Biosynthesis of Methionine
Chapter
33
RONALD C. GREENE
Methionine was first discovered by Mueller (124) as a new sulfur- containing amino acid during a search for components of protein hydrolysates that would support the growth of streptococci. Shortly thereafter, Odake (130) described the isolation of methylthioadenosine from yeast cells, but its importance was not appreciated till many years later, when Cantoni discovered S-adenosylmethionine (25). Methionine is a relatively minor component of proteins that might not deserve special consideration if that were its only role. However, its other functions are central to the metabolism of the cell. N-Formylmethionyl-tRNAfMet is the initiator for peptide synthesis (2, 196), and S-adenosylmethionine, formed by reaction with ATP, is the donor of most methyl groups (25), the source of the propylamino group of spermidine (62), and, as recently discovered, the source of the cyclopentenediol group of the tRNA wobble base queuine (173, 174). Recent reviews of the biosynthesis and regulation of methionine in Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) have been written by workers at the Institut Pasteur and their colleagues (32, 132, 158); the earlier work is well covered in a review by Flavin (50).
As diagrammed in Fig. 1, the pathways of methionine biosynthesis and metabolism are branched, with both convergent and divergent segments. Methionine has been classified as a member of the aspartate family of amino acids since the atoms of its four-carbon chain are derived from aspartic acid. The first three reactions are shared with the biosynthetic pathways of several other metabolites, including lysine, threonine, and the branched-chain amino acids. The other reactions of the pathway, which convert homoserine and methylenetetrahydrofolate to methionine, are uniquely concerned with methionine synthesis. The loss of any or all of the enzymes that catalyze them merely creates a nutritional requirement for methionine. Methionyl-tRNA synthetase (MetRS) and methionine adenosyltransferase are essential to the cell, and complete loss of their activities is lethal.
The chemistry and enzymology of the reactions diagrammed in Fig. 1 will be individually considered with the exception of transmethylations, which will be described as they arise in other sections, and the synthesis of spermidine, which is described in chapter 25. In addition to these reactions, the available information on methionine transport systems will be discussed.
Finally, the organization, transcription, and control of expression of each of the met regulon genes will be described.
Homoserine is an intermediate in the synthesis of threonine and the branched-chain amino acids as well as in the synthesis of methionine. Aspartate semialdehyde is a lysine precursor. Thus, it is not surprising that there are several enzymes to catalyze these reactions. This part of the pathway is discussed in more detail by Patte in chapter 32. In short, E. coli K-12 has an aspartokinase encoded by lysC and two bifunctional enzymes with aspartokinase-homoserine dehydrogenase activity. Aspartokinase I-homoserine dehydrogenase I, the product of the thrA gene, accounts for most of the homoserine dehydrogenase activity in cells grown in minimal medium. The activity of this enzyme is inhibited by threonine. Aspartokinase II-homoserine dehydrogenase II, specified by metL, supplies a considerably smaller amount of activity but enough to allow a thrA mutant to grow on minimal medium. The enzyme is not subject to feedback inhibition. The metL coding sequence is downstream from that of metB in the metBL operon. The MetL protein from E. coli B lacks homoserine dehydrogenase activity. Thus, thrA mutants of this strain have a nutritional requirement for homoserine or threonine and methionine, which eased their isolation and characterization. Studies of metL were first done in a strain of E. coli K-12 which carried a thrA mutation that had originally been isolated in E. coli B (137).
Strangely, there is a single aspartate semialdehyde dehydrogenase, product of the asd gene, to catalyze the reduction of β-aspartyl-phosphate, the central reaction of the pathway.
The first reaction unique to the homocysteine synthetic pathway is O succinylation of homoserine catalyzed by homoserine transsuccinylase (EC 2.3.1.46), the product of the metA gene. Early studies by Rowbury (155) showed that ATP, coenzyme A, and succinate were required for conversion of homoserine and cysteine to cystathionine by extracts of a metC mutant. The reaction was clarified when Flavin and coworkers synthesized O-succinylhomoserine and showed it to be a substrate for the MetB enzyme (51, 85). The enzyme was assayed in various ways in the early work, but Savin et al. (161) found problems with all of them. They obtained much higher activities by a coupled assay in which the MetB enzyme rapidly converted O-succinylhomoserine to α-ketobutyrate. As might be expected for the catalyst of the first committed step of a pathway, the enzyme is inhibited by either S-adenosylmethionine or methionine and even more strongly by a mixture of the two (100, 161).
The metA gene of E. coli has been cloned, and its sequence has been determined (45, 117, 118, 204). Its product is predicted to be a 309-residue peptide of 35,673 Da (45), which correlates well with the size of a labeled peptide in minicells carrying a cloned metA gene (117). The enzyme has only been slightly purified (100, 153), and detailed structural and mechanistic studies have not been done.
When either E. coli or S. typhimurium is shifted from 37 to 44°C, a slowing of growth rate occurs which can be prevented by methionine supplementation (150, 152, 153). This phenomenon appears to result from temperature sensitivity of homoserine transsuccinylase. The inactivation of the enzyme is rapidly reversible in whole cells or in partially purified extracts (∼30-fold), but the mechanism has not been studied.
Displacement of the succinyl moiety of O-succinyl-l-homoserine by l-cysteine to yield l-cystathionine and succinate is catalyzed by cystathionine γ-synthase (EC 4.2.99.9), the product of the metB gene (85). Hydrogen sulfide or methyl mercaptan can serve as a nucleophile to yield homocysteine or methionine, respectively, as a product (52). These reactions are slow, but the direct synthesis of homocysteine from O-succinylhomoserine and hydrogen sulfide may explain the slow growth of metC mutants on methionine-free medium. In the absence of a suitable nucleophile, the enzyme catalyzes a gamma-elimination reaction to yield α-ketobutyrate, succinate, and ammonia. This reaction is often used to assay the enzyme. The enzyme reacts slowly with O-acetylhomoserine. Since cells are permeable to this compound, it can be used as a growth supplement for metA mutants.
Simon and Hong (172) isolated revertants of metC strains of E. coli that are able to grow on minimal medium in the absence of cystathionase activity. The products of the metA, metB, and metE or metH genes are required for growth of these strains, so the only step bypassed is the cleavage of cystathionine. The mutations affect a locus called metQ, which is remote from other known met genes (133). The nature of the mutations is unknown, but they may lead to accumulation of a sulfur source that can be used for direct synthesis of homocysteine or a compound that is readily converted to homocysteine.
Flavin and associates purified the enzyme from S. typhimurium (86, 87) and conducted numerous studies on its properties and reaction mechanism (reviewed by Flavin [50]). The metB gene of E. coli was cloned in φ80 by Press et al. (143) and in λ by Johnson et al. (78). The metJBLF gene clusters from these phage were subcloned into plasmids (206). Using DNA from the λ transducing phage as a template, Krueger et al. (95) demonstrated in vitro synthesis of cystathionine synthetase.
Since the presence of metJ interferes with the high-level expression of the other met genes carried on these plasmids, Holbrook et al. (74) constructed modified plasmids lacking that gene. A strain carrying one of these plasmids was used for preparation of large quantities of the enzyme. The enzymes from both organisms are tetramers of 4 × 104-Da subunits, in accord with the value of 41,503 Da calculated from the sequence of the E. coli metB gene (44). Each tetramer contains four molecules of tightly bound pyridoxal phosphate.
An abbreviated reaction scheme deduced from the mechanistic studies of Brzovic et al. (18) is shown in Fig. 2. This work shows a pyridoxamine derivative (compound I) to be the partitioning intermediate linking the two branches of the pathway, instead of compound III proposed by Johnston et al. (79).
Hydrogen exchanges catalyzed by the enzyme have been exploited by Homer et al. (76) for the preparation of a number of selectively deuterated amino acids. These compounds are useful in nuclear magnetic resonance studies of protein structure.
A straightforward β elimination that converts l-cystathionine to l-homocysteine, pyruvate, and ammonia is catalyzed by cystathionine β-lyase (cystathionase) (EC 4.4.1.8), the product of the metC gene. Although little work has been done on its mechanism, the reaction is similar to that catalyzed by many pyridoxal phosphate enzymes such as serine dehydrase (195).
The enzyme purified from E. coli has 1 mol of bound pyridoxal phosphate per subunit (48). The subunit molecular mass estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (8, 48) is close to the value of 43 × 103 Da predicted from the coding sequence (8). The sequence of the S. typhimurium gene predicts a peptide of the same size, with 54 mostly conservative replacements.
The molecular weight of the native enzyme from E. coli was estimated to be 2.8 × 105 by size exclusion chromatography (48). This result is consistent with a hexameric structure, but more recently, C. Bouthier de la Tour (as cited in reference 132) has reported that the E. coli enzyme is a tetramer.
In addition to cystathionine cleavage, cystathionase rapidly catalyzes β-elimination reactions of several other sulfur amino acids, including cystine, lanthionine, and djenkolic acid. An interesting aside is the recent observation (56) that the extracellular β-cystathionase of Bordetella avium functions as a potent cytotoxin for osteogenic cells, presumably by the formation of toxic sulfur compounds from the S-thiocysteine produced by the β-elimination reaction of cystine.
The metF gene product catalyzes reduction of N 5,N 10-methylenetetrahydrofolate to N 5-methyltetrahydrofolate (see chapter 36). The enzyme was first purified from E. coli by Katzen and Buchanan (88), who reported that reduced flavin adenine dinucleotide was the electron donor. Recently, C. Sheppard and R. Matthews (personal communication; chapter 36) have isolated the homogenous enzyme with bound flavin and found that it can be directly reduced with NADH. Katzen and Buchanan showed the equilibrium of the reaction with reduced flavin to be far in the direction of methyltetrahydrofolate synthesis (estimated K eq, about 3,400). Thus, in the low-potential environment of the cell, the reaction is functionally irreversible. If a high-potential electron acceptor such as menadione is present, the equilibrium is in the direction of methyltetrahydrofolate oxidation. While probably not important in vivo, the reverse reaction is convenient for enzyme assay, since it is easy to measure production of labeled formaldehyde from labeled methyltetrahydrofolate.
The metF genes from both E. coli and S. typhimurium have been cloned (78, 181, 206). The coding sequences of these genes (157, 181) predict peptides with subunit molecular masses of about 33 kDa, consistent with the value measured by SDS-polyacrylamide gel electrophoresis (157). The two peptides are almost identical, and both show substantial homology to a portion of the human enzyme and to yeast gene RAD1 (61).
Both E. coli and S. typhimurium have two enzymes that catalyze transfer of the methyl group from 5-methyltetrahydropteroryl glutamates to the sulfur of homocysteine to form methionine. These are the cobalamin-dependent methionine synthase (EC 2.1.1.13), the product of the metH gene, and the cobalamin-independent methionine synthase (EC 2.1.1.14), the product of the metE gene.
The existence of parallel enzymes explains the behavior of mutants described by Davis and Mingioli (40) that will grow on medium supplemented with either methionine or cobalamin. These strains have lesions in metE and can synthesize methionine if supplied with the cofactor for the metH gene product. S. typhimurium can synthesize cobalamin under anaerobic conditions (77, 154), but such activity has not been detected in E. coli. Strains with defects in metH have no growth requirement unless they also lack metE. The phenotype of doubly mutant strains is the same as that of metF mutants.
The cobalamin-dependent methionine synthase has many similarities to the enzyme of mammals, in which it is used to recycle homocysteine produced after methyl transfer. Early studies on the purification, properties, and reaction mechanism of this enzyme have been reviewed by Taylor and Weissbach (184). The metH genes of both E. coli and S. typhimurium have been cloned (6, 131, 189). The homogeneous enzyme has been prepared from an overproducing E. coli strain (6) and thoroughly studied (42). The reactivity of the enzyme is due to the uniquely potent nucleophilicity of cob(I)alamin. The synthesis of this form of the enzyme and the mechanism of the methyl transfer reaction are described in detail in chapter 36. The MetH enzymes can use methyltetrahydrofolate as well as its polyglutamyl derivatives.
Nucleotide sequences of the metH genes have been determined (6, 102, 131, 191). To resolve inconsistencies between the expected and observed compositions of fragments of the E. coli enzyme, Drummond et al. (43) reexamined the sequence and corrected several errors. The new results predict a 1,227-residue peptide. The molecular size of the native enzyme is about 136 kDa, near the expected size of the monomer. The long peptide chain appears to fold into several functional domains.
The cobalamin-independent methionine synthase (product of the metE gene) from E. coli was first purified and characterized by Whitfield et al. (198). The enzyme does not react with methyltetrahydropteroyl derivatives containing fewer than three glutamate residues. Except for stimulatory effects by magnesium, phosphate, and dithiothreitol, no other cofactors appear to be involved (see reference 184 for a review of the early work). The metE gene from E. coli has been cloned (110), its sequence has been determined (60, 110), and its product has been overproduced for quick, efficient purification (60). The metE reading frame specifies a 753-residue peptide with a calculated molecular weight of 84,654. This agrees well with molecular weight of the native enzyme measured by Whitfield et al. (198), and so the enzyme functions as a monomer. The metE gene of S. typhimurium has been cloned (168), but its sequence has not been determined.
The enzyme from E. coli has been well studied, but the mechanism of the transfer reaction is less clear than that of the MetH enzyme. Details about the MetE enzyme are also given in chapter 36. The enzyme is sluggish, with a catalytic turnover number less than 1/100 that of the cobalamin-dependent methionine synthase. Because of its low activity, a large amount of enzyme is required to carry out sufficient methionine synthesis. The enzyme accounts for several percent of the total soluble protein in cells grown on minimal medium.
To fulfill many of its essential roles, methionine must first be converted to S-adenosylmethionine. The synthetic reaction involves condensation of methionine and ATP, with the concomitant production of PPi and Pi, catalyzed by methionine adenosyltransferase. The major methionine adenosyltransferase is the product of the metK gene.
The metK gene from E. coli has been cloned (14). An enzyme-overproducing strain carrying a metK plasmid was used as the source for preparation of homogeneous E. coli methionine adenosyltransferase (108). The native enzyme (molecular mass of 180 kDa) is a tetramer of 43-kDa subunits, which correlates well with the value of 42 kDa calculated from the metK coding sequence (reference 107, as corrected by G. D. Markham [personal commununication]). The enzyme (108) has an absolute requirement for a divalent cation, usually magnesium, but manganous and cobaltous ions are also active. The rate of the reaction is strongly stimulated by several monovalent cations. Potassium and ammonium are the best, but thallium works well and binds more tightly to the enzyme. The enzyme will also react with l-selenomethionine to produce the corresponding adenosyl derivative.
Studies by Markham and colleagues (101, 104, 105, 106, 108, 208) using imidophosphate derivatives of ATP and tightly binding cations (thallous, manganous, vanadyl) led to proposal of the following mechanism. One ATP, one methionine, one monovalent cation, and two divalent cations are bound by each subunit, followed by reaction to form one S-adenosylmethionine and one tripolyphosphate. The binding energies of S-adenosylmethionine and tripolyphosphate are large enough to pull the equilibrium far in the direction of the products. Hydrolysis of the bound polyphosphate is necessary for a reasonable rate of product release.
Although many of the methionine analog-resistant metK mutants have very low levels of methionine adenosyltransferase (as little as 1 to 3% of the derepressed level), none have complete (149).loss of activity and none show severe depletion of S-adenosylmethionine (64, 66). The most deficient of these E. coli metK strains grown on LB medium were found to have higher methionine adenosyltransferase activities than cells of the same strains grown on minimal medium (64). Satishchandran et al. (159) used different selection conditions to isolate temperature-sensitive metK mutants with extremely low methionine adenosyltransferase activity at the nonpermissive temperature. When cells growing on minimal medium are switched to the nonpermissive temperature, enzyme activities fall to very low levels, the pools of S-adenosylmethionine are depleted, and growth stops. In contrast, mutant cells grown on LB medium are not markedly different from wild type at either temperature. They present evidence for the existence of a second adenosylmethionine synthetase encoded by the metX gene. The product of the metK gene is postulated to be produced in cells grown on minimal medium, while that of the metX gene is supposedly found only in cells grown on rich medium. In a more recent report, Satishchandran et al. (160) describe attempts to map the metX locus by hybridization and by study of kanamycin insertion mutations. Unfortunately, the insertion mutations were unstable and could not be well characterized. The sequence of the E. coli chromosome in the vicinity of metK has been determined (see chapter 109), the position tentatively assigned to metX is occupied by galP (149). Further work is required to locate metX and to characterize its product.
S-Adenosylhomocysteine remains after transfer of the methyl group, and methylthioadenosine is produced during transfer or elimination of the side chain. These compounds must be further metabolized.
E. coli andS. typhimurium differ from many other organisms in that they lack S-adenosylhomocysteine hydrolase. Work by Duerre (46) and Duerre and Walker (47) has shown that degradation of S-adenosylhomocysteine is initiated by cleavage of the nucleoside linkage to yield adenine and S-ribosylhomocysteine. As shown in Fig. 1B, the S-ribosylhomocysteine is then degraded to homocysteine and a carbonyl compound reported to be 4,5-dihydroxy-2,3-pentanedione (47, 121). The mechanism of the reaction was not studied, but it is probably similar to that of S-adenosylhomocysteinase from beef liver (134). The genes that specify these enzymes have not been identified.
The metabolism of methylthioadenosine has been studied in many organisms (see the review by Schlenk [163]). In most bacteria, including E. coli and S. typhimurium, the first step in methylthioadenosine metabolism is hydrolytic, yielding 5-methylthioribose (MTR) catalyzed by the same enzyme that cleaves adenosylhomocysteine (47). The further metabolism of MTR is not well known in these species, but its conversion to methionine in the related organism Klebsiella pneumoniae has been thoroughly studied.
The first step in conversion of MTR to methionine by K. pneumoniae is phosphorylation to yield 5-methylthioribose-α1-phosphate (58, 126). An isomerase then catalyzes the formation of 5-methylthioribulose-1-phosphate, which is then enzymatically dehydrated to give 2,3-diketo-5-methylthio-1-phosphopentane (55). The diketone is acted upon by an enzyme designated E-1 (enolase-phosphatase) by Myers et al. (127) to form a keto enolate anion. In rat liver, an enzyme (E-3) (200) catalyzes the reaction of this compound with oxygen to yield formate and α-keto-γ-methiobutyrate, but no such enzyme has been found in K. pneumoniae, in which the reaction appears to be spontaneous. Methionine can be formed by transamination of the keto acid. Wray and Abeles (199) have purified from K. pneumoniae an enzyme, called E-2, that catalyzes oxidation of the keto enolate to produce β-methiopropionate, formate, and carbon monoxide. The importance of this last reaction in the metabolism of MTR is not clear, but E-2 has been shown to be present in extracts of E. coli JM109.
A number of observations suggest that an MTR recycling pathway is either missing or present at a very low level in E. coli. Davis and Mingioli (40) found that methionine auxotrophs (later shown to be metE) of E. coli W could not use methylthioadenosine as a growth supplement. Schroeder et al. (165, 166) showed that E. coli B accumulates significant amounts of MTR (as much as 0.7 nmol/109 cells) during growth on minimal medium. They also state that after 2 h of incubation with E. coli cells, about 40% of added 35S-labeled MTR was converted to other, unspecified sulfur compounds, indicating slow metabolism.
In their studies on the metabolism of MTR in K. pneumoniae and other bacteria, Gianotti et al. showed that 5-trifluoromethylthioribose is highly toxic to cells that have the MTR recycling pathway (58). The 50% inhibitory concentration for K. pneumoniae is about 40 nM. They propose that the pathway converts the trifluoromethyl derivative to one or more toxic compounds. These workers have screened numerous bacterial species for MTR kinase and find that its presence correlates well with the ability to use MTR as a nutritional source of methionine and with sensitivity to 5-trifluoromethylthioribose. Among the strains examined were 10 clinical isolates of E. coli and strain LE392. None of these have demonstrable MTR kinase, and all are relatively resistant to 5-trifluoromethylthioribose, with 50% inhibitory concentrations about 100 times greater than that observed for K. pneumoniae (M. Riscoe, personal communication). Thus, it would appear that E. coli has little if any capacity to convert MTR back to methionine, possibly because of a deficiency of MTR kinase. However, the residual toxicity of the trifluoromethylthioribose may indicate a low level of recycling.
Since methionine plays a dual role in protein synthesis, there are two types of methionine tRNAs, tRNAfMet, which is converted to N-formylmethionyl-tRNAfMet for initiation of peptide synthesis, and tRNAMet, for incorporation of methionine into a growing peptide chain. Methionine is coupled to either of these types of tRNA by a single MetRS, the product of the metG gene. The structure and mechanism of the E. coli enzyme have been extensively studied. Much of this work is described in recent reviews (113, 145, 162, 167).
A series of S. typhimurium methionine auxotrophs, classified as metG (177), were first shown by Gross and Rowbury (68) to have modified MetRSs. Blumenthal (10) described similar mutants of E. coli. Some metG strains, i.e., those that require a high concentration of methionine for adequate functioning of defective MetRSs, are useful for the isolation of methionine regulatory mutants. Secondary mutations that cause overproduction of methionine can suppress the growth requirement of such strains (28).
The E. coli metG gene has been cloned, and its sequence has been determined (7, 36, 38). The product of the cloned gene was shown to be a 76-kDa subunit by SDS-gel electrophoresis, in good agreement with the size of the 676-residue peptide predicted by the sequence. The native protein is a dimer (94). An active 551-residue monomer, obtained by limited proteolysis (27), has been crystallized, and its structure has been determined (17, 207). A phagemid vector (57) with a modified metG gene specifies a truncated product (547-residue amino-terminal portion) which is a stable, active monomer (116). Such clones have been used to produce many mutant derivatives of MetRS.
MetRS is a class I aminoacyl-tRNA synthetase. Members of this class have highly homologous amino terminal domains and more variable carboxy-terminal domains. The N-terminal domain (∼360 amino acids) has β strands and α helices organized into a nucleotide fold. This domain has the ATP binding site (17) as well as the catalytic activity for synthesis of methionyl-adenylate and transfer of the methionyl residue to the 3' end of tRNA. The C-terminal domain of the truncated enzyme (∼187 residues) is a cluster of α-helical segments interspersed with loops. The final carboxy-terminal region (from F-527 to K-547) folds back to associate with the amino-terminal domain and form part of the catalytic site.
In contrast to many other aminoacyl-tRNA synthetases, interactions with the anticodon sequence are predominant in tRNA selectivity by MetRS. Without crystal structures, the details of the complexes of the enzyme with tRNAs are unknown, but numerous experiments including specific mutagenesis have yielded information about the most important interactions (57, 90, 91, 92, 109, 112, 114, 115, 164, 167). Several residues (especially W-461) located near the end of a cluster of α helices in the carboxy- terminal domain are important for anticodon recognition.
Each subunit of E. coli MetRS contains one molecule of zinc tightly bound to a four-cysteine cluster in the amino-terminal domain near the active site (53, 54, 97, 203). The zinc atom is required for activity, but its exact function has not been established.
There is relatively little information about regulation of metG expression. Cassio (26) had previously observed that in vivo inhibition of MetRS synthetase caused as much as a threefold increase in the rate of metG expression. Dardel et al. (38) observed as much as twofold differences in expression of a fused metG-lacZ coding sequence under different growth conditions. The lowest levels of expression were seen in cells that overproduced a functional MetRS, conditions that would be expected to reduce the fraction of unacylated tRNA. These workers (37, 38) found a divergently transcribed gene (mrp) 132 nucleotides upstream from metG. In addition to promoters in the intergenic region, there is another promoter within mrp where transcription is initiated in the direction of metG. Most of the transcription from this promoter is terminated in the upstream part of the intergenic sequence, but some reads through metG. It is not clear whether this second promoter is involved in regulation of metG expression, perhaps by some regulatory system that can alter the frequency of readthrough.
Methionine is taken up by cells of E. coli or S. typhimurium by a high-affinity transport system (Km of ∼ 0.1 mM) and one or more low-affinity systems (Km of ∼20 to 40 mM) (4, 5, 80, 83). The locus for the high-affinity transport system of E. coli was called metD, since mutants deficient in this system are unable to utilize d-methionine (80). The comparable locus of S. typhimurium had been designated metP by Ayling and Bridgeland (4), leading to confusion since Kadner (80) used metP for the low-affinity transport system of E. coli. Ayling and coworkers (69, 169) have recently accepted the E. coli nomenclature for use in S. typhimurium.
Ayling and coworkers (33, 69) have constructed a fine-structure map of the S. typhimurium metD locus. They have found evidence for the existence of four or more complementation groups (69, 169). If this result is indicative of the presence of at least four coding sequences, the high-affinity methionine transport system is probably a multiprotein complex. Results of Kadner and Winkler (84) suggest that ATP is the primary energy source for methionine transport in E. coli. These observations are consistent with the hypothesis that the high-affinity methionine uptake system is an ABC transporter, but compelling evidence is lacking.
The metD system also functions as a low-affinity glutamine transporter in S. typhimurium (P. D. Ayling, personal communication).
Kadner (80, 81, 82) has shown that growth conditions can cause severalfold changes in the amount and activity of the high-affinity transport system of E. coli. However, except for the observation that metD expression is not directly controlled by the metJ system, little is known about the regulatory mechanisms.
Except for the observation that methionine transport still occurs in metD mutants (5, 83), little is known about the low-affinity system(s). Part of such uptake appears to result from methionine transport by a leucine transporter (5). Kadner and Watson (83) described an E. coli strain deficient in both the high- and low-affinity methionine transporters. This strain was shown to be metD, but its other mutations were not well characterized.
The ability of E. coli to use methionine analogs has practical value. Of particular current interest is the replacement of methionine residues in proteins with selenomethionine in tolerant strains (30, 35). Under appropriate conditions, nearly 100% of the methionine residues of a protein can be replaced with selenomethionine. Hendrickson et al. (71) have described a method for solving the crystallographic phase problem by comparing the multiwavelength anomalous dispersion of selenomethionyl protein crystals with those of methionyl protein crystals. This procedure is proving to be very useful in the determination of protein structures. Preliminary results by Boles et al. (12) suggest that telluromethionine replacement can be achieved, which would give an even stronger signal.
Norleucine can efficiently replace methionine in protein synthesis in E. coli (89). This replacement can be a desired goal or an unexpected nuisance. A number of proteins exhibit full activity when their methionine residues have been completely replaced with norleucine (3, 59, 128). Such modified proteins may be more stable to oxidative degradation than their normal counterparts. Conversely, if synthesis of a protein is induced in minimal medium, the supply of methionine may become limiting and endogenous norleucine (synthesized by the branched-chain amino acid pathway) may replace a significant fraction of the methionine residues (11, 146). Such replacement may cause problems such as giving spurious peaks in nuclear magnetic resonance spectra.
E. coli and S. typhimurium can use a number of methionine derivatives for growth. These include N-acyl derivatives such as acetyl and formyl but not benzoyl, methionine methyl ester, small methionine peptides, α-keto-γ-methiobutyrate, and methionine sulfoxide, but not methionine sulfone (49). Utilization of d-methionine or d-methionine sulfoxide can be rate limiting in methionine auxotrophs, allowing prolonged methionine-limited growth (41, 63) without a chemostat.
Except for the feedback inhibition of homoserine transsuccinylase (metA gene product) by methionine and S-adenosylmethionine, control of methionine biosynthesis is accomplished by regulating the quantities of the biosynthetic enzymes at the level of transcription. Cohen and Jacob (31) first described a mutant strain of E. coli in which methionine synthesis was not repressible. Later, Lawrence et al. (99) isolated mutants of S. typhimurium that are resistant to growth inhibition by several analogs of methionine. These strains had mutations at one of three loci, metI, metJ, or metK. The metI mutations were subsequently found to be in metA, yielding an enzyme that had lost sensitivity to feedback inhibition (29).
The metJ and metK mutants had elevated levels of methionine biosynthetic enzymes and were defective in repression (99). Mutations at the metJ and metK loci of E. coli were isolated during studies on the regulation of S-adenosylmethionine synthetase (64, 66, 75, 183). Results from both strains were consistent with the hypothesis that metK is the structural gene for S-adenosylmethionine synthetase and that metJ codes for an aporepressor (64, 66, 73, 161).
The peptide specified by the metJ gene product was identified (175), and the protein was purified to homogeneity from strains carrying a metJB plasmid (176). The sequences of the metJ genes of E. coli and S. typhimurium predict peptides of 104 residues (156, 187), which agrees with the measured subunit molecular mass of 12 kDa. The protein is a 24-kDa dimer in solution (176).
The pure MetJ protein was shown to protect a promoter- containing region between the metB and metJ genes from cleavage by DNase I (93, 176). Shoeman et al. (170, 171) showed that pure MetJ protein inhibited in vitro transcription of the metB, metF, metJ, and metL genes and that S-adenosylmethionine enhanced the repression. Belfaiza et al. (8) noticed the presence of tandemly repeated octameric sequences at the 5' ends of four met genes (including the region of the metB-metJ footprint) and suggested that they are the binding sites for MetJ repressor. Subsequent results (see below) have supported this conclusion. Table 1 shows the octameric repeats of most members of the met regulon. The consensus sequence 5'-AGACGTCT-3' is called the MET box. Old et al. (132) showed that each of the 12 possible symmetrical mutations of a tandem pair of MET boxes in a 32-bp nucleotide causes a reduction in the affinity for MetJ protein.
Table 1Sequences of contiguous octameric repeats near the transcription start sites of met regulon genesa |
Because of cooperative interactions between the bound repressor molecules, the number of adjacent octamers is an important parameter in determining the affinity of the MetJ protein for a given operator. Highly repressible transcription units (e.g., metB and metF) have more repeats than a poorly repressible one (metC) (64, 75, 96).
The three-dimensional structures of the MetJ repressor protein and its complexes have been solved at 2.8-Å (1 Å = 0.1 nm) resolution in the laboratory of Phillips (144, 179). This work has been described in several reviews (132, 138, 139, 140, 180), which should be consulted for more detail about the structures. Each subunit of the dimeric repressor has three helical segments. Near the amino termini, residues 20 through 28 of each subunit are intertwined to form an antiparallel two-stranded β ribbon. One molecule of S-adenosylmethionine binds to each subunit with displacement of the side chain of a phenylalanine residue and some movement of flexible loops, but otherwise the binding has little effect on the structure.
The crystal structure of a repressor-adenosylmethionine complex bound to a 19-bp oligonucleotide containing two adjacent MET boxes (5'-TTAGACGTCTAGACGTCTA-3') shows two molecules of bound repressor dimer, one centered over each MET box. A diagram of the entire complex is shown in Fig. 3. Figure 4 shows a cutaway diagram of residues 10 through 58 of a repressor dimer bound to a single MET box, to give a better view of the position of the β ribbon in the major groove.
The repressor dimer is positioned on the DNA double helix by interactions of several residues with phosphate groups of the nucleotide backbone. The β ribbon is inserted into the major groove where the ε amino groups of lysines 23 and 23' and the hydroxyl groups of threonines 25 and 25' (of the two strands) make specific hydrogen bonds with bases. This structure has been called a ribbon-helix-helix DNA binding motif by Phillips (138) or the (βαα)2 fold (147). The DNA double helix is bent about 25° at the center of each MET box, which improves the contacts between functional groups of the protein and the DNA. Protein-protein interactions make a substantial contribution to the binding energy of the met repressor to the operator sequences. As shown in Fig. 3, adjacent bound repressor dimers make contacts primarily through the A helices. Hydrophobic interactions and a network of water-mediated hydrogen bonds connect the two dimers.
He et al. (70) examined the effects of a number of single amino acid substitutions on the affinity of MetJ repressor for a 16-residue oligonucleotide consisting of two MET boxes, as judged by a gel shift assay. The authors concluded that the structure of the complex in the crystal is a reasonable representation of its structure in solution.
Only a few proteins are known to use the ribbon-helix-helix DNA binding motif. In addition to MetJ, the best studied are the Arc and Mnt repressors that control the expression of ant in bacteriophage P22 (147). The structure of the Arc repressor and of a 79-residue fragment of the Mnt repressor in solution has been determined by nuclear magnetic resonance spectroscopy (15, 19, 20), and the crystallographic structure of two Arc dimers complexed with a 21-bp operator has been solved at 2.6-Å resolution (148). The subunits of these proteins range in size from 53 (Arc) to 104 (MetJ) residues, but the positions of the atoms of the peptide backbones of the DNA binding elements can be superimposed with relatively small deviation between the different molecules. The effects of numerous mutations on the stability and function of the Arc repressor have been studied by Sauer and colleagues (16, 119, 120). Examination of sequence homology and secondary structure predictions suggest that the TraY proteins of F and related episomes (13, 129) and auxin-induced gene products of pea (1) have ribbon-helix-helix structures, but detailed structural information is lacking.
Although metJ mutants show increased amounts of most of the met regulon gene products, there were several indications that other regulatory elements were at play, such as vitamin B12 effects that could not be easily explained by a simple MetJ repressor system (67, 96). The discovery of the metR gene by Urbanowski et al. (194) provided an explanation of some of these phenomena. metR mutants grow rapidly when fed methionine but only very slowly on medium supplemented with vitamin B12. The product of the gene is required for a useful level of metE expression, and it stimulates the expression of metH.
The metR genes of S. typhimurium and E. coli have been cloned (110, 194), and their sequences have been determined (111, 141). The Salmonella coding sequence predicts a 276-residue polypeptide (molecular mass of 31 kDa), whereas that of E. coli predicts one of 317 amino acids (molecular mass of 35.6 kDa). Both proteins have been purified (21, 111), and both have a subunit molecular mass of about 34 kDa as estimated by SDS-polyacrylamide gel electrophoresis. Thus, it seems likely that the two proteins are the same size and that there is an error in one of the sequences. Maxon et al. (111) reported that the native protein from E. coli has a molecular mass of 68 kDa and propose that it is a dimer. They propose that a leucine zipper motif functions in the dimerization.
The MetR proteins are members of a large set of bacterial activator proteins known as the LysR family (72). The proteins of this family have helix-turn-helix DNA binding domains and appear to function by binding to specific sequences near promoters and stimulating transcription. The MetR protein binds to DNA near the promoters of metE and metH and stimulates transcription (102, 110, 191, 192, 194), but in addition, it has effects on the expression of several other genes. The MetR protein represses its own transcription (110, 190), stimulates the expression of glyA (142) and of metA (103), and counters the MetJ repression of metF (34). DNase I footprints of MetR binding and the effects of several mutations on the response to MetR protein have identified TGAANN(T/A)NNTTCA as the consensus MetR target sequence.
Derepression of metA in metJ strains was first shown in S. typhimurium (98). Indications of the involvement of factors other than simple metJ S-adenosylmethionine system were seen in the anomalous behavior of a metE strain grown in a chemostat (161). Reasonable levels of metA expression were obtained when vitamin B12 was the limiting growth supplement, while little derepression was seen when the limiting nutrient was methionine. This behavior may result from a more recently discovered effect of homocysteine on metA expression (103). A metA-lacZ fusion was shown to require a functional metR gene for full expression. The MetR-mediated activation appears to be reversed by homocysteine, which would be expected to accumulate during methionine-limited growth of a metE strain. The homocysteine effect on metA expression should function to coordinate the activities of the two branches of the synthetic pathway.
The metA gene from E. coli has been cloned (118, 204), and its sequence has been determined (45, 117). The sequence of 69 codons and 464 5'-flanking bases of the S. typhimurium metA gene has been determined (103). Figure 5 shows the first two codons and the 5' regulatory sequences of these genes. Michaeli et al. (117) detected two transcripts of this region in E. coli. The positions of the origins of transcription are shown by the small dots above the sequence, and the associated promoters are overlined. The P1 transcript, which is present in much higher quantity than P2, is controlled by the metJ regulatory system. A series of four MET boxes beginning one base after the –10 sequence of P1 are marked with vertical lines. In S. typhimurium, Mares et al. (103) could detect only the transcript corresponding to the one initiated at P1 even though sequences similar to the P2 promoter are present. The –35 and –10 sequences of the major promoters are identical in the two strains, and there is only one base difference between the MET boxes. The region of S. typhimurium DNA protected from DNase I digestion by bound MetR protein is overlined. A MetR-binding sequence within the protected region is marked by asterisks beneath the bases. The E. coli DNA has not been tested for MetR binding, but the recognition sequence is present.
Ron and associates (9, 151) have shown a marked increase in transcription of metA when E. coli is exposed to elevated temperatures. Transcription stimulation does not occur in rpoH mutants, indicating that metA is a heat shock gene. The heat shock transcripts begin at the normal P1 initiation site, but the promoter region has only partial homology to the σ 32 consensus sequence.
The metB and metL genes comprise the only known operon of the met regulon (65). The sequence of the entire operon of E. coli has been determined (44, 205); only two bases (AA) separate the terminal TAA of metB and the initiating ATG of metL. The sequences of the metJ genes of E. coli (156) and S. typhimurium (187) have been determined. The coding sequences are divergently transcribed on either side of an intergenic region, 276 bp long in E. coli and 264 bp long in S. typhimurium. These regions contain one promoter for transcription of metBL and three promoters for transcription of metJ (93, 186, 188).
In vivo expression of metB, metL, and metJ was shown to be subject to control by the metJ regulatory system (75, 99, 137, 156). The in vitro expression of the metB, metL, and metJ genes was shown to be repressible by the MetJ protein and adenosylmethionine (171). The extended MetJ-binding site (176) contains five MET boxes marked by the vertical lines in Fig. 6. The cluster of MET boxes overlaps the –35 sequence of the metBL promoter and both the –35 and –10 sequences of the first metJ promoter. The MET box region ends a few bases from the –35 region of the second metJ promoter, but since under strongly repressing conditions MetJ binding extends out from the cluster of MET boxes (T. W. Kirby, B. R. Hindenach, and R. C. Greene, unpublished data), this sequence may sometimes be covered. Transcription from all of these promoters is subject to met repression, whereas that from the third metJ promoter, the weakest of them all, is not repressible (93). Mutations that allow partially constitutive β-galactosidase synthesis from a fused metB-lacZ coding sequence are shown in Fig. 6B (186). They all lie in MET boxes closest to the metB promoter, verifying the participation of these sequences in the regulatory process.
The sequences of the promoters and the MetJ target sequences of the metC genes of E. coli and S. typhimurium are shown in Fig. 7 (8, 135). Each sequence has only a pair of MET boxes located immediately downstream from the –10 sequences of the promoters and overlapping the start site of the transcript. The small number of MET boxes may account for the relatively poor repressibility of the metC gene, usually between 4- and 20-fold in the different strains. Park and Stauffer (135) studied regulation by measuring β-galactosidase synthesized from a metC-lacZ fusion gene. The base changes of two cis dominant mutations that produce elevated levels of enzyme in a strain with two functional copies of metJ (136) are shown below the sequence in Fig. 7B. Although both of these mutants show reduced repression by methionine, their primary effects are elevation of gene expression under nonrepressing conditions (e.g., in a metJ strain), suggesting that some other phenomenon may be involved. The wild type and one of the mutant constructs show almost a twofold difference in β-galactosidase activity when grown on an adequate or limiting methionine source in a metJ strain. These results are similar to those of Greene et al. (67), who showed that metC expression in metJ strains was slightly increased by vitamin B12 supplementation and decreased by methionine supplementation, with a total difference of about threefold between cells grown under the two conditions. Cells of a metJ metF double mutant had the lower enzyme activity regardless of the presence of vitamin B12 or of methionine. This behavior is suggestive of a role for homocysteine in lowering the expression of metC and is similar to observations of Stauffer and colleagues for MetR-mediated regulation (e.g., metA). However, metC and its flanking regions lack a MetR recognition sequence, so an explanation of these effects remains to be found.
The sequences of the metF genes of both E. coli and S. typhimurium have been determined (44, 157, 181). The regulatory regions of these two genes are shown in Fig. 8. The start sites of the transcripts are indicated by dots over the sequences, and the proposed –35 and –10 promoter sequences are overlined. Expression of metF is known to be regulated by the metJ system (96, 170). The MetJ-binding site, located by DNase I footprinting (39), contains five MET boxes delineated by the vertical lines in Fig. 8A. Davidson and Saint-Girons (39) constructed a large number of derivatives with mutations in the MET boxes. Substitutions that reduce operator function by 50% or more are indicated by letters below the sequence. Three single-base insertions and a 7-bp deletion also markedly reduced operator function (not shown). Four mutations that reduce repressibility of a metF-lacZ fusion in S. typhimurium are shown in Fig. 8B (182). These substitutions lie within the MET box sequences.
More recently, Cowan et al. (34) have studied the effect of the metR system on metF expression. The results suggest that the MetR protein reduces repression by the MetJ protein. Three MetR-binding sites, located by DNase I footprinting, are marked by asterisks above the consensus sequences in Fig. 8B. The furthest upstream site has a markedly higher affinity for MetR protein than the other two. Four base changes that eliminate the effect of MetR on metF expression are shown below the consensus sequences. Although the interaction of MetR with the E. coli metF gene has not been investigated, similar potential MetR binding sequences are present (also marked with asterisks above the sequence).
Like most members of the met regulon, metE is subject to repression by the metJ system (96, 110, 202). In contrast, its response to the metR system is unique. Transcription of metE has a nearly absolute requirement for the MetR protein, and so metR mutants require methionine for growth (23, 192, 194). Homocysteine is required for the full stimulation of metE expression by the metR gene product (24, 110, 193), helping to coordinate the activities of the two branches of the methionine biosynthetic pathway. MetJ repression is the primary control of metR expression, but the MetR protein also slightly represses transcription (23, 110, 190). Thus, the MetJ repressor controls expression of metE both directly and indirectly by repressing synthesis of the MetR activator protein.
The metE and metR genes of both E. coli and S. typhimurium are divergently transcribed from overlapping promoters with overlapping control elements. The sequence of the 236-base intergenic region from E. coli (110) and that of the 248-base region from S. typhimurium (141) are shown in Fig. 9. The transcriptional start sites are indicated by dots above or below the sequences (23, 141). The –10 and –35 sequences of the promoters associated with these initiation sites are overlined or underlined. Sets of MET boxes (shown by vertical lines in Fig. 9), which overlap the promoters or transcription start sites of both genes, were located by DNase footprinting of MetJ complexes in E. coli (23) and from the sites of mutations causing reduced sensitivity to metJ repression in S. typhimurium (202). The function of the second MetJ-binding site found by footprinting in E. coli is not known. The MetR-binding sites were demonstrated in both organisms by footprinting of protein-DNA complexes (23, 202). The consensus MetR target sequences are marked by asterisks over the bases.
Vitamin B12 has been shown to cause repression of metE that is not entirely due to the metJ regulatory system (22, 67, 96, 122). A functional metH gene and N 5-methyltetrahydropteroyl glutamates are required for the repression (22, 96). Weissbach and Brot postulated that the methylated form of the holo-cobalamin-dependent methionine synthase acts as a repressor of metE (reviewed in reference 197), but this hypothesis is controversial. Wu et al. (201) report that homocysteine supplementation can overcome vitamin B12 repression of a metE-lacZ fusion and that overproduction of the cobalamin-independent methionine synthase can repress expression of the fusion even in the absence of vitamin B12. They interpret these results to mean that vitamin B12-mediated repression results from decrease of the intracellular homocysteine pool by activation of the cobalamin-dependent methionine synthase. In contrast, Cai et al. (22) report that vitamin B12 supplementation can cause ∼95% repression of the single chromosomal metE gene while causing only a small reduction in the intracellular homocysteine pool. They question whether the minor change in homocysteine concentration is sufficient to cause a major reduction in metE expression. This question remains unresolved, but there is no direct evidence for interaction of the purified MetH protein with metE DNA (197).
Transfer of E. coli cells from anaerobic to aerobic conditions causes a rapid increase in metE expression, and transfer from aerobic to anaerobic conditions causes a rapid decrease in expression (178). The mechanism of these rapid changes in the rate of synthesis of the cobalamin-independent methyltransferase is unknown, but since this enzyme may represent 5% of the soluble cellular protein, its synthesis requires a considerable energy investment.
Unlike the genes for the other methionine biosynthetic enzymes, metH does not appear to be directly regulated by the metJ system, and no recognizable MET boxes can be found in the 5'-flanking sequence of either the E. coli or S. typhimurium gene. Efficient metH expression requires the product of the metR gene, but the basal expression in the absence of MetR protein is higher than that of metE (23, 194). Thus, although metR mutants have a reduced level of cobalamin-dependent methionine synthase, enough is present to allow slow growth in the presence of vitamin B12. The 5'-flanking sequences of the E. coli and Salmonella metH genes, the start sites for their transcription, and the proposed promoter sequences are shown in Fig. 10 (102, 191). The MetR target sequences are marked by asterisks above the bases (21, 102). Divergently transcribed iclR genes lie upstream from the metH genes in both organisms. Although the coding sequences of these genes are highly homologous, the intergenic regions are very different, with the S. typhimurium sequence being about 100 bases longer than that of E. coli.
Byerly et al. (21) measured the effects of several single-base substitutions in the MetR recognition sequence (shown below the sequence) on the expression of a metH-lacZ fusion and on the in vitro binding of MetR protein. All of the mutations caused a decrease in β-galactosidase synthesis. Even the conversion of CTCA to the same sequence found in metE (TTCA), which would be expected to increase the activation, caused about a 30% decrease. The leftmost two mutations and the rightmost two mutations each cause a substantial decrease in MetR binding, while the central three mutations have little effect.
E. coli lacks the promoter sequences of S. typhimurium (102), and transcription is initiated 324 bases upstream from the coding sequence, within the iclR gene. Even though the MetR-binding site is downstream from the start site of transcription, the MetR protein stimulates transcription. Further studies on the mechanism of stimulation will be of interest.
In contrast to its stimulatory effect on metE expression, homocysteine supplementation causes about a threefold reduction in β-galactosidase synthesis from a metH-lacZ fusion (193). However, homocysteine has little or no effect on the in vitro stimulation of metH expression by MetR protein (24, 185). Thus, the in vivo effect of homocysteine on metH expression appears to be indirect.
Various workers have shown that metK is derepressed in metJ mutants (73, 75, 161), but little work has been done on the molecular mechanism of its regulation. The E. coli metK sequence published by Markham et al. (107) had no obvious promoter sequence or MET boxes. Moore and Boyle (123) reported the sequence of the divergently transcribed E. coli speA gene, including the 795 bases between the two coding sequences. Examination of the intergenic region reveals a good promoter (overlined in Fig. 11) ending 146 bases upstream from the coding sequence and an adjacent cluster of three moderately good MET boxes (marked by the vertical lines). Although these are probably the promoter and MetJ-binding sites, there is no direct evidence to support the hypothesis.
Although methionine is important as the initiating residue in protein synthesis and as a component of proteins, it mostly behaves as a nonpolar amino acid that can usually be replaced by norleucine with retention of function. Rarely, the thioether group plays a specific role, such as serving as a ligand for the heme iron in cytochromes c. The thioether moiety is, of course, essential for the synthesis of S-adenosylmethionine, which functions as an activated donor in many transfer reactions. The reactions of the pathways of methionine biosynthesis and metabolism are interesting. Despite the fact that the genes for most of the enzymes have been cloned and it is technically feasible to produce them in large amounts, only a few have been well studied.
The pathways seem to be primarily controlled by regulation of gene expression, since the only enzyme known to be subject to feedback inhibition is homoserine transsuccinylase. The apparent failure of methionine metabolites to inhibit methylenetetrahydrofolate reductase is surprising, since excess activity of this enzyme should cause trapping of folates and the pool of one carbon metabolites as the nonutilizable N 5-methyl derivative (the methyl trap). It is possible that activities of the various enzymes of the pathways are more subtly modulated than is evident from the meager information that is presently available.
At present, two proteins are known to be involved in regulation of met gene expression, the products of the metJ and metR genes. The MetJ protein is a global repressor that inhibits transcription of met genes with different efficiencies. The most marked effect of the MetR protein is stimulation of expression of the two genes for methionine synthase (metE and metH), but it has smaller effects on other genes, some inhibitory and some stimulatory. It seems likely that other elements will be found to influence the expression of met genes and flow of metabolites through the biosynthetic pathway.
References
1. Abel, S., P. W. Oeller, and A. Theologis. 1994. Early auxin-induced genes encode short-lived nuclear proteins. Proc. Natl. Acad. Sci. USA 91:326–330.
2. Adams, J. M., and M. R. Cappecchi. 1966. N-Formylmethionyl-sRNA as the initiator of protein synthesis. Proc. Natl. Acad. Sci. USA 55:147–155.
3. Anfinsen, C. B., and L. G. Corley. 1969. An active variant of staphylococcal nuclease containing norleucine in place of methionine. J. Biol. Chem. 244:5149–5152.
4. Ayling, P. D., and E. S. Bridgeland. 1972. Methionine transport in wild-type and transport-defective mutants of Salmonella typhimurium. J. Gen. Microbiol. 73:127–141.
5. Ayling, P. D., T. Mojica-a, and T. Klopotowski. 1979. Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system. J. Gen. Microbiol. 114:227–246.
6. Banerjee, R. V., N. L. Johnston, J. K. Sobeski, P. Datta, and R. G. Matthews. 1989. 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. J. Biol. Chem. 264:13888–13895.
7. Barker, D. G., J.-P. Ebel, R. Jakes, and C. J. Bruton. 1982. Methionyl-tRNA synthetase from Escherichia coli: primary structure of the active crystallized tryptic fragment. Eur. J. Biochem. 127:449–457.
8. 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.
9. Biran, D., N. Brot, H. Weissbach, and E. Z. Ron. 1995. Heat shock-dependent transcriptional activation of the metA gene of Escherichia coli. J. Bacteriol. 177:1374–1379.
10. Blumenthal, T. 1972. P1 transduction: formation of heterogenotes upon cotransduction of bacterial genes with a P2 prophage. Virology 47:76–93.
11. Bogosian, G., B. N. Violand, E. J. Dorward-King, W. E. Workman, P. E. Jung, and J. F. Kane. 1989. Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J. Biol. Chem. 264:531–539.
12. Boles, J. O., K. Lewinski, M. Kunkle, J. D. Odom, R. B. Dunlap, L. Lebioda, and M. Hatada. 1994. Bio-incorporation of telluromethionine into buried residues of dihydrofolate reductase. Nat. Struct. Biol. 1:283–284.
13. Bowie, J. U., and R. T. Sauer. 1990. TraY proteins of F and related episomes are members of the arc and mnt repressor family. J. Mol. Biol. 211:5–6.
14. Boyle, S. M., G. D. Markham, E. W. Hafner, J. M. Wright, H. Tabor, and C. W. Tabor. 1984. Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK). Gene 30(1–3):129–136.
15. Breg, J. N., J. H. J. van Opheusden, M. J. M. Burgering, R. Boelens, and R. Kaptein. 1990. Structure of Arc repressor in solution: evidence for a family of β-sheet DNA-binding proteins. Nature (London) 346:586–589.
16. Brown, B. M., M. E. Milla, T. L. Smith, and R. T. Sauer. 1994. Scanning mutagenesis of the Arc repressor as a functional probe of operator recognition. Nat. Struct. Biol. 1:164–168.
17. Brunie, S., C. Zelwer, and J. L. Risler. 1990. Crystallographic study at 2.5 Å resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP. J. Mol. Biol. 216:411–424.
18. Brzovic, P., E. L. Holbrook, R. C. Greene, and M. F. Dunn. 1990. Reaction mechanism of Escherichia coli cystathionine γ-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent γ-elimination and γ-replacement reactions. Biochemistry 29:442–451.
19. Burgering, M. J., R. Boelens, M. Caffrey, J. N. Breg, and R. Kaptein. 1993. Observation of inter-subunit nuclear Overhauser effects in a dimeric protein. Application to the Arc repressor. FEBS Lett. 330:105–109.
20. Burgering, M. J. M., R. Boelens, D. E. Gilbert, J. N. Breg, K. L. Knight, R. T. Sauer, and R. Kaptein. 1994. Solution structure of dimeric Mnt repressor (1–76). Biochemistry 33:15036–15045.
21. Byerly, K. A., M. L. Urbanowski, and G. V. Stauffer. 1991. The MetR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation. J. Bacteriol. 173:3547–3553.
22. Cai, X. Y., H. Jakubowski, B. Redfield, B. Zaleski, N. Brot, and H. Weissbach. 1992. Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid. Biochem. Biophys. Res. Commun. 182:651–658.
23. Cai, X. Y., M. E. Maxon, B. Redfield, R. Glass, N. Brot, and H. Weissbach. 1989. Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression. Proc. Natl. Acad. Sci. USA 86:4407–4411.
24. Cai, X. Y., B. Redfield, M. Maxon, H. Weissbach, and N. Brot. 1989. The effect of homocysteine on MetR regulation of metE, metR and metH expression in vitro. Biochem. Biophys. Res. Commun. 163:79–83.
25. Cantoni, G. L. 1953. S-adenosylmethionine: a new intermediate formed enzymatically from l-methionine and adenosinetriphosphate. J. Biol. Chem. 204:403–416.
26. Cassio, D. 1975. Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12. J. Bacteriol. 123:589–597.
27. Cassio, D., and J. P. Waller. 1971. Modification of methionyl-tRNA synthetase by proteolytic cleavage and properties of the trypsin-modified enzyme. Eur. J. Biochem. 20:283–300.
28. Chater, K. F., D. A. Lawrence, R. J. Rowbury, and T. S. Gross. 1970. Suppression of methionyl transfer RNA synthetase mutants of Salmonella typhimurium by methionine regulatory mutations. J. Gen. Microbiol. 63:121–131.
29. Chater, K. F., and R. J. Rowbury. 1970. A genetical study of the feedback-sensitive enzyme of methionine synthesis in Salmonella typhimurium. J. Gen. Microbiol. 63:111–120.
30. Coch, E. H., and R. C. Greene. 1971. The utilization of selenomethionine by Escherichia coli. Biochim. Biophys. Acta 230:223–236.
31. Cohen, G. N., and F. Jacob. 1959. Sur la répression de la synthèse des enzymes intervenant dans la formation du tryptophane chez E. coli. C. R. Acad. Sci. 248:3490.
32. Cohen, G. N., and I. Saint-Girons. 1987. Biosynthesis of threonine, lysine, and methionine, p. 429–444. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1. American Society for Microbiology, Washington, D.C.
33. Cottam, A. N., and P. D. Ayling. 1989. Genetic studies of mutants in a high-affinity methionine transport system in Salmonella typhimurium. Mol. Gen. Genet. 215:358–363.
34. Cowan, J. M., M. L. Urbanowski, M. Talmi, and G. V. Stauffer. 1993. Regulation of the Salmonella typhimurium metF gene by the MetR protein. J. Bacteriol. 175:5862–5866.
35. Cowie, D. B., and G. N. Cohen. 1957. Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur. Biochim. Biophys. Acta 26:252–261.
36. Dardel, F., G. Fayat, and S. Blanquet. 1984. Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthetase gene. J. Bacteriol. 160:1115–1122.
37. Dardel, F., M. Panvert, S. Blanquet, and G. Fayat. 1991. Locations of the metG and mrp genes on the physical map of Escherichia coli. J. Bacteriol. 173:3273.
38. Dardel, F., M. Panvert, and G. Fayat. 1990. Transcription and regulation of expression of the Escherichia coli methionyl-tRNA synthetase gene. Mol. Gen. Genet. 223:121–133.
39. Davidson, B. E., and I. Saint-Girons. 1989. The Escherichia coli regulatory protein MetJ binds to a tandemly repeated 8 bp palindrome. Mol. Microbiol. 3:1639–1648.
40. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17–28.
41. Dawes, J., and M. A. Foster. 1971. Vitamin B12 and methionine synthesis in Escherichia coli. Biochim. Biophys. Acta 237:455–464.
42. Drennan, C. L., R. G. Matthews, and M. L. Ludwig. 1994. Cobalamin-dependent methionine synthase—the structure of a methylcobalamin-binding fragment and implications for other B12-dependent enzymes. Curr. Opin. Struct. Biol. 4:919–929.
43. Drummond, J. T., R. R. Ogorzalek Loo, and R. G. Matthews. 1993. Electrospray mass spectrometric analysis of the domains of a large enzyme: observation of the occupied cobalamin-binding domain and redefinition of the carboxyl terminus of methionine synthase. Biochemistry 32:9282–9289.
44. Duchange, N., M. M. Zakin, P. Ferrara, I. Saint-Girons, I. Park, S. V. Tran, M. C. Py, and G. N. Cohen. 1983. Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5'- and 3'-flanking regions of the metBL operon. J. Biol. Chem. 258:14868–14871.
45. Duclos, B., J. C. Cortay, F. Bleicher, E. Z. Ron, C. Richaud, I. Saint Girons, and A. J. Cozzone. 1989. Nucleotide sequence of the metA gene encoding homoserine trans-succinylase in Escherichia coli. Nucleic Acids Res. 17:2856.
46. Duerre, J. A. 1962. A hydrolytic nucleosidase acting on S-adenosylhomocysteine and on 5'-methylthioadenosine. J. Biol. Chem. 237:3737–3741.
47. Duerre, J. A., and R. D. Walker. 1977. Metabolism of adenosylhomocysteine, p. 43–57. In F. Salvatore, E. Borek, V. Zappia, H. G. Williams-Ashman, and F. Schlenk (ed.), The Biochemistry of Adenosylmethionine. Columbia University Press, New York.
48. Dwivedi, C. M., R. C. Ragin, and J. R. Uren. 1982. Cloning, purification, and characterization of β-cystathionase from Escherichia coli. Biochemistry 21:3064–3069.
49. Ejiri, S. I., H. Weissbach, and N. Brot. 1979. Reduction of methionine sulfoxide to methionine by Escherichia coli. J. Bacteriol. 139:161–164.
50. Flavin, M. 1975. Methionine biosynthesis, p. 457–503. In D. M. Greenberg (ed.), Metabolism of Sulfur Compounds, 3rd ed., vol. 7. Academic Press, New York.
51. Flavin, M., and C. Slaughter. 1965. Synthesis of the succinic ester of homoserine, a new intermediate in the bacterial biosynthesis of methionine. Biochemistry 4:1370–1375.
52. Flavin, M., and C. Slaughter. 1967. Enzymatic synthesis of homocysteine or methionine directly from O-succinyl-homoserine. Biochim. Biophys. Acta 132:400–405.
53. Fourmy, D., F. Dardel, and S. Blanquet. 1993. Methionyl-tRNA synthetase zinc binding domain. Three-dimensional structure and homology with rubredoxin and gag retroviral proteins. J. Mol. Biol. 231:1078–1089.
54. Fourmy, D., T. Meinnel, Y. Mechulam, and S. Blanquet. 1993. Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase. J. Mol. Biol. 231:1068–1077.
55. Furfine, E. S., and R. H. Abeles. 1988. Intermediates in the conversion of 5'-S-methylthioadenosine to methionine in Klebsiella pneumoniae. J. Biol. Chem. 263:9598–9606.
56. Gentry-Weeks, C. R., J. M. Keith, and J. Thompson. 1993. Toxicity of Bordetella avium β-cystathionase toward MC3T3-E1 osteogenic cells. J. Biol. Chem. 268:7298–7314.
57. Ghosh, G., H. Pelka, and L. H. Schulman. 1990. Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase. Biochemistry 29:2220–2225.
58. Gianotti, A. J., P. A. Tower, J. H. Sheley, P. A. Conte, C. Spiro, A. J. Ferro, J. H. Fitchen, and M. K. Riscoe. 1990. Selective killing of Klebsiella pneumoniae by 5-trifluoromethylthioribose. Chemotherapeutic exploitation of the enzyme 5-methylthioribose kinase. J. Biol. Chem. 265:831–837.
59. Gilles, A. M., P. Marliere, T. Rose, R. Sarfati, R. Longin, A. Meier, S. Fermandjian, M. Monnot, G. N. Cohen, and O. Barzu. 1988. Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase. J. Biol. Chem. 263:8204–8209.
60. Gonzalez, J. C., R. V. Banerjee, S. Huang, J. S. Sumner, and R. G. Matthews. 1992. Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem. Biochemistry 31:6045–6056.
61. Goyette, P., J. S. Sumner, R. Milos, A. M. V. Duncan, D. S. Rosenblatt, R. G. Matthews, and R. Rozen. 1994. Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification. Nat. Genet. 7:195–200.
62. Greene, R. C. 1957. Incorporation of the carbon chain of methionine into spermidine. J. Am. Chem. Soc. 79:3929.
63. Greene, R. C. 1973. Methionine limitation in Escherichia coli K-12 by growth on the sulfoxides of d-methionine. J. Bacteriol. 116:230–234.
64. Greene, R. C., J. S. V. Hunter, and E. H. Coch. 1973. Properties of metK mutants of Escherichia coli K-12. J. Bacteriol. 115:57–67.
65. Greene, R. C., and A. A. Smith. 1984. Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for a metBL operon. J. Bacteriol. 159:767–769.
66. Greene, R. C., C.-H. Su, and C. T. Holloway. 1970. S-Adenosylmethionine synthetase deficient mutants of Escherichia coli K-12 with impaired control of methionine biosynthesis. Biochem. Biophys. Res. Commun. 38:1120–1126.
67. Greene, R. C., R. D. Williams, H.-F. Kung, C. Spears, and H. Weissbach. 1973. Effects of methionine and vitamin B12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12. Arch. Biochem. Biophys. 158:249—256.
68. Gross, T. S., and R. J. Rowbury. 1969. Methionyl transfer RNA synthetase mutants of Salmonella typhimurium which have normal control of the methionine biosynthetic enzymes. Biochim. Biophys. Acta 184:233–236.
69. Grundy, C. E., and P. D. Ayling. 1992. Fine structure mapping and complementation studies of the metD methionine transport system in Salmonella typhimurium. Genet. Res. 60:1–6.
70. He, Y.-Y., T. McNally, I. Manfield, O. Navratil, I. G. Old, S. E. V. Phillips, I. Saint-Girons, and P. G. Stockley. 1992. Probing met repressor-operator recognition in solution. Nature (London) 359:431–433.
71. Hendrickson, W. A., J. R. Horton, and D. M. LeMaster. 1990. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9:1665–1672.
72. Henikoff, S., J. C. Wallace, and J. P. Brown. 1990. Finding protein similarities with nucleotide sequence databases. Methods Enzymol. 183:111–132.
73. Hobson, A. C., and D. A. Smith. 1973. S-Adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium. Mol. Gen. Genet. 126:7–18.
74. Holbrook, E. L., R. C. Greene, and J. H. Krueger. 1990. Purification and properties of cystathionine γ-synthase from overproducing strains of Escherichia coli. Biochemistry 29:435–442.
75. Holloway, C. T., R. C. Greene, and C.-H. Su. 1970. Regulation of S-adenosylmethionine synthetase in Escherichia coli. J. Bacteriol. 104:734–747.
76. Homer, R. J., M. S. Kim, and D. M. LeMaster. 1993. The use of cystathionine γ-synthase in the production of α and chiral β deuterated amino acids. Anal. Biochem. 215:211–215.
77. Jeter, R. M., B. M. Olivera, and J. R. Roth. 1984. Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions. J. Bacteriol. 159:206–213.
78. Johnson, J. R., R. C. Greene, and J. H. Krueger. 1977. Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster. J. Bacteriol. 131:795–800.
79. Johnston, M., P. Marcotte, J. Donovan, and C. Walsh. 1979. Mechanistic studies with vinylglycine and β-haloaminobutyrates as substrates for cystathionine γ-synthetase from Salmonella typhimurium. Biochemistry 18:1729–1738.
80. Kadner, R. J. 1974. Transport systems for l-methionine in Escherichia coli. J. Bacteriol. 117:232–241.
81. Kadner, R. J. 1975. Regulation of methionine transport activity in Escherichia coli. J. Bacteriol. 122:110–119.
82. Kadner, R. J. 1977. Transport and utilization of d-methionine and other methionine sources in Escherichia coli. J. Bacteriol. 129:207–216.
83. 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.
84. Kadner, R. J., and H. H. Winkler. 1975. Energy coupling for methionine transport in Escherichia coli. J. Bacteriol. 123:985–991.
85. Kaplan, M. M., and M. Flavin. 1965. Enzymatic synthesis of l-cystathionine from the succinic ester of l-homoserine. Biochim. Biophys. Acta 104:390–396.
86. Kaplan, M. M., and M. Flavin. 1966. Cystathionine γ-synthetase of Salmonella. Structural properties of a new enzyme in bacterial methionine biosynthesis. J. Biol. Chem. 241:5781–5789.
87. Kaplan, M. M., and M. Flavin. 1966. Cystathionine γ-synthetase of Salmonella. Catalytic properties of a new enzyme in bacterial methionine biosynthesis. J. Biol. Chem. 241:4463–4471.
88. Katzen, H. M., and J. M. Buchanan. 1965. Enzymatic synthesis of the methyl group of methionine. VIII. Repression-derepression, purification, and properties of 5,10-methylenetetrahydrofolate reductase from Escherichia coli. J. Biol. Chem. 240:825–835.
89. Kerwar, S. S., and H. Weissbach. 1970. Studies on the ability of norleucine to replace methionine in the initiation of protein synthesis in E. coli. Arch. Biochem. Biophys. 141:525–532.
90. Kim, H. Y., H. Pelka, S. Brunie, and L. H. Schulman. 1993. Two separate peptides in Escherichia coli methionyl-tRNA synthetase form the anticodon binding site for methionine tRNA. Biochemistry 32:10506–10511.
91. Kim, S., L. R. de Pouplana, and P. Schimmel. 1993. Diversified sequences of peptide epitope for same-RNA recognition. Proc. Natl. Acad. Sci. USA 90:10046–10050.
92. Kim, S., L. R. de Pouplana, and P. Schimmel. 1994. An RNA binding site in a tRNA synthetase with a reduced set of amino acids. Biochemistry 33:11040–11045.
93. 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.
94. Koch, G. L., and C. J. Bruton. 1974. The subunit structure of methionyl-tRNA synthetase from Escherichia coli. FEBS Lett. 40:180–182.
95. Krueger, J. H., J. R. Johnson, and R. C. Greene. 1978. In vitro synthesis of cystathionine γ-synthetase in Escherichia coli K-12. J. Bacteriol. 133:1351–1357.
96. Kung, H.-F., C. Spears, R. C. Greene, and H. Weissbach. 1972. Regulation of the terminal reactions in methionine biosynthesis by vitamin B12 and methionine. Arch. Biochem. Biophys. 150:23–31.
97. Landro, J. A., and P. Schimmel. 1993. Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold. Proc. Natl. Acad. Sci. USA 90:2261–2265.
98. Lawrence, D. A. 1972. Regulation of the methionine feedback-sensitive enzyme in mutants of Salmonella typhimurium. J. Bacteriol. 109:8–11.
99. Lawrence, D. A., D. A. Smith, and R. J. Rowbury. 1968. Regulation of methionine synthesis in Salmonella typhimurium: mutants resistant to inhibition by analogues of methionine. Genetics 58:473–492.
100. Lee, L.-W., J. M. Ravel, and W. Shive. 1966. Multimetabolite control of a biosynthetic pathway by sequential metabolites. J. Biol. Chem. 241:5479–5480.
101. Ma, Q. F., G. L. Kenyon, and G. D. Markham. 1990. Specificity of S-adenosylmethionine synthetase for ATP analogues mono- and disubstituted in bridging positions of the polyphosphate chain. Biochemistry 29:1412–1416.
102. Marconi, R., J. Wigboldus, H. Weissbach, and N. Brot. 1991. Transcriptional start and MetR binding sites on the Escherichia coli metH gene. Biochem. Biophys. Res. Commun. 175:1057–1063.
103. Mares, R., M. L. Urbanowski, and G. V. Stauffer. 1992. Regulation of the Salmonella typhimurium metA gene by the MetR protein and homocysteine. J. Bacteriol. 174:390–397.
104. Markham, G. D. 1981. Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase. J. Biol. Chem. 256:1903–1909.
105. Markham, G. D. 1984. Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance. Biochemistry 23:470–478.
106. Markham, G. D. 1986. Characterization of the monovalent cation activator binding site of S-adenosylmethionine synthetase by 205Tl NMR of enzyme-bound Tl+. J. Biol. Chem. 261:1507–1509.
107. Markham, G. D., J. DeParasis, and J. Gatmaitan. 1984. The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli. J. Biol. Chem. 259:14505–14507.
108. Markham, G. D., E. W. Hafner, C. W. Tabor, and H. Tabor. 1980. S-Adenosylmethionine synthetase from Escherichia coli. J. Biol. Chem. 255:9082–9092.
109. Martinis, S. A., and P. Schimmel. 1992. Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine. Proc. Natl. Acad. Sci. USA 89:65–69.
110. Maxon, M. E., B. Redfield, X. Y. Cai, R. Shoeman, K. Fujita, W. Fisher, G. Stauffer, H. Weissbach, and N. Brot. 1989. Regulation of methionine synthesis in Escherichia coli: effect of the MetR protein on the expression of the metE and metR genes. Proc. Natl. Acad. Sci. USA 86:85–89.
111. Maxon, M. E., J. Wigboldus, N. Brot, and H. Weissbach. 1990. Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein. Proc. Natl. Acad. Sci. USA 87:7076–7079.
112. Meinnel, T., Y. Mechulam, S. Blanquet, and G. Fayat. 1991. Binding of the anticodon domain of tRNAfMet to Escherichia coli methionyl-tRNA synthetase. J. Mol. Biol. 220:205–208.
113. Meinnel, T., Y. Mechulam, F. Dardel, J. Schmitter, C. Hountondji, S. Brunie, P. Dessen, G. Fayat, and S. Blanquet. 1990. Methionyl-tRNA synthetase from E. coli—a review. Biochimie 72:625–632.
114. Meinnel, T., Y. Mechulam, C. Lazennec, S. Blanquet, and G. Fayat. 1993. Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase. J. Mol. Biol. 229:26–36.
115. Meinnel, T., Y. Mechulam, D. Le Corre, M. Panvert, S. Blanquet, and G. Fayat. 1991. Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site. Proc. Natl. Acad. Sci. USA 88:291–295.
116. Mellot, P., Y. Mechulam, D. Le Corre, S. Blanquet, and G. Fayat. 1989. Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition. J. Mol. Biol. 208:429–443.
117. Michaeli, S., M. Mevarech, and E. Z. Ron. 1984. Regulatory region of the metA gene of Escherichia coli K-12. J. Bacteriol. 160:1158–1162.
118. Michaeli, S., E. Z. Ron, and G. Cohen. 1981. Construction and physical mapping of plasmids containing the metA gene of Escherichia coli K-12. Mol. Gen. Genet. 182:349–354.
119. Milla, M. E., B. M. Brown, and R. T. Sauer. 1994. Protein stability effects of a complete set of alanine substitutions in Arc repressor. Nat. Struct. Biol. 1:518–523.
120. Milla, M. E., and R. T. Sauer. 1995. Critical side-chain interactions at a subunit interface in the Arc repressor dimer. Biochemistry 34:3344–3351.
121. Miller, C. H., and J. A. Duerre. 1968. S-ribosylhomocysteine cleavage enzyme from Escherichia coli. J. Biol. Chem. 243:92–97.
122. Milner, L., C. Whitfield, and H. Weissbach. 1969. Effect of l-methionine and vitamin B12 on methionine biosynthesis in Escherichia coli. Arch. Biochem. Biophys. 133:413–419.
123. Moore, R. C., and S. M. Boyle. 1990. Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli. J. Bacteriol. 172:4631–4640.
124. Mueller, J. H. 1922. A new sulphur-containing amino acid isolated from casein. Proc. Soc. Exp. Biol. Med. 19:161–163.
125. Mulligan, M. E., D. K. Hawley, R. Entriken, and W. R. McClure. 1984. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 12:789–800.
126. Myers, R. W., and R. H. Abeles. 1989. Conversion of 5-S-ethyl-5-thio-d-ribose to ethionine in Klebsiella pneumoniae. Basis for the selective toxicity of 5-S-ethyl-5-thio-d-ribose. J. Biol. Chem. 264:10547–10551.
127. Myers, R. W., J. W. Wray, S. Fish, and R. H. Abeles. 1993. Purification and characterization of an enzyme involved in oxidative carbon-carbon bond cleavage reactions in the methionine salvage pathway of Klebsiella pneumoniae. J. Biol. Chem. 268:24785–24791.
128. Naider, F., Z. Bohak, and J. Yariv. 1972. Reversible alkylation of a methionyl residue near the active site of β-galactosidase. Biochemistry 11:3202–3208.
129. Nelson, W. C., B. S. Morton, E. E. Lahue, and S. W. Matson. 1993. Characterization of the Escherichia coli F factor traY gene product and its binding sites. J. Bacteriol. 175:2221–2228.
130. Odake, S. 1925. Über das Vorkommen einer Schwefelhaltigen aminosäure im alkoholischen Extrakt der Hefe. Biochem. Z. 161:446–455.
131. Old, I. G., D. Margarita, R. E. Glass, and I. Saint Girons. 1990. Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2. Gene 87:15–21.
132. Old, I. G., S. E. V. Phillips, P. G. Stockley, and I. Saint-Girons. 1991. Regulation of methionine biosynthesis in the Enterobacteriaceae. Prog. Biophys. Mol. Biol. 56:145–185.
133. Old, I. G., I. Saint Girons, and C. Richaud. 1993. Physical mapping of the scattered methionine genes on the Escherichia coli chromosome. J. Bacteriol. 175:3689–3691.
134. Palmer, J. L., and R. H. Abeles. 1979. The mechanism of action of S-adenosylhomocysteinase. J. Biol. Chem. 254:1217–1226.
135. Park, Y. M., and G. V. Stauffer. 1989. DNA sequence of the metC gene and its flanking regions from Salmonella typhimurium LT2 and homology with the corresponding sequence of Escherichia coli. Mol. Gen. Genet. 216:164–169.
136. Park, Y. M., and G. V. Stauffer. 1989. Salmonella typhimurium metC operator-constitutive mutations. FEMS Microbiol. Lett. 51:137–141.
137. Patte, J. C., G. Le Bras, and G. N. Cohen. 1967. Regulation by methionine of the synthesis of a third aspartokinase and of a second homoserine dehydrogenase in Escherichia coli K 12. Biochim. Biophys. Acta 136:245–247.
138. Phillips, S. E. V. 1991. Specific β-sheet interactions. Curr. Opin. Struct. Biol. 1:89–98.
139. Phillips, S. E. V. 1994. The β-ribbon DNA recognition motif. Annu. Rev. Biophys. Biomol. Struct. 23:671–701.
140. Phillips, S. E. V., C. W. G. Boys, Y.-Y. He, I. Manfield, T. McNally, O. Navratil, I. G. Old, K. Phillips, J. B. Rafferty, W. S. Somers, S. Strathdee, I. Saint-Girons, and P. G. Stockley. 1993. E. coli Met repressor: DNA recognition by β-strands, p. 28–45. In F. L. Eckstein and D. M. J. Lilley (ed.), Nucleic Acids and Molecular Biology, vol. 7. Springer-Verlag, Berlin. Heidelberg.
141. Plamann, L. S., and G. V. Stauffer. 1987. Nucleotide sequence of the Salmonella typhimurium metR gene and the metR-metE control region. J. Bacteriol. 169:3932–3937.
142. Plamann, M. D., and G. V. Stauffer. 1989. Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine. J. Bacteriol. 171:4958–4962.
143. Press, R., N. Glansdorff, P. Miner, J. De Vries, R. Kadner, and W. K. Maas. 1971. Isolation of transducing particles of φ80 bacteriophage that carry different regions of the Escherichia coli genome. Proc. Natl. Acad. Sci. USA 68:795–798.
144. Rafferty, J. B., W. S. Somers, I. Saint-Girons, and S. E. V. Phillips. 1989. Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor. Nature (London) 341:705–710.
145. RajBhandary, U. L. 1994. Initiator transfer RNAs. J. Bacteriol. 176:547–552.
146. Randhawa, Z. I., H. E. Witkowska, J. Cone, J. A. Wilkins, P. Hughes, K. Yamanishi, S. Yasuda, Y. Masui, P. Arthur, C. Kletke, and F. Bitsch. 1994. Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-csf, 4–153) expressed in Escherichia-coli—structural analysis. Biochemistry 33:4352–4362.
147. Raumann, B. E., B. M. Brown, and R. T. Sauer. 1994. Major groove DNA recognition by β-sheets: the ribbon-helix-helix family of gene regulatory proteins. Curr. Opin. Struct. Biol. 4:36–43.
148. Raumann, B. E., M. A. Rould, C. O. Pabo, and R. T. Sauer. 1994. DNA recognition by β-sheets in the Arc repressor-operator crystal structure. Nature (London) 367:754–757.
149. Roberts, P. E. 1992. Ph.D. thesis. University of Cambridge, Cambridge, England.
150. Ron, E. Z. 1975. Growth rate of Enterobacteriaceae at elevated temperatures: limitation by methionine. J. Bacteriol. 124:243–246.
151. Ron, E. Z., S. Alajem, D. Biran, and N. Grossman. 1990. Adaptation of Escherichia coli to elevated temperatures: the metA gene product is a heat shock protein. Antonie van Leeuwenhoek 58:169–174.
152. Ron, E. Z., and B. D. Davis. 1971. Growth rate of Escherichia coli at elevated temperatures: limitation by methionine. J. Bacteriol. 107:391–396.
153. Ron, E. Z., and M. Shani. 1971. Growth rate of Escherichia coli at elevated temperatures: reversible inhibition of homoserine trans-succinylase. J. Bacteriol. 107:397–400.
154. Roth, J. R., J. G. Lawrence, M. Rubenfield, S. Kieffer-Higgins, and G. M. Church. 1993. Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 175:3303–3316.
155. Rowbury, R. J. 1962. A succinyl derivative of homoserine as a precursor of methionine in Escherichia coli. J. Gen. Microbiol. 28:v-vi.
156. 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.
157. 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 of its control region. Nucleic Acids Res. 11:6723–6732.
158. Saint-Girons, I., C. Parsot, M. M. Zakin, O. Barzu, and G. N. Cohen. 1988. Methionine biosynthesis in Enterobacteriaceae: biochemical, regulatory, and evolutionary aspects. Crit. Rev. Biochem. 23(Suppl. 1):S1–S42.
159. Satishchandran, C., J. C. Taylor, and G. D. Markham. 1990. Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis. J. Bacteriol. 172:4489–4496.
160. Satishchandran, C., J. C. Taylor, and G. D. Markham. 1993. Isozymes of S-adenosylmethionine synthetase are encoded by tandemly duplicated genes in Escherichia coli. Mol. Microbiol. 9:835–846.
161. Savin, M. A., M. Flavin, and C. Slaughter. 1972. Regulation of homocysteine biosynthesis in Salmonella typhimurium. J. Bacteriol. 111:547–556.
162. Schimmel, P., R. Giegé, D. Moras, and S. Yokoyama. 1993. An operational RNA code for amino acids and possible relationship to genetic code. Proc. Natl. Acad. Sci. USA 90:8763–8768.
163. Schlenk, F. 1983. Methylthioadenosine. Adv. Enzymol. Relat. Areas Mol. Biol. 54:195–265.
164. Schmitt, E., T. Meinnel, M. Panvert, Y. Mechulam, and S. Blanquet. 1993. Two acidic residues of Escherichia coli methionyl-tRNA synthetase act as negative discriminants towards the binding of non-cognate tRNA anticodons. J. Mol. Biol. 233:615–628.
165. Schroeder, H. R., C. J. Barnes, R. C. Bohinski, and M. F. Mallette. 1973. Biological production of 5-methylthioribose. Can. J. Microbiol. 19:1347–1354.
166. Schroeder, H. R., C. J. Barnes, R. C. Bohinski, R. O. Mumma, and M. F. Mallette. 1972. Isolation and identification of 5-methylthioribose from Escherichia coli B. Biochim. Biophys. Acta 273:254–264.
167. Schulman, L. H. 1991. Recognition of tRNAs by aminoacyl-tRNA synthetases. Prog. Nucleic Acid Res. Mol. Biol. 41:23–87.
168. Schulte, L. L., L. T. Stauffer, and G. V. Stauffer. 1984. Cloning and characterization of the Salmonella typhimurium metE gene. J. Bacteriol. 158:928–933.
169. Shaw, N. A., and P. D. Ayling. 1991. Cloning of high-affinity methionine transport genes from Salmonella typhimurium. FEMS Microbiol. Lett. 78:127–131.
170. 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.
171. 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.
172. Simon, M., and J. S. Hong. 1983. Direct homocysteine biosynthesis from O-succinylhomoserine in Escherichia coli: an alternate pathway that bypasses cystathionine. J. Bacteriol. 153:558–561.
173. Slany, R. K., M. Bösl, P. F. Crain, and H. Kersten. 1993. A new function of S-adenosylmethionine: the ribosyl moiety of AdoMet is the precursor of the cyclopentenediol moiety of the tRNA wobble base queuine. Biochemistry 32:7811–7817.
174. Slany, R. K., M. Bosl, and H. Kersten. 1994. Transfer and isomerization of the ribose moiety of Adomet during the biosynthesis of Queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli. Biochimie. 76:389–393.
175. 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.
176. 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.
177. Smith, D. A., and J. D. Childs. 1966. Methionine genes and enzymes of Salmonella typhimurium. Heredity 21:265–286.
178. Smith, M. W., and F. C. Neidhardt. 1983. Proteins induced by aerobiosis in Escherichia coli. J. Bacteriol. 154:344–350.
179. Somers, W. S., and S. E. Phillips. 1992. Crystal structure of the met repressor-operator complex at 2.8 Å resolution reveals DNA recognition by β-strands. Nature (London) 359:387–393.
180. Somers, W. S., J. B. Rafferty, K. Phillips, S. Strathdee, Y. Y. He, T. McNally, I. Manfield, O. Navratil, I. G. Old, I. Saint-Girons, P. G. Stockley, and S. E. V. Phillips. 1994. The Met repressor-operator complex: DNA recognition by β-strands. Ann. N. Y. Acad. Sci. 726:105–117.
181. Stauffer, G. V., and L. T. Stauffer. 1988. Cloning and nucleotide sequence of the Salmonella typhimurium LT2 metF gene and its homology with the corresponding sequence of Escherichia coli. Mol. Gen. Genet. 212:246–251.
182. Stauffer, G. V., and L. T. Stauffer. 1988. Salmonella typhimurium LT2 metF operator mutations. Mol. Gen. Genet. 214:32–36.
183. Su, C.-H., and R. C. Greene. 1971. Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ +/metJ – diploid. Proc. Natl. Acad. Sci. USA 68:367–371.
184. Taylor, R. T., and H. Weissbach. 1973. N 5-Methyltetrahydrofolate-homocysteine methyltransferases, p. 121–165. In P. D. Boyer (ed.), The Enzymes, 3rd ed., vol. IX, part B. Group Transfer. Academic Press, New York.
185. Urbanowski, M., and G. V. Stauffer. 1993. An indirect role for homocysteine in activation of the S. typhimurium metH gene, abstr. H-175. In Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993. American Society for Microbiology, Washington, D.C.
186. Urbanowski, M. L., L. S. Plamann, and G. V. Stauffer. 1987. Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2. J. Bacteriol. 169:126–130.
187. Urbanowski, M. L., and G. V. Stauffer. 1985. Nucleotide sequence and biochemical characterization of the metJ gene from Salmonella typhimurium LT2. Nucleic Acids Res. 13:673–685.
188. Urbanowski, M. L., and G. V. Stauffer. 1986. Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene. J. Bacteriol. 165:740–745.
189. Urbanowski, M. L., and G. V. Stauffer. 1986. The metH gene from Salmonella typhimurium LT2: cloning and initial characterization. Gene 44:211–217.
190. Urbanowski, M. L., and G. V. Stauffer. 1987. Regulation of the metR gene of Salmonella typhimurium. J. Bacteriol. 169:5841–5844.
191. Urbanowski, M. L., and G. V. Stauffer. 1988. The control region of the metH gene of Salmonella typhimurium LT2: an atypical met promoter. Gene 73:193–200.
192. Urbanowski, M. L., and G. V. Stauffer. 1989. Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium. J. Bacteriol. 171:5620–5629.
193. Urbanowski, M. L., and G. V. Stauffer. 1989. Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J. Bacteriol. 171:3277–3281.
194. Urbanowski, M. L., L. T. Stauffer, L. S. Plamann, and G. V. Stauffer. 1987. A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 169:1391–1397.
195. Walsh, C. 1979. Enzymatic Reaction Mechanisms. W. H. Freeman and Co., San Francisco.
196. Webster, R. E., D. L. Engelhardt, and N. D. Zinder. 1966. In vitro protein synthesis: chain initiation. Proc. Natl. Acad. Sci. USA 55:155–161.
197. Weissbach, H., and N. Brot. 1991. Regulation of methionine synthesis in Escherichia coli. Mol. Microbiol. 5:1593–1597.
198. 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.
199. Wray, J. W., and R. H. Abeles. 1993. A bacterial enzyme that catalyzes formation of carbon monoxide. J. Biol. Chem. 268:21466–21469.
200. Wray, J. W., and R. H. Abeles. 1995. The methionine salvage pathway in Klebsiella pneumoniae and rat liver: identification and characterization of two novel dioxygenases. J. Biol. Chem. 270:3147–3153.
201. Wu, W.-F., M. L. Urbanowski, and G. V. Stauffer. 1992. Role of the MetR regulatory system in vitamin Bl2-mediated repression of the Salmonella typhimurium metE gene. J. Bacteriol. 174:4833–4837.
202. Wu, W. F., M. L. Urbanowski, and G. V. Stauffer. 1993. MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region. FEMS Microbiol. Lett. 108:145–150.
203. Xu, B., G. A. Krudy, and P. R. Rosevear. 1993. Identification of the metal ligands and characterization of a putative zinc finger in methionyl-tRNA synthetase. J. Biol. Chem. 268:16259–16264.
204. Yamamoto, M., and M. Nomura. 1976. Isolation of λ transducing phages carrying rRNA genes at the metA-purD region of the Escherichia coli chromosome. FEBS Lett. 72:256–261.
205. Zakin, M. M., N. Duchange, P. Ferrara, and G. N. Cohen. 1983. Nucleotide sequence of the metL gene of Escherichia coli. Its product, the bifunctional aspartokinase II-homoserine dehydrogenase II, and the bifunctional product of the thrA gene, aspartokinase I-homoserine dehydrogenase I, derive from a common ancestor. J. Biol. Chem. 258:3028–3031.
206. Zakin, M. M., R. C. Greene, A. Dautry-Varsat, N. Duchange, F. P., M. C. Py, D. Margarita, and G. N. Cohen. 1982. Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12. Mol. Gen. Genet. 187:101–106.
207. Zelwer, C., J. L. Risler, and S. Brunie. 1982. Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 Å resolution. J. Mol. Biol. 155:63–81.
208. Zhang, C., G. D. Markham, and R. LoBrutto. 1993. Coordination of vanadyl(IV) cation in complexes of S-adenosylmethionine synthetase: multifrequency electron spin echo envelope modulation study. Biochemistry 32:9866–9873.
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