A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.     

Cloning and identification of methionine synthase gene from Pichia pastoris

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5‘ and 3‘ regions were further cloned by 5‘- and 3‘-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.     

Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid.

The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression. Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.     

Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy

X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.     

Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation

The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of L-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants. Of nine candidate ORFs (four of which were known previously), seven ORFs (cg0754 (metX), cg0755 (metY), cg1290 (metE), cg1702 (metH), cg2383 (metF), cg2536 (aecD), and cg2687 (metB)) were demonstrated to be part of the pathway while two others (cg0961 and cg3086) could be excluded. C. glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine. C. glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction. For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine beta-lyase in C. glutamicum, while Cg3086 can act neither as cystathionine gamma-synthase nor as cystathionine beta-lyase. Finally, MetE and MetH, which catalyze the conversion of L-homocysteine to L-methionine, could be newly identified, together with MetF which provides the necessary N(5)-methyltetrahydrofolate.     

Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions.

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.     

New Methionine Structural Gene in Salmonella typhimurium

Eight metH mutants in Salmonella typhimurium with closely linked sites of mutation which could grow only on methionine were isolated from a metE mutant deficient in N(5)-methyltetrahydropteroyltriglutamate-homocysteine transmethylase; their deficiency in cobalamin-dependent N(5)-methyltetrahydrofolate-homocysteine transmethylase was supported by the results of enzyme studies of one of them. Cotransduction of metH and metA (homoserine O-transsuccinylase) mutants was obtained, thus revealing linkage between a second pair of the six known methionine structural genes. One metH mutant clearly differed from the rest in that it reverted at a higher frequency, was temperature sensitive, complemented all other metH mutants, and was located farthest from the metA gene.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Salmonella typhimurium metE operator-constitutive mutations

We used a metE-lacZ fusion phage (lambda Elac) to select for mutants with operator-constitutive mutations in the Salmonella typhimurium metE control region. All of the mutations identified were found to lie within a region containing tandemly-repeating 8-bp palindromes with the consensus sequence 5'-AGACGTCT-3', previously proposed to be the binding region for the metJ-encoded repressor. Lysogens carrying mutant lambda Elac phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. Although repression of metE expression by vitamin B12 is not disrupted in metJ+ lysogens, vitamin B12 repression is disrupted in lysogens lacking an active MetJ repressor. These results suggest that there is an interaction between the metJ-encoded repressor and the vitamin B12 repression system mediated by the metH gene product.     

Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (βα)₈ barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)₃Zn site in the related enzymes, MetH and betaine–homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E·Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.     

The effect of homocysteine on MetR regulation of metE, metR and metH expression in vitro

An Escherichia coli S-30 DNA directed protein synthesis system was used to study the effect of homocysteine on the in vitro expression of the metE, metH and metR genes. In the presence of purified MetR protein, which is known to regulate the expression of these genes, homocysteine activates metE expression and inhibits both metR and metH expression. These findings support the recent in vivo results of Urbanowski, M.L. and Stauffer, G.V. (1989), J. Bacteriol. 171, 3277-3281.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Investigation of the regulation of the Escherichia coli btuB gene using operon fusions

Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor. Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated. Mutations in metH, metE and ompR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.     

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.     

Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response.

Using an Escherichia coli lac deletion strain lysogenized with a lambda phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression.