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Escherichia coli is known
to possess two enzymes (methionine synthases) that catalyze the last
step in methionine biosynthesis. The metE gene product, a
zinc-containing monomer (9) with a molecular mass of 85 kDa
(37), transfers the methyl group of N5-methyl-tetrahydrofolate to the thiolate of
homocysteine. The second enzyme, the metH gene product, is
also a monomer but with a size of 136 kDa (8); it catalyzes
methyl transfer from N5-methyl-tetrahydrofolate to the
cob(I)alamine coenzyme and from there to homocysteine. MetH exhibits a
distinct structure of four domains, which can be correlated with the
partial reactions catalyzed: an N-terminal homocysteine binding domain
carrying a zinc ion, an N5-methyl-tetrahydrofolate binding
domain, a cobalamine binding domain, and a C-terminal domain for
S-adenosylmethionine binding involved in reactivation of the
oxidized cob(II)alamine form (6, 10, 11). Since E. coli cannot synthesize corrinoids, MetH is only active when
cobalamine is present in the medium. MetH is about 100-fold more active
than MetE, which is, however, compensated by the very strong expression
of the metE gene (37).
Cloning and sequencing of the gene for a selenocysteine
methyltransferase (smtA) from Astragalus
bisulcatus involved in selenium tolerance and comparison of the
sequence with entries in the databases revealed that E. coli
possesses a gene (yagD) whose derived product shares 40%
amino acid sequence identity with SmtA (25). In addition, YagD shows low sequence similarities to the amino-terminal domain of
MetH and to human betaine:homocysteine methyltransferase (data not
shown). The regions that are conserved among these proteins comprise
the GGCC motif containing Cys310 and Cys311 and the GLNCA motif around
Cys247 (numbering refers to MetH from E. coli). These cysteine residues were identified as putative ligands of the zinc cofactor of MetH (10, 26). In accordance with this, human betaine:homocysteine methyltransferase was shown to also contain zinc,
which presumably is ligated by cysteine residues (24). These
findings indicate that YagD may be a zinc-dependent methyltransferase which uses a catalytical mechanism similar to that of MetH (10, 26). The YagD protein was purified and shown to synthesize
methionine from S-methylmethionine or
S-adenosylmethionine and homocysteine (25), an
activity previously described for crude extracts from this organism
(2) and for enriched protein preparations of Saccharomyces cerevisiae (31, 32) and
Canavalia ensiformis (1). Formally, therefore,
YagD constitutes a third methionine synthase in E. coli. In
this communication we report on its physiological role in the sulfur
metabolism of this organism.
Physiological role of the homocysteine methyltransferase.
To
elucidate the role of the YagD protein in methionine metabolism, a
mutant was constructed with an in-frame deletion in both the
metE and metH genes. For this purpose,
chromosomal copies of the two genes were amplified by PCR. After
deletion of residues 493 to 1665 of metE and residues 1198 to 1554 of metH, the mutant genes were re-introduced into
wild-type strain KL19 (20) by homologous recombination
according to the method of Hamilton et al. (13). Stationary
cells (see Fig. 1) of the resulting strain, MTD23, were then used to
inoculate minimal medium containing 15 µM (each) different
supplements to an optical density at 600 nm (OD600) of
0.05. Throughout this work M9 minimal medium (30) containing
0.8% glucose and the supplement indicated was used. L-S-methylmethionine was from Acros Organics (Geel,
Belgium). All other compounds were purchased from Sigma (Deisenhofen,
Germany). After 18 h of aerobic incubation at 37°C, the cell
densities of the cultures were measured. The OD600 values
(± standard deviations) obtained were 0.056 (± 0.003) without any
supplement, 0.280 (± 0.002) with DL-methionine sulfoxide,
0.380 (± 0.006) with L-methionine, 0.074 (± 0.002) with
S-adenosyl-L-methionine, 0.771 (± 0.001) with
DL-S-methylmethionine, and 0.679 (± 0.005) with
L-S-methylmethionine. These results demonstrate
that L-methionine, DL-methionine sulfoxide, and
S-methylmethionine complement auxotrophy of the strain. YagD does not contribute to the utilization of methionine sulfoxide, because
no transfer of the methyl group of this compound to homocysteine takes
place (data not shown). Presumably, methionine sulfoxide is converted
to methionine via reduction (7). The lower OD value obtained
for this substrate may be due to its slower utilization in comparison
to methionine (7, 12). With
S-adenosyl-L-methionine (AdoMet), only marginal
growth could be observed, which probably resulted from utilization of
impurities contained in the AdoMet preparation. This finding is in
accordance with previous observations, namely that E. coli
K-12 and its derivatives are largely impermeable to AdoMet
(14). Growth yield with the L form of
S-methylmethionine and the racemate was about twice that
obtained with L-methionine.
The YagD homocysteine methyltransferase does not utilize the
D stereoisomer of S-methylmethionine as a
substrate (25). It was, therefore, surprising that the
DL racemate yielded the same cell mass as the L
form. This result was supported in an experiment in which a series of
different concentrations of
DL-S-methylmethionine and
L-methionine were supplemented (Fig.
1). The data indicate that
D-methylmethionine is converted to the L form
prior to its utilization, possibly via the same pathway as the
conversion of D-methionine to L-methionine
(5).
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FIG. 1.
Effects of  yagD and ykfD
mutations on the utilization of S-methylmethionine. Cells of
strains MTD23 (metE metH), MTD123 (yagD
metE metH), and MTD234 (metE metH ykfD)
were grown in minimal medium containing 100 µM
L-methionine. After 18 h of aerobic incubation at
37°C, they were washed, resuspended in 0.9% NaCl to an
OD600 of 10, and transferred into minimal medium containing
different concentrations of L-methionine (met) or
DL-S-methylmethionine (methyl-met),
respectively. The initial OD600 was 0.05. After 18 h
of aerobic incubation at 37°C, the resulting cell densities were
determined. The data shown represent the mean values of at least two
independent growth experiments.
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To assess the involvement of the yagD gene product in
S-methylmethionine metabolism, an in-frame deletion
(residues 270 to 548) was introduced into the corresponding gene of
strain MTD23 according to the method of Hamilton et al.
(13), yielding the triple mutant MTD123 (yagD
metE metH). An immunoblotting analysis using antiserum
directed against homocysteine methyltransferase confirmed that there
was no cross-reacting material (data not shown). The results of the
growth experiment shown in Fig. 1 prove that metabolism of
S-methylmethionine requires a functional YagD protein, since
inactivation of the yagD gene abolishes the capacity to grow
on S-methylmethionine.
ykfD, a gene coding for a putative
S-methylmethionine permease.
The results described
above point to a role for the YagD homocysteine methyltransferase in
the utilization of external S-methylmethionine as a source
for methionine. Since there is no evidence in the literature that
E. coli is able to synthesize S-methylmethionine, there might be a transport system for its uptake from the medium. The
results presented previously do not exclude that
S-methylmethionine uses the methionine uptake system
(16, 17).
An inspection of the genomic sequence of E. coli in the
vicinity of yagD revealed that upstream of yagD
there is an open reading frame of 1,533 bp (ykfD) coding for
a putative protein and overlapping with the yagD reading
frame by 14 bp (4) (Fig. 2).
Its amino acid sequence shares significant sequence similarity with
that of amino acid permeases (data not shown). This similarity and the
overlapping reading frames might be an indication that ykfD codes for a S-methylmethionine transporter which is
cotranscribed with the yagD gene.
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FIG. 2.
Chromosomal organization of the ykfD and
yagD open reading frames. The restriction sites used in this
study are shown. Putative MET boxes in the 5' region of ykfD
are depicted as boxed nucleotide sequences. The degrees of identity
with the MET box consensus sequence 5'-AGACGTCT-3' are
indicated. Possible translational start codons are marked by boldface
letters.
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To test this assumption we introduced an in-frame deletion into
ykfD (residues 304 to 1089) and combined this mutation with the metE metH lesions of strain MTD23 according to the
method of Hamilton et al. (13). The resulting triple mutant,
MTD234 (metE metH ykfD), was tested for growth on
different supplements. Figure 1 demonstrates that the ykfD
lesion prevents growth on S-methylmethionine but not on
methionine. The control strain MTD23, in contrast, is able to utilize
S-methylmethionine. Immunoblotting analysis of cell lysates
from strain MTD234 grown in minimal medium supplemented with 20 µM
L-methionine confirmed that the yagD gene was
expressed (data not shown). Thus, the mutation in ykfD did not abolish YagD formation by any polarity effect. At
S-methylmethionine concentrations of higher than 50 µM
slow but significant growth of strain MTD234 could be observed, which
may indicate nonspecific transport of the compound via other systems
(data not shown).
Regulation.
The phenotypes of the ykfD and
yagD mutants described above indicate a physiological role
for the gene products in the acquisition of external
S-methylmethionine and in its conversion into methionine. This could imply that the two genes are subject to regulation by the
MetJ-S-adenosyl-methionine system. MetJ is a homodimer of
12-kDa promoters (35, 36). It binds to the MET box
(5'-AGACGTCT-3') in the upstream region of genes
(metA, metBL, metC, metF,
metJ, metR, and metE) whose products
are involved in methionine biosynthesis and acts as a transcriptional
repressor (3, 18, 28, 29). Between two and five of these MET
boxes are organized in tandem repeats. Because of cooperative
interactions between the repressor molecules bound, their number and
match with the consensus sequence determines the level of
repressibility. S-adenosylmethionine, which is synthesized
from methionine and adenosine triphosphate by the metK gene
product (21), acts as a corepressor (27, 33, 34)
and thereby mediates methionine regulation of the met regulon.
Upstream of yagD, no sequence similar to that of the MET box
motif can be found. In contrast, there are four of these motifs upstream of ykfD (Fig. 2). Depending on which of the two ATG
codons of the ykfD gene is functioning for the start of the
translation, the four MET boxes overlap or immediately precede the
start of the reading frame.
To analyze whether the ykfD yagD putative transcriptional
unit is indeed under control of methionine, cultures of E. coli KL19 (wild type) were grown in minimal medium supplemented
with different concentrations of L-methionine or
DL-S-methylmethionine. After 230 min of growth,
cells were harvested and analyzed for the formation of YagD protein by
immunoblotting (Fig. 3). It is evident
that the amount of YagD protein was strongly reduced when 40 µM
L-methionine or higher concentrations were present in the medium. Supplementation of S-methylmethionine only caused a
slight reduction in the amount of YagD, which might be due to its
intracellular conversion into methionine. Similar results were obtained
when synthesis of YagD was followed by measuring enzyme activity
(results not shown).
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FIG. 3.
Influence of methionine and
S-methylmethionine supplementation on the expression of
yagD. Minimal medium containing different concentrations of
L-methionine (A) or
DL-S-methylmethionine (B) was inoculated to an
OD600 of 0.05 with stationary cells (see legend to Fig. 1;
grown without L-methionine) of wild-type strain E. coli KL19. After 230 min of aerobic incubation at 37°C, the
cells were harvested and lysed. The proteins were separated on a 15%
polyacrylamide gel in the presence of sodium dodecyl sulfate
(19). The detection of YagD was performed by immunoblotting
analysis using anti-YagD antiserum (obtained from Eurogentech, Belgium,
by custom immunization of a rabbit with purified YagD) in a 1:4,000
dilution, protein A-horseradish peroxidase conjugate (Bio-Rad, Munich,
Germany), and chemiluminscence blotting substrate from Boehringer
(Mannheim, Germany).
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The results described here identify the function of two unassigned open
reading frames from E. coli. Their products are involved in
the uptake of S-methylmethionine and in the methyl transfer to homocysteine, rendering two molecules of methionine. Such an activity has been described for crude extracts from E. coli
cells by Balish and Shapiro (2).
S-methylmethionine is a compound synthesized and stored by
many plant species (15, 23), and expression of
ykfD and yagD enables E. coli to
utilize this compound. The abilities of other methionine auxotrophic
bacterial species to use S-methylmethionine as a source of
methionine (22) might be based on similar systems. In
agreement with this physiological role, ykfD and
yagD are subject to control of expression by methionine, thus constituting two more genes belonging to the methionine regulon. More detailed studies, however, are required to confirm that MetJ is
involved as a regulatory protein. On the basis of the results obtained
in this study we propose the new gene designation mmu (S-methylmethionine utilization). Accordingly, the gene
coding for the putative S-methylmethionine permease
(ykfD) is renamed mmuP, and the
S-methylmethionine:homocysteine methyltransferase gene
(yagD) is renamed mmuM.
This work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
| 1.
|
Abrahamson, L., and S. K. Shapiro.
1965.
The biosynthesis of methionine: partial purification and properties of homocysteine methyltransferase of jack bean meal.
Arch. Biochem. Biophys.
109:376-382.
|
| 2.
|
Balish, E., and S. K. Shapiro.
1966.
Methionine biosynthesis in Escherichia coli: induction and repression of methylmethionine (or adenosylmethionine): homocysteine methyltransferase.
Arch. Biochem. Biophys.
119:62-68.
|
| 3.
|
Belfaiza, J.,
C. Parsot,
A. Martel,
C. Bouthier de la Tour,
D. Margarita,
G. N. Cohen, and I. Saint-Girons.
1986.
Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region.
Proc. Natl. Acad. Sci. USA
83:867-871[Abstract/Free Full Text].
|
| 4.
|
Blattner, F. R.,
G. Plunkett III,
C. A. Bloch,
N. T. Perna,
V. Burland,
M. Riley,
J. Collado-Vides,
J. D. Glasner,
C. K. Rode,
G. F. Mayhew,
J. Gregor,
N. W. Davis,
H. A. Kirkpatrick,
M. A. Goeden,
D. J. Rose,
B. Mau, and Y. Shao.
1997.
The complete genome sequence of Escherichia coli K-12.
Science
277:1453-1474[Abstract/Free Full Text].
|
| 5.
|
Cooper, S.
1966.
Utilization of D-methionine by Escherichia coli.
J. Bacteriol.
92:328-332[Abstract/Free Full Text].
|
| 6.
|
Drummond, J. T.,
S. Huang,
R. M. Blumenthal, and R. G. Matthews.
1993.
Assignment of enzymatic function to specific protein regions of cobalamine-dependent methionine synthase from Escherichia coli.
Biochemistry
32:9290-9295[Medline].
|
| 7.
|
Ejiri, S.-I.,
H. Weissbach, and N. Brot.
1979.
Reduction of methionine sulfoxide to methionine by Escherichia coli.
J. Bacteriol.
139:161-164[Abstract/Free Full Text].
|
| 8.
|
Frasca, V.,
R. V. Banerjee,
W. R. Dunham,
R. H. Sands, and R. G. Matthews.
1988.
Cobalamine-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme.
Biochemistry
27:8458-8465[Medline].
|
| 9.
|
Gonzalez, J.,
K. Peariso,
J. E. Penner-Hahn, and R. G. Matthews.
1996.
Cobalamine-independent methionine synthase from E. coli: a zinc metalloenzyme.
Biochemistry
35:12228-12234[Medline].
|
| 10.
|
Goulding, C. W., and R. G. Matthews.
1997.
Cobalamine-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation.
Biochemistry
36:15749-15757[Medline].
|
| 11.
|
Goulding, C. W.,
D. Postigo, and R. G. Matthews.
1997.
Cobalamine-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolat, cobalamine, and adenosylmethionine.
Biochemistry
36:8082-8091[Medline].
|
| 12.
|
Greene, R. C.
1973.
Methionine limitation in Escherichia coli K-12 by growth on the sulfoxides of D-methionine.
J. Bacteriol.
116:230-234[Abstract/Free Full Text].
|
| 13.
|
Hamilton, C. M.,
M. Aldea,
B. K. Washburn,
P. Babitzke, and S. R. Kushner.
1989.
New method for generating deletions and gene replacements in Escherichia coli.
J. Bacteriol.
171:4617-4622[Abstract/Free Full Text].
|
| 14.
|
Holloway, C. T.,
R. C. Greene, and C.-H. Su.
1970.
Regulation of S-adenosylmethionine synthetase in Escherichia coli.
J. Bacteriol.
104:734-747[Abstract/Free Full Text].
|
| 15.
|
James, F.,
K. D. Nolte, and A. D. Hanson.
1995.
Purification and properties of S-adenosyl-L-methionine:L-methionine S-methyltransferase from Wollastonia biflora leaves.
J. Biol. Chem.
270:22344-22350[Abstract/Free Full Text].
|
| 16.
|
Kadner, R. J.
1974.
Transport systems for L-methionine in Escherichia coli.
J. Bacteriol.
117:232-241[Abstract/Free Full Text].
|
| 17.
|
Kadner, R. J., and W. J. Watson.
1974.
Methionine transport in Escherichia coli: physiological and genetic evidence for two uptake systems.
J. Bacteriol.
119:401-409[Abstract/Free Full Text].
|
| 18.
|
Kirby, T. W.,
B. R. Hindenach, and R. C. Greene.
1986.
Regulation of in vivo transcription of the Escherichia coli K-12 metJBLF gene cluster.
J. Bacteriol.
165:671-677[Abstract/Free Full Text].
|
| 19.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 20.
|
Low, B.
1968.
Formation of merodiploids in matings with a class of rec recipient strains of Escherichia coli K12.
Proc. Natl. Acad. Sci. USA
60:160-167[Free Full Text].
|
| 21.
|
Markham, G. D.,
E. W. Hafner,
C. White Tabor, and H. Tabor.
1980.
S-adenosylmethionine synthetase from Escherichia coli.
J. Biol. Chem.
255:9082-9092[Abstract/Free Full Text].
|
| 22.
|
McRorie, R.,
A. Margaret,
R. Glazener,
C. G. Skinner, and W. Shive.
1954.
Microbiological activity of the methylsulfonium derivative of methionine.
J. Biol. Chem.
211:489-497[Free Full Text].
|
| 23.
|
McRorie, R. A.,
G. L. Sutherland,
S. Margaret,
A. D. Barton,
Margaret R. Glazener, and W. Shive.
1954.
Isolation and identification of a naturally occurring analog of methionine.
J. Am. Chem. Soc.
76:115-118.
|
| 24.
|
Millian, N. S., and T. A. Garrow.
1998.
Human betaine-homocysteine methyltransferase is a zinc metalloenzyme.
Arch. Biochem. Biophys.
356:93-98[Medline].
|
| 25.
|
Neuhierl, B.,
M. Thanbichler, and A. Böck.
1998.
A family of S-methyl-methionine-dependent thiol/selenol methyltransferases: role in selenium tolerance and evolutionary relation.
(submitted)
|
| 26.
|
Peariso, K.,
C. W. Goulding,
S. Huang,
R. G. Matthews, and J. E. Penner-Hahn.
1998.
Characterization of the zinc binding site in methionine synthase enzymes of Escherichia coli: The role of zinc in the methylation of homocysteine.
J. Am. Chem. Soc.
120:8410-8416.
|
| 27.
|
Saint-Girons, I.,
J. Belfaiza,
Y. Guillou,
D. Perrin,
N. Guiso,
O. Barzu, and G. N. Cohen.
1986.
Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine.
J. Biol. Chem.
261:10936-10940[Abstract/Free Full Text].
|
| 28.
|
Saint-Girons, I.,
N. Duchange,
G. N. Cohen, and M. M. Zakin.
1984.
Structure and autoregulation of the metJ regulatory gene in Escherichia coli.
J. Biol. Chem.
259:14282-14285[Abstract/Free Full Text].
|
| 29.
|
Saint-Girons, I.,
N. Duchange,
M. M. Zakin,
I. Park,
D. Margarita,
P. Ferrara, and G. N. Cohen.
1983.
Nucleotide sequence of metF, the E. coli structural gene for 5-10 methylene tetrahydrofolate reductase and its control region.
Nucl. Acids Res.
11:6723-6732[Abstract/Free Full Text].
|
| 30.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 31.
|
Shapiro, S. K.,
A. Almenas, and J. F. Thomson.
1965.
Biosynthesis of methionine in Saccharomyces cerevisiae.
J. Biol. Chem.
240:2512-2518[Free Full Text].
|
| 32.
|
Shapiro, S. K.,
D. A. Yphantis, and A. Almenas.
1964.
Biosynthesis of methionine in Saccharomyces cerevisiae.
J. Biol. Chem.
239:1551-1556[Free Full Text].
|
| 33.
|
Shoeman, R.,
B. Redfield,
T. Coleman,
R. Greene,
A. Smith,
N. Brot, and H. Weissbach.
1985.
Regulation of methionine synthesis in E. coli: effect of the metJ gene product and S-adenosylmethionine on the expression of the metF gene.
Proc. Natl. Acad. Sci. USA
82:3601-3605[Abstract/Free Full Text].
|
| 34.
|
Shoeman, R.,
B. Redfield,
T. Coleman,
R. Greene,
A. Smith,
I. Saint-Girons,
N. Brot, and H. Weissbach.
1985.
Regulation of methionine synthesis in Escherichia coli: effect of the metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.
Biochem. Biophys. Res. Commun.
133:731-739[Medline].
|
| 35.
|
Smith, A. A., and R. C. Greene.
1984.
Cloning of the methionine regulatory gene, metJ, of Escherichia coli K12 and identification of its product.
J. Biol. Chem.
259:14279-14281[Abstract/Free Full Text].
|
| 36.
|
Smith, A. A.,
R. C. Greene,
T. W. Kirby, and B. R. Hindenach.
1985.
Isolation and characterization of the product of the methionine regulatory gene, metJ, of Escherichia coli K12.
Proc. Natl. Acad. Sci. USA
82:6104-6108[Abstract/Free Full Text].
|
| 37.
|
Whitfield, C. D.,
E. J. J. Steers, and H. Weissbach.
1970.
Purification and properties of 5-methyltetrahydropteroyltriglutamate-homocysteine transmethylase.
J. Biol. Chem.
245:390-401[Abstract/Free Full Text].
|
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