arg Lys Glu Ala Leu Gly Glu 385 390 395 400
* * * * *