Methionine
ELISE R. HONDORP1 AND ROWENA G. MATTHEWS1,2*
[SECTION EDITOR: GEORGES COHEN]
Posted April 28, 2006
Life Sciences Institute1 and Department of Biological Chemistry and Biophysics Research Division,2 University of Michigan, Ann Arbor, MI 48109
*Corresponding author. Phone: 734-764-9459, Fax: 734-763-6492, E-mail:
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Methionine was first isolated in 1922 and in the ensuing years has become most recognized for its role as one of the basic building blocks of proteins (190). Yet, methionine also contributes to several other essential processes in Escherichia coli and Salmonella. Formylated methionine is required to initiate the translation of all proteins, and reaction of methionine with ATP to form S-adenosylmethionine (AdoMet) provides the major methyl donor found in the cell (1, 39, 296). AdoMet also participates in a wide variety of other processes, ranging from gene regulation and RNA modifications to biosynthetic pathways and chemotactic control.
The de novo biosynthesis of methionine is quite costly (requiring 7 ATPs and 8 NADPHs) and involves several reactions (Fig. 1) (129). In E. coli and Salmonella, the sulfur of methionine comes from cysteine, which in turn obtains its sulfur from sulfate via the sulfate assimilation pathway (see Chapter Biosynthesis of Cysteine). The carbon skeleton is derived from aspartate (see the introductory Biosynthesis chapter, Amino Acid Metabolism and Fluxes), while the methyl group is acquired from serine via one-carbon metabolism (see Chapter Regulation of Serine, Glycine, and One-Carbon Biosynthesis).
The biosynthesis of methionine is complex—it is interconnected with other biosynthetic pathways and contains both convergent and divergent branch points. Since many of those reactions are described elsewhere, this chapter focuses on the steps unique to methionine biosynthesis, namely the conversion of homoserine to methionine (Fig. 2). Homoserine is activated by succinylation, followed by reaction with cysteine to generate cystathionine. Cystathionine is then cleaved to form homocysteine, which receives a methyl group from methyltetrahydrofolate to produce methionine. Note that this pathway is not the same in mammals (where methionine cannot be synthesized de novo), and therefore many of the bacterial biosynthetic enzymes have been considered to be attractive drug targets.
The past decade has provided a wealth of information concerning the details of methionine metabolism and this chapter focuses on providing a comprehensive overview of the field, emphasizing more recent findings. Details of methionine biosynthesis are addressed along with key cellular aspects, including regulation, uptake, utilization, AdoMet, the methyl cycle, and growing evidence that inhibition of methionine biosynthesis occurs under stressful cellular conditions. The last print edition of Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., remains an excellent resource, and in instances where there has not been additional progress the reader is directed there, as well as to other insightful reviews (82, 99, 200, 234, 248).
The first unique step in methionine biosynthesis is catalyzed by the metA gene product, homoserine transsuccinylase (HTS, or homoserine O-succinyltransferase, EC 2.3.1.46). Succinylation of homoserine serves to activate the molecule for subsequent condensation with cysteine to form cystathionine (Fig. 2). The gene encoding the enzyme has been cloned and overexpressed to produce a protein of 35.6 kDa (27, 182, 183). An apparent molecular weight of 86 kDa has been observed by gel filtration analysis, suggesting that HTS exists as a dimer in solution (27, 182).
Within the past decade, the first detailed mechanistic studies of the recombinant protein were performed (27). Though the reaction is reversible, the equilibrium is estimated to lie far toward O-succinylhomoserine (Keq, ~170). Both D- and L-homoserine were found to be substrates of HTS, with only minimal differences in their kinetics of utilization. The data are consistent with a ping-pong mechanism, where succinyl-CoA reacts to form a succinylated enzyme intermediate, which is then transferred to the γ-oxygen of homoserine. Biochemical evidence suggests that a cysteine thiolate (cysteine 142 of E. coli HTS) forms the succinylated intermediate (27), but a succinylated lysine residue (lysine 46 of E. coli HTS) was recently isolated in HTS excised from two-dimensional gels (which had been treated with dithiothreitol [DTT] and iodoacetamide) (230). Our attempts to identify and align HTS sequences using BLAST indicated that cysteine 142 is completely conserved, whereas lysine 46 does not appear to be conserved among more distant homologues.
As may be expected with the first committed step in methionine biosynthesis, HTS is subject to extensive control. Both methionine and AdoMet act alone or synergistically to inhibit the enzyme allosterically and, like the other methionine biosynthetic genes, metA expression is transcriptionally regulated by the MetJ and MetR proteins (161, 240). In addition, heat shock appears to activate transcription of HTS, and the protein itself is reported to be unstable and susceptible to heat-induced inactivation, as well as aggregation and proteolysis (22, 23, 107, 226, 227, 228, 229). The regulation of HTS is further discussed in "Regulation of Methionine Biosynthesis" and "Methionine Biosynthesis under Stress Conditions" (see below).
Cystathionine γ-synthase (CGS) (EC 2.5.1.48), the metB gene product, catalyzes an attack on the γ-carbon of O-succinyl-L-homoserine (L-OSHS) by L-cysteine to form L-cystathionine and succinate (Fig. 2). Early structural and mechanistic studies indicated that the active enzyme is a homotetramer, where each 40-kDa subunit binds a pyridoxal phosphate (PLP) cofactor that is linked to the protein via Schiff base formation with a specific lysine residue (119, 143, 144, 172, 276). The ability to obtain high levels of recombinant CGS has provided significant advances in the characterization of the protein, including a 1.5-Å resolution crystal structure (48). The tetramer was thus found to consist of two dimers, each of which contains two active sites located at the subunit interface.
Although the γ-replacement reaction is the only physiological reaction of CGS, the enzyme can also catalyze a γ-elimination reaction to form α-ketobutyrate and ammonia. However, the rate of this side reaction was determined to be only ~1.5% that of the substitution reaction, as may be expected from the role of CGS in the methionine biosynthetic pathway (3). Detailed stopped-flow kinetic studies identified an α-imino β,γ-unsaturated pyridoxamine derivative as the partitioning intermediate between the substitution and elimination reactions (Fig. 3) (29). The development of novel continuous assays for CGS activity has provided new insights into the steady-state kinetic parameters of the enzyme; the reader may wish to refer to references 2 and 3 for a detailed discussion of the enzyme kinetics.
Numerous studies have investigated the various reactions that can be catalyzed by this rather promiscuous enzyme. In vitro, CGS can catalyze nonphysiological β-replacement, β-elimination, and proton-exchange reactions at the α or β position of amino acids (105, 106, 216). O-Acetyl-L-homoserine can replace O-succinyl-L-homoserine as a substrate for CGS, but with decreased efficiency (111, 143, 300). Thus, the specificity of the enzyme in vivo is thought to depend on the supply of substrate rather than on strict enzyme specificity (2, 111). Hydrogen sulfide and methyl mercaptan can substitute for L-cysteine to generate directly L-homocysteine and L-methionine, respectively. The former reaction provides a potential bypass to synthesis of cystathionine in vivo and has been suggested as an explanation for the leakiness of metC mutants. However, it is unlikely to provide a significant source of L-homocysteine due to the high concentration of sulfide required (83).
The metC gene product, cystathionine β-lyase (CBL) (EC 4.4.1.8), catalyzes the penultimate step in methionine biosynthesis, the β-elimination of L-cystathionine to produce L-homocysteine, pyruvate, and ammonia (Fig. 2). Both the E. coli and Salmonella proteins have been cloned, overexpressed, and purified (18, 78, 203). The active enzyme is a homotetramer with 43-kDa subunits, each of which binds a PLP cofactor (46, 172). In addition to the physiological hydrolysis of L-cystathionine, CBL is capable of acting on a wide variety of substrates, including L-cystine, L-cysteine, L-homolanthionine, L-meso-lanthionine, and L-djenkolic acid (78). Enzyme inhibitor studies combined with a 1.83-Å resolution crystal structure have led to identification of critical active site residues and a straightforward proposal of the reaction mechanism (46, 47). CBL has been found to be important in Salmonella virulence and in B. avium has been implicated in toxicity towards certain tissues (79, 93).
Early characterizations indicated that CBL was highly homologous to CGS (the metB gene product) (18). The two proteins were found to copurify in many systems; in fact, CBL was originally purified as a byproduct of the CGS purification (104). Both proteins are dimers of dimers and appear to be almost identical upon superposition of their structures (46, 48). Though they share the same active-site residues, variations in substrate-binding characteristics are believed to account for the different enzyme activities (48).
Recently, a second protein in E. coli has been discovered to have β-cystathionase activity, the product of the malY gene (314). MalY is a 43-kDa monomeric PLP-containing protein that is more homologous to aminotransferases than it is to CBL (49, 314). Nevertheless, MalY is able to catalyze the same reactions as CBL with similar kinetic parameters. This activity is functional in vivo, as constitutive expression of MalY can complement the methionine auxotrophy of metC mutants (314).
As its name implies, MalY was originally identified because of its role in maltose metabolism. MalY directly interacts with MalT to repress transcription of the mal genes, which are important for the metabolism of maltose and maltodextrins (243). However, the regulatory role of MalY appears to be independent of its β-cystathionase activity (314). While MalY can substitute for CBL in vivo, this reaction is likely to be only indicative of the type of chemistry performed by MalY on an unknown physiological substrate (49).
In 1983, Simon and Hong identified a mutation in a gene they called metQ, which allowed E. coli deficient in the metC gene product to grow in the absence of methionine (252). The observation that cystathionine could rescue metB-deficient cells, but not metBCQ, indicated that metQ did not result in the induction of another cystathionase. Instead, metQ was believed to be responsible for increasing the supply of sulfide, which could then permit the direct synthesis of L-homocysteine via the metB gene product. The metQ gene was subsequently mapped to 35.9 minutes on the E. coli chromosome, between uidAR and tyrS (199, 200). This is the same location as the malI gene, which encodes a protein that represses the expression of malY (223). Induction of MalY, a protein with β-cystathionase activity, could account for the relief of the methionine auxotrophy. In fact, all of the revertants characterized by Zdych et al. were mapped to malI (314). Note that the metQ gene described here is distinct from the methionine permease yaeE, which was renamed metQ by Merlin et al. (181) and Gál et al. (110) (see "Methionine Uptake," below).
One-carbon units are provided for methionine biosynthesis via the action of methylenetetrahydrofolate reductase (MTHFR) (EC 1.5.1.20), the product of the metF gene. MTHFR catalyzes the conversion of methylenetetrahydrofolate (CH2-H4folate) to methyltetrahydrofolate (CH3-H4folate; Fig. 4), which subsequently transfers its methyl group to homocysteine to form methionine (Fig. 1 and Fig. 2). MTHFR activity in crude preparations was initially described in the 1960s, and the gene was cloned relatively soon thereafter (112, 136, 146, 313). Yet it was several decades before the active E. coli enzyme was purified and rigorously characterized (251).
The native protein is a tetramer composed of identical 33-kDa subunits that each contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor (103, 112, 146, 235, 251, 261). NADH is the source of reducing equivalents and transfers its electrons to FAD, which in turn reduces CH2-H4folate. The enzyme functions via a ping-pong mechanism whereby NAD+ release precedes CH2-H4folate binding (277). NADH is the preferred pyridine nucleotide, but NADPH has minimal activity (112, 146, 251). In addition to the physiological reaction, MTHFR can use menadione to catalyze the oxidation of either NADH or CH3-H4folate, which provides a convenient means to assay activity aerobically. Mechanistic studies have detailed the kinetic parameters of the enzyme as well as the role of active-site residues (251, 277, 278, 279).
Initial estimates of the equilibrium constant for CH3-H4folate formation suggested that the reaction was completely irreversible. However, careful remeasurement of the redox potential for the CH2-H4folate/CH3-H4folate couple showed that the value was actually more negative (−200 mV) than previously calculated (−120 mV) (305). As a result, ΔGo' increased 3.8 kcal/mol to −5.4 kcal/mol (and Keq decreased 500-fold to 6,000 at pH 7). Although the equilibrium still favors the formation of CH3-H4folate, the reaction is less irreversible than was previously believed. This is significant in E. coli, in which NADH to NAD+ ratios can be quite low (25, 63). Therefore, it may be possible for the reverse reaction to occur under certain circumstances. This is in contrast to mammalian systems where MTHFR utilizes NADPH and the NADPH to NADP+ ratios are much higher (59, 174).
Crystal structures of E. coli MTHFR revealed that the enzyme is a β8α8 barrel with a novel FAD-binding site (103). The pyridine nucleotide was found to bind the enzyme in an unusual folded conformation (211). The β8α8 barrels associate to form a planar tetramer, where each subunit interacts with one neighbor significantly more than with the other (Fig. 5). Dissociation into dimers and loss of the FAD cofactor was found to occur readily upon dilution of the enzyme (103). Thus, maintaining high protein concentrations during purification proved critical to obtaining active MTHFR; the propensity toward FAD loss and enzyme inactivation may explain prior difficulties in purifying the protein (251). Additional studies have investigated the interactions influencing the oligomeric state and activity of the enzyme (187). Folate binding appears to protect the enzyme from FAD loss and inactivation (103). Since E. coli MTHFR is highly homologous to the N-terminal catalytic domain of the human enzyme (30% identity), it has been successfully used as a model for a human polymorphism that leads to elevated homocysteine levels, a risk factor for multiple disease states (103, 311).
The terminal step of de novo methionine biosynthesis is catalyzed by either cobalamin (B12)-dependent methionine synthase (MetH) (EC 2.1.1.13), the metH gene product, or B12-independent methionine synthase (MetE) (EC 2.1.1.14), the metE gene product (84). Both enzymes transfer a methyl group from CH3-H4folate to the sulfur of L-homocysteine to form L-methionine (Fig. 2). Thus, this reaction connects the methyl cycle to the one-carbon pathway, which supplies one-carbon units for de novo purine biosynthesis and other cellular functions (Fig. 1).
Although the genomes of E. coli and Salmonella contain both metE and metH, they are differentially expressed. Functional MetH is available only in the presence of B12, which also represses MetE expression (61, 152, 186). In the absence of exogenously supplied B12, MetE is the only enzyme that catalyzes the final step in de novo methionine synthesis. Salmonella can synthesize B12 under anaerobic conditions; however, E. coli cannot do this and must rely on exogenously supplied cobalamin (231). Thus, in nature, E. coli MetH is likely to be functional in the gut, where the intestinal flora and/or components of the diet are able to supply the necessary B12 cofactor. The regulation of methionine synthase expression will be discussed further in the next section.
Extensive studies have been performed on the B12-dependent enzyme. For reviews of the early work, see references 13, 102, 135, 173, and 274. The difficult chemistry is performed by MetH with the aid of the powerful cobalamin nucleophile. During turnover, CH3-H4folate donates its methyl group to the cob(I)alamin of MetH to form methylcobalamin and H4folate. The methyl group is subsequently transferred to L-homocysteine to produce methionine and regenerate cob(I)alamin (Fig. 6) (15). Occasionally, the cob(I)alamin is oxidized to the inactive cob(II)alamin form. To return to primary turnover, the enzyme must be reductively methylated through the action of flavodoxin and AdoMet (88, 167, 202).
MetH is a large (136 kDa) modular protein composed of four domains that are responsible for the various catalytic activities of the enzyme (Fig. 7) (73). The N-terminal domain binds and activates L-homocysteine, while the second module contains the determinants for CH3-H4folate binding. The B12 cofactor is bound within the third domain, and the C-terminal module binds AdoMet (98). During turnover each substrate module must interact with the cobalamin cofactor in turn, requiring conformational changes involving rearrangements of the protein domains. The conformational heterogeneity of MetH may be responsible for the difficulties encountered in attempts to crystallize the full-length protein. However, structures for each of the domains have now been determined, and studies have begun to dissect the mechanisms governing the conformational transitions of MetH (11, 12, 70, 72, 80).
Although there is scant sequence homology between the cobalamin-dependent and -independent proteins, current research suggests that the enzymes employ similar strategies for substrate activation (175). Protonation of the N5 of bound CH3-H4folate serves to make it a better methyl donor. Likewise, in both cases homocysteine is activated for methyl transfer by coordination to a zinc ion, which serves to lower the pKa of the thiol (95, 97, 207). The catalytically essential zinc is coordinated to the homocysteine-binding module of MetH by three cysteines and a displaceable water and to MetE via two cysteines, a histidine, and a glutamate (206, 210, 317).
Performing the same reaction in the absence of the potent B12 cofactor is chemically much more challenging because the reaction involves direct attack of homocysteine on CH3-H4folate and thiols are less reactive than cob(I)alamin as nucleophiles. Thus it is not surprising that the cobalamin-independent enzyme is less efficient, with a turnover number approximately 50 times lower than that of MetH (95). MetE is a large (84 kDa) monomeric protein. The enzyme is distinct from all other folate-dependent enzymes because it requires a polyglutamylated CH3-H4folate substrate compared to the monoglutamyl form accepted by other proteins (84, 96, 299). Divalent cations (such as Mg2+ and Mn2+) and inorganic phosphate have been found to stimulate the activity of MetE (299). The crystal structure of the MetE homolog in Thermotoga maritima (60% similar to E. coli MetE) has recently been solved. The protein contains two β8α8 barrels arranged in a unique orientation, where the C termini of the β-strands point toward each other (Fig. 8). The active site is in a cleft formed between the two barrel domains. The zinc-coordinating residues are all found within the C-terminal half of the protein and position the metal at the top of the second barrel. In contrast, the residues that provide the folate-binding determinants are found in both domains of MetE (210).
MetE levels can be quite high in E. coli, where MetE comprises approximately 3 to 5% of the total soluble protein in cells growing aerobically under steady-state conditions in glucose minimal medium (208, 287, 299). Recent studies suggest that MetE activity, and thus methionine availability, may be sensitive to stress conditions (121). The details and physiological implications of this will be discussed further in the final section of this chapter.
Although there is considerable industrial demand for methionine, large-scale production of the amino acid has not yet been achieved in microbial systems, in part due to the strong regulation of its biosynthesis (126, 193). In general, control of the methionine biosynthetic pathway is accomplished at the transcriptional level. However, as described earlier, the first enzyme in the pathway, homoserine transsuccinylase (HTS, the product of the metA gene), is subject to feedback inhibition by methionine and AdoMet (161, 240). In addition, the activities of HTS and MetE appear to be modulated under various stress conditions, the mechanisms of which are considered in "Methionine Biosynthesis under Stress Conditions," below.
It has long been known that addition of methionine to the growth medium represses the biosynthetic enzymes (50, 301). Control of the methionine biosynthetic genes involves regulation of multiple promoters because the met genes are not all in one operon (as is the case for the trp genes) and are scattered throughout the E. coli and Salmonella chromosomes (199). Transcriptional regulation is accomplished by three different means: via the MetJ repressor, the MetR activator, and the presence of B12. The general aspects of each system are presented here, with emphasis on recent understandings of the mechanism of MetJ-mediated repression. For details concerning the transcriptional regulation of each met gene, the reader is referred to Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., which contains an excellent discussion summarizing available knowledge (99); the only promoter region detailed here is that of metE, which includes updated information.
Transcription of the methionine biosynthetic genes (with the exception of metH) is repressed by the product of the metJ gene (MetJ) and its corepressor, AdoMet. Thus, mutations in either metJ or metK (which is responsible for AdoMet synthesis from methionine) have been found to result in upregulation of proteins involved in methionine biosynthesis (153). The MetJ protein has been overexpressed, purified, and characterized extensively. It is a 104-amino-acid (12-kDa) protein, which is a dimer in solution (233, 255, 256, 282). MetJ binds two molecules of AdoMet per dimer noncooperatively, increasing the affinity of the repressor for its cognate DNA (51, 125, 213, 221, 234, 236). The MetJ repressor recognizes and binds to an eight-nucleotide consensus sequence with dyad symmetry (AGACGTCT) called a "met-box" (18, 200, 213, 234). Operators of genes that are repressed by MetJ contain between two and five met-boxes positioned in tandem. A perfect consensus sequence is rarely present in natural operators; the sequence variability and number of met-boxes is believed to control the degree of MetJ-mediated repression. In general, increased homology to the consensus sequence is apparent in operators containing fewer met-boxes, and less variation is found toward the center of longer operators (213, 302). Binding of the holorepressor to operator DNA is highly cooperative, explaining the rationale for multiple adjacent met-boxes (125, 213). The affinity of MetJ for a single met-box is rather low; however, the stability of the complex is greatly increased upon binding of a second repressor. Two tandem met-boxes appear to be the minimum operator size for repression by MetJ (212, 213).
Crystal structures of repressor-DNA complexes have provided insight into the structural basis for MetJ specificity and cooperativity (91, 258). The archetypal member of the ribbon-helix-helix (RHH) class of DNA-binding proteins, MetJ interacts with the major groove of DNA via a β-ribbon (114). Sequence specificity is achieved through both direct and indirect readout mechanisms: MetJ binding forms direct hydrogen bond contacts with four base pairs of a met-box and also depends on the conformational flexibility of the target sequence (91, 258). The repressor binds to the center of a met-box and slightly kinks the DNA (~25 degrees) to narrow the minor groove, tightening the DNA around the protein dimer. Cooperativity is accomplished through interactions between the helices of adjacent MetJ proteins. This results in an array of repressor proteins that wrap around the DNA to form a left-handed superhelix (258).
Structures have been determined for the apo-, holo-, and DNA-bound forms of MetJ (221, 258). Surprisingly, there is very little change upon binding to either corepressor or DNA. AdoMet binds to MetJ on the side opposite that which interacts with the operator, and, instead of directing a conformational change, appears to exert a long-range electrostatic effect. The positive charge on the tertiary sulfur propagates through the repressor to interact with the negative phosphodiester backbone. (In contrast, adenosylhomocysteine [which lacks the positive charge and methyl group] competitively binds to MetJ without increasing the affinity for DNA.) This unusual means of control has been termed the "electric genetic switch" (154, 204, 212).
The mechanism and specificity of MetJ repression has received considerable scrutiny, as it involves protein-small molecule, protein-DNA, and protein-protein interactions. Thermodynamic and kinetic parameters have been examined for wild-type and mutant proteins using a variety of biophysical techniques (51, 115, 125, 154, 263). MetJ appears to bind to DNA loosely and nonspecifically; then, via a sliding mechanism, the repressor is able to locate a specific operator site where it is more tightly bound (154). In vitro evolution experiments (SELEX) indicate that the tandem met-box consensus sequence derived from natural operators is in fact the preferred binding site for MetJ (113). A bioinformatics approach was recently employed to identify potential MetJ-binding sites within the E. coli genome by taking into account the primary sequence information as well as the sequence-dependent conformation of the DNA (166). In addition to all of the known MetJ-regulated genes, several putative operators were found. Among these were metK (AdoMet synthetase) and two genes coding for the transport of methionine and S-methylmethionine, abc (renamed metN) and ykfD (renamed mmuP), respectively. Recent experiments suggest that transcription of these genes is indeed regulated by MetJ (see sections on methionine uptake and metabolism of S-methylmethionine), although the repressor-binding sites have not yet been verified (110, 120, 181, 275, 315). Other novel sites include ahpC (alkyl hydroperoxide reductase), hemG (involved in heme biosynthesis), atpA (α subunit of ATP synthase), yhaQ (renamed tdcG, serine deaminase 3), mgtA (Mg2+ and Ni2+ transporter, ATPase), dmsB (chain B of dimethyl sulfoxide reductase), and several proteins of unknown function (yafD, ybdH, and ycbK). ybdH (encoding a putative oxidoreductase) is divergently transcribed from ybdL, which encodes a protein that was recently crystallized and found to be a PLP-dependent methionine aminotransferase (71). A role for MetJ binding has not been investigated for any of these genes, and they may represent false positives. However, in view of recent studies that suggest inhibition of methionine biosynthesis under stress conditions (discussed in the final section of this chapter), it will be interesting to see whether these genes may in fact be repressed by MetJ.
In addition to repression by MetJ, several met genes are also subject to regulation by a second protein, the product of the metR gene (MetR). MetR acts in trans to activate expression of metA and the methionine synthases (metE and metH), while opposing MetJ-mediated repression of metF (56, 168, 286). Strains lacking metR have the same phenotype as those containing mutations in metE, suggesting that MetR is absolutely required for metE expression (286). Growth of metR mutant strains depends on exogenously supplied methionine; addition of B12 to the medium allows slow growth, presumably via low-level expression of metH, the B12-dependent methionine synthase.
The metR gene encodes a 36-kDa protein that has been found to exist as a homodimer in solution (36, 176). MetR is a member of the LysR family of transcriptional regulators and is therefore expected to contain a helix-turn-helix DNA-binding domain at its N terminus (242). However, although the MetR protein has been purified, its structure has not yet been determined (35, 177).
While MetR generally serves as an activator protein, it has also been found to repress its own transcription (177, 283). The metR and metE genes are divergently transcribed and contain overlapping promoter elements (Fig. 9) (214). Thus, the apparent repression of metR may simply be a consequence of steric hindrance incurred upon binding of MetR and RNA polymerase to the metE promoter (285). This arrangement also provides a rationale for MetJ-mediated repression of both metR and metE genes (283, 307). As a result, metE is dually repressed by MetJ, which acts directly to repress metE expression and indirectly by repression of the synthesis of the MetR activator.
Homocysteine serves as a coactivator of MetR-mediated activation of metE and metF expression (and ostensibly as a corepressor of metR transcription) (35, 283, 284). In contrast, expression of the metA and metH genes is negatively impacted by homocysteine; however, this appears to be an indirect effect due to decreased MetR levels, since homocysteine does not influence MetR activation of metH transcription in vitro (35, 168, 284) (M. L. Urbanowski and G. V. Stauffer, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, abstr. H-175). Nevertheless, there is clearly a disparity in the effects of homocysteine on the MetR-mediated regulation of different met genes, and it has been suggested that the spacing between the MetR-binding site and the −35 region of the gene may determine the role that homocysteine plays (168, 285). Experiments indicate that homocysteine binding does not alter the affinity of MetR for its cognate DNA, and the mechanism by which homocysteine modulates MetR activity is unclear (168, 285). Truncation mutations of MetR demonstrated that amino acids 88 to 182 are required for activation of metE but not metH, which was taken as evidence that homocysteine-binding determinants are likely located within this region (176). E. coli strains expressing mutant MetR proteins that no longer respond to homocysteine have also been generated, but further biochemical studies have not yet been reported (31).
The MetR-binding sites within each met operator have been elucidated from footprinting experiments and mutational analyses (32, 36, 168, 285). A consensus-binding site has been identified, which consists of the interrupted palindrome TGAA – – T/A – – TTCA. Interactions between MetR and the α-CTD of RNA polymerase appear to be involved in activation of metE and metH, and subtle modifications may contribute to differences in activation of these genes (86, 132, 133). However, the mechanism by which MetR modulates expression of the met genes is only beginning to be unraveled.
Recent studies have shown that a second MetR-binding site (site 2) is located within the promoter region of the divergently transcribed metR and metE genes in Salmonella (Fig. 9) (308). (A homologous binding site is also apparent within the sequence of the E. coli intergenic region.) MetR was found to bind to site 1 (which has a perfect match to the consensus sequence) with high affinity, but independently of homocysteine. In contrast, binding to site 2, which has a 7-of-9-base-pair match to the consensus sequence, is much weaker and depends on homocysteine. MetR binding at site 1 augments binding to the adjacent lower-affinity site, reminiscent of the cooperative mechanism employed by other LysR-type activators (242). Although both sites appear to be necessary for transcription, evidence suggests that activation occurs primarily from site 2. Mutation of site 2 to the consensus sequence resulted in a site where MetR binding was independent of both homocysteine and site 1. This suggests that the homocysteine dependence may originate from the deviation of site 2 from the consensus sequence, while site 1 serves to enhance MetR binding to the weaker, homocysteine-dependent site (308). However, MetR activation of metH expression appears to occur by a different mechanism (as evidenced by a single MetR-binding site and the disparate interactions with RNA polymerase), and further understanding of the subtle differences that account for these variations will provide insight into the means by which the bacteria controls methionine production.
MetR also serves as an activator of glyA (encoding serine hydroxymethyltransferase) expression and recently has been found to affect hmp transcription (Hmp is a flavohemoglobin involved in the detoxification of nitric oxide) (180, 215). Further details concerning MetR-mediated regulation of these genes (as well as the methionine biosynthetic genes) may be found in Chapter Regulation of Serine, Glycine, and One-Carbon Biosynthesis. The MetR homologue in V. harveyi reportedly represses the lux genes that are responsible for bioluminescence (without affecting autoinducer levels), suggesting a possible role for MetR in quorum sensing (41).
The final means of transcriptional control of the met genes involves B12. Addition of B12 to the medium serves to repress metE and, to a limited extent, metF (61, 152, 186). Though it has been known for some time, the mechanism responsible for the severe repression by B12 is still unclear. Repression of metE has been shown to require formation of the MetH-B12 holoenzyme as well as CH3-H4folate, and thus the product of the metF gene, MTHFR, is necessary to supply the folate derivative (37, 152, 186, 191). As reported in the previous edition, it is possible that the efficient production of methionine by B12-MetH serves to deplete the homocysteine pool, thus removing the coactivator of MetE expression (306). However, direct measurements of the homocysteine pool size and other physiological experiments argue against such a mechanism (37, 100). The expression of MetE homologues in other species of bacteria appears to be regulated by B12 riboswitches (224). (Riboswitches are RNA aptamers found in the leader sequences of genes, which selectively bind metabolites, such as B12, and modulate gene expression accordingly.) However, no evidence has been found for riboswitch-mediated control of metE expression in E. coli or Salmonella, even though B12 riboswitches appear to modulate expression of cobalamin biosynthetic and transport genes in these bacteria (192, 197). Nevertheless, the observed repression by B12 allows for reciprocal control of the two methionine synthase proteins: when B12 is available, the more efficient B12-dependent enzyme (MetH) is responsible for methionine synthesis, while expression of the slower B12-independent protein (MetE) is repressed. However, when B12 is unavailable, MetE is able to provide necessary methionine. Therefore, in the absence of B12, MetE is the rate-limiting step in de novo methionine biosynthesis, but in the presence of B12, MTHFR assumes this role (61).
The E. coli metA, metB, metE, and metF genes were also reported to be mildly repressed by Lrp, the leucine-responsive regulatory protein, in a global analysis of Lrp-regulated proteins (271). Similarly, microarray analysis has hinted that under anaerobic conditions, metL (and presumably metB since they are in the same operon) may be activated by Fnr, the fumarate nitrate reduction regulatory protein (237). Putative-binding sites for Fur, the ferric uptake regulator protein, have been identified in the metJ and metH promoters, suggesting that Fur may repress transcription of these genes (264). However, further experiments addressing regulation by Lrp, Fnr, or Fur do not appear to have been performed, and it is unclear whether these effects are direct or indirect.
Both E. coli and Salmonella are able to accumulate methionine against a concentration gradient. The rate of methionine uptake was shown to be regulated by the intracellular methionine pool (138). Low methionine levels lead to an increased rate of import, while ample supplies of methionine result in decreased uptake. Methionine transport is also sensitive to elevated temperature, arsenate, and osmotic shock (55, 141, 290). Early studies found that uptake of L-methionine is accomplished by two different systems: a high-affinity transporter (Km, ~0.1 μM) encoded by the metD locus, and at least one low-affinity transporter (Km, ~20 to 40 μM) called MetP (8, 9, 137, 140). (The high-affinity system in Salmonella was initially referred to as MetP, but to avoid confusion with the E. coli system [where MetP describes the low-affinity transporter] the nomenclature was eventually changed to reflect the E. coli classification.) MetD (but not MetP) can also transport D-methionine and other methionine analogues, such as N-acetylmethionine and methionine sulfoxide (7, 139). Intracellular conversion to L-methionine thus allows for slow growth of methionine auxotrophs on these analogues (52).
Recent identification and characterization of the genes constituting the metD locus in E. coli confirmed earlier hints that the high-affinity system is a multiprotein ATP-binding cassette (ABC) transporter (110, 181). The high-affinity MetD transporter consists of three proteins: an ATPase (the abc gene product), a permease (the yaeE gene product), and a substrate-binding protein (the yaeC gene product), which were renamed metN, metI, and metQ, respectively (Fig. 10). (This is not to be confused with the metQ gene initially described by Simon and Hong [252], which appears to refer to malI [see "Homocysteine Synthesis," above].) The 343-amino-acid MetN sequence contains a classical ATPase motif; hydrolysis of ATP provides the energy to drive transport (141, 181, 315). The MetI permease contains 217 amino acids and includes five putative transmembrane domains (181, 315). MetQ was determined to function as the substrate-binding receptor, and sequence analysis suggests that it is a 271-amino-acid membrane-anchored lipoprotein, which is unusual for gram-negative bacteria. A second protein, NlpA (lipoprotein 28, the product of the nlpA gene) may also serve as a receptor for methionine, but much less efficiently (315).
Strains lacking metQ show greatly reduced transport of both L- and D-methionine, suggesting that MetQ binds both isomers (315). Transport of D-methionine (by the MetD transport system) is competitively inhibited by the L- isomer; however, L-methionine uptake is only weakly inhibited by D-methionine (139, 315). The preferential transport of L-methionine may be based on a difference in the relative affinities for the isomers: the Km for L-methionine is ~0.1 μM compared to ~1.2 μM for D-methionine (137, 139, 315).
Expression of the high-affinity MetD transport system was found to be regulated by the MetJ repressor (110, 181, 315). Three met-boxes (with 50, 62.5, and 100% identity to the consensus sequence) are located upstream of the metD locus, overlapping the predicted −10 and −35 regions of the promoter (110, 315). However, the MetJ-binding site has not yet been experimentally determined.
Phylogenetic analysis suggests that the MetD permease is the founding member of a new family of transporters within the ABC superfamily, named the methionine uptake transporter (MUT) family (315). Proteins within the MUT family are predicted to transport various sulfur compounds, and interestingly, several appear to also be virulence factors.
In the absence of the high-affinity MetD system, the low-affinity transporter(s), termed metP, is able to provide the cell with L-methionine (140). However, much less is known about this means of methionine transport, which is genetically uncharacterized. In contrast to MetD, the low-affinity transporter appears to be specific for L-methionine or Se-methionine and does not transport D-methionine (140). It has been suggested that the major function of at least one of the low-affinity systems may actually be to transport leucine (9).
Early studies established that E. coli methionine auxotrophs are able to utilize S-methylmethionine as a source of methionine (10). Yet, it was not until much later that the enzyme responsible for methionine production via methyl transfer from S-methylmethionine to homocysteine was identified (Fig. 11a) (194, 275). In strains lacking metE and metH, the mmuM gene product (for S-methylmethionine utilization, originally yagD), provides for growth on exogenously supplied S-methylmethionine (275). Thus, the S-methylmethionine: homocysteine methyltransferase, MmuM, formally represents a third methionine synthase (in addition to MetE and MetH). Although MmuM does not play a role in de novo methionine biosynthesis, it apparently allows the bacteria to take advantage of the S-methylmethionine produced by many plant species.
MmuM was initially identified by its homology (40% identical) to the Astragalus bisulcatus selenocysteine methyltransferase, the smtA gene product, which is involved in selenium detoxification (194, 275). SmtA catalyzes the methylation of selenocysteine using S-methylmethionine to form methylselenocysteine (Fig. 11b), which is not incorporated into proteins and is therefore nontoxic. E. coli MmuM is able to catalyze this reaction, but much less efficiently, and only confers a slight increase in selenium tolerance compared with that of SmtA. Hence, although MmuM and SmtA catalyze similar reactions, their substrate specificity and physiological roles appear to be quite disparate.
In addition to S-methylmethionine, MmuM also accepts S-adenosylmethionine (AdoMet) as a methyl donor (Fig. 11c) (194). However, S-methylmethionine appears to be the predominant substrate in vivo, which seems sensible from a physiological viewpoint. Copious utilization of endogenous AdoMet to produce methionine, a substrate for AdoMet synthesis, would produce futile cycling. MmuM may be specific for the nonphysiological S(+) isomer of AdoMet, and it has been suggested that the enzyme could play a role in removing any of this isomer that was produced by spontaneous racemization.
The E. coli MmuM protein has been overexpressed, purified, and found to be a monomer of ~35 kDa (194). It has low sequence homology to the N-terminal domain of MetH and appears to contain the three conserved cysteine residues that serve as zinc ligands (275). This suggests that MmuM may employ a zinc ion to catalyze methyl transfer in a manner similar to that of the other methionine synthases, though detailed studies have not yet been performed.
An S-methylmethionine permease, the product of the mmuP gene (originally ykfD), appears to lie upstream of (and slightly overlapping with) the mmuM gene (275). MmuP displays homology to other amino acid transporters, and E. coli lacking mmuP are no longer able to metabolize S-methylmethionine. Four tandem met-boxes (with 62.5, 75, 75, and 62.5% identity to the consensus sequence) are located upstream of mmuP, suggesting that expression is regulated by MetJ and AdoMet. Both the S-methylmethionine methyltransferase activity and expression of mmuM have been shown to be modulated by methionine levels (10). These observations are consistent with MetJ-mediated repression of the genes, but further studies are necessary to confirm the role of MetJ in regulation of S-methylmethionine metabolism.
Methionine serves as a central player in several fundamental cellular processes. In its most well-known capacity, methionine provides one of the twenty amino acid building blocks of proteins. Its flexible thioether side chain is relatively unreactive and tends to participate in hydrophobic interactions. Protein methionyl residues contribute to structural features, nonpolar recognition surfaces, and occasionally metal (Fe2+ and Cu2+) ligation (92, 145, 291). Yet, methionine is used relatively sparingly in proteins; in fact, only tryptophan occurs less often (150). However, methionine plays an added role in protein synthesis because formylated methionine is required to initiate the translation of all proteins (1, 296).
Several met genes are devoted to protein translation processes. The metG gene product (methionyl-tRNA synthetase, MetRS) charges tRNAs with methionine, which are used for either elongation (methionyl-tRNAMet) or initiation (methionyl-tRNAfMet) (288). The duplicate metT and metU genes both code for tRNAMet (21). There are two initiator methionine tRNAs, a major form (tRNAfMet1 encoded by triplicate genes, metV, metW, and metZ) and a minor form (tRNAfMet2 encoded by metY) (127, 148). The fmt gene product transfers the formyl group of 10-formyl-H4folate to a methionyl-tRNAfMet to generate formylmethionyl-tRNAfMet, which is used to initiate translation (288). For further discussion of the methionyl-tRNA synthetase, the reader is directed to Chapter Aminoacyl-tRNA Synthetases in the Bacterial World; additional details concerning translation initiation can be found in Chapter Translation Initiation.
Methionine also serves as the precursor of S-adenosylmethionine, which is an essential molecule employed in numerous biological processes (as discussed in the next sections). In addition, some strains of E. coli (most notably strain SPAO) have been reported to utilize methionine as a substrate for the production of ethylene (218, 219). The pathway appears to proceed via the formation of 2-keto-4-methylthiobutyric acid (KMBA), the transaminated derivative of methionine. An unidentified oxidoreductase is then believed to oxidize KMBA to generate ethylene, methanethiol, and carbon dioxide. Reference 90 provides a comprehensive review of the studies describing ethylene synthesis from methionine.
Methionine can be activated by condensation with ATP to form S-adenosyl-L-methionine (AdoMet or SAM), which plays many roles within the cell (detailed below). The synthesis of AdoMet is catalyzed by S-adenosylmethionine synthetase (MAT) (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6), the metK gene product. In an unusual reaction, the triphosphate of ATP is replaced by methionine to form the sulfonium, AdoMet (Fig. 12). Then, in the second step, the tripolyphosphate (PPPi) is hydrolyzed to produce pyrophosphate (PPi) and orthophosphate (Pi). Thus, MAT catalyzes two reactions at opposite ends of the phosphate chain, using a single active site.
E. coli MAT has been overexpressed, purified, and extensively characterized. The enzyme is a homotetramer with 42-kDa subunits (170). Crystal structures revealed that the "peanut-shaped" tetramer is composed of two spherical dimers (270). Each dimer contains two active sites formed from residues supplied by both subunits. Two divalent cations (usually Mg2+) per monomer are required for catalysis, and a monovalent cation (generally K+) stimulates AdoMet formation (170).
A wide variety of biochemical techniques have been employed to gain insight into the mechanism of catalysis (see reference 151 and references therein). The reaction proceeds via an SN2 attack on ATP by the sulfur of methionine to displace PPPi (Fig. 12). Then, in a closely coupled but chemically distinct reaction, PPPi is hydrolyzed between the β- and γ-phosphates prior to AdoMet release. The products PPi and Pi bind more weakly to the enzyme than the PPPi intermediate; therefore the PPPiase activity of the enzyme (which is stimulated by the presence of AdoMet) helps provide for efficient turnover. In depth kinetic analyses have determined the rate constants for each step of the reaction, and, together with mutagenesis and crystallographic studies, a detailed mechanism has been proposed (151, 178, 269, 273).
Both AdoMet formation and PPPi hydrolysis appear to occur without substantial rearrangement of the substrates within the enzyme active site (151). However, significant conformational changes of the enzyme are observed during catalysis and were traced to the movement of a flexible loop near the active site (151, 178, 272, 273). Only when the loop is open can substrates and Mg2+ enter the active site. This loop is among the least conserved region of an enzyme that has high homology to MAT proteins from other species. Yet, even though the active-site residues are strictly conserved, the rate of catalysis varies widely (by as much as 100-fold) between species; in fact, the E. coli enzyme has an activity greater than that measured for other MAT proteins. Moreover, mutations in the loop region have dramatic effects on AdoMet formation, suggesting that the sequence variability within the loop may explain the differences in rate (178, 273).
Early experiments suggested that E. coli contained a second homologous AdoMet synthetase encoded by the metX gene, which was proposed to be a gene duplication of metK (238, 239). However, further analysis of the genome indicated that metX does not exist, and that the metK gene product provides the only route to synthesis of AdoMet (195, 297). The availability of AdoMet appears to be essential for the cell, even though individual AdoMet-dependent reactions have not been found to be indispensable in E. coli (195, 297). Strains containing deletions in metK require the presence of a rescue plasmid (containing a functional metK), since E. coli cannot utilize exogenous AdoMet (297). Decreased levels of AdoMet have been found to result in hypomethylation of DNA and severe defects in cell division (195, 297).
Expression of metK appears to be repressed by MetJ complexed with its corepressor, AdoMet. As with the methionine biosynthetic genes, addition of methionine represses metK transcription and derepression occurs in mutant metJ strains (120). The transcription start site of metK that was proposed in the previous edition based on computer analysis has now been experimentally demonstrated (297). Putative met-boxes have been identified, but detailed studies have not yet been reported (99, 166). In addition to MetJ-mediated repression, evidence suggests that the expression of metK is repressed by the global regulator Lrp (the leucine-responsive regulatory protein) and induced by leucine (which antagonizes the effects of Lrp) (195, 271). This is particularly apparent in the metK84 mutant strain, which requires high concentrations of leucine for normal growth due to a mutation in the −10 region that leads to decreased levels of MAT (195, 297). Moreover, leaky metK mutants tend to accumulate suppression mutations in lrp (280). However, in-depth characterizations of the mechanism of metK regulation do not appear to have been performed.
AdoMet, the "active [form of] methionine," was initially discovered by Cantoni in 1953, and has since been found to participate in a great diversity of cellular processes ranging from biosynthesis to signal transduction and gene regulation (38). Much of AdoMet’s utility lies within its unique structure. The positively charged sulfonium ion makes AdoMet reactive toward a host of nucleophiles, and in principle, each group attached to the sulfonium moiety may be donated. Transfer of the methyl group accounts for the majority of reactions; however, all parts of the molecule are exploited by E. coli and Salmonella. AdoMet provides the adenosyl radical for radical SAM enzymes, the ribosyl group for queuosine biosynthesis, and aminobutyryl derivatives that contribute to spermidine synthesis, tRNA modification and biotin synthesis (Fig. 13).
AdoMet is a potent methylating agent and, as such, serves as the principle methyl group donor within the cell (Fig. 13A). Preference for AdoMet is based on the highly favorable energetics of transfer. The ΔG°' for methyl transfer from AdoMet to homocysteine (AdoMet + Hcy → AdoHcy + Met) is −17 kcal mol−1 compared with −7.8 kcal mol−1 for ATP hydrolysis (ATP → ADP + Pi) (4, 14, 77, 89, 189). Moreover, AdoMet is estimated to be approximately 1,000-fold more reactive (toward most substrates) than methyltetrahydrofolate, the second most frequently used methyl group donor (39).
E. coli and Salmonella contain AdoMet-dependent methyltransferases (MTases) capable of methylating a wide variety of substrates, including nucleotides, proteins, small molecules, and lipids at either oxygen, nitrogen, sulfur, or carbon centers. Table 1 details many of the AdoMet-dependent MTases that have assigned genes. Methylation of adenine and cytosine residues within DNA by the dam and dcm gene products is important in gene regulation and replication of DNA. Numerous MTase reactions are involved in production of modified rRNAs and tRNAs (see Chapters Modified Nucleosides of Escherichia coli Ribosomal RNA and Transfer RNA Modification, respectively). AdoMet-dependent methyl transfers are also central to the control of chemotaxis (cheR) and the biosynthesis of numerous molecules, including siroheme (cysG), menaquinone (ubiE), ubiquinone (ubiE and ubiG), and cobalamin (cbiE, cbiT, cbiF, cbiH, and cbiL in Salmonella). In addition, cyclopropane fatty acids obtain a methylene group from AdoMet; cyclopropane fatty acid synthase (the cfa gene product) appears to transfer the methyl group from AdoMet to an unsaturated fatty acid, which then loses a proton and is cyclized to form the cyclopropane product, which is primarily produced during stationary phase (131).
Table 1AdoMet-dependent MTases with assigned genes in E. coli and Salmonella. |
The crystal structures for many AdoMet-dependent MTases have been solved, and thus far divide into five structural groups (246). E. coli possess enzymes from four of the five classes. Further information concerning structural aspects of AdoMet-dependent MTases may be found in excellent recent reviews (43, 44, 246).
AdoMet plays a central role in an emerging class of proteins known as radical SAM enzymes. These proteins utilize an adenosyl radical to catalyze a broad assortment of reactions (85, 134, 155, 171). Notable examples include HemN (involved in anaerobic heme biosynthesis), BioB (which catalyzes the final step in biotin biosynthesis), lipoate synthase (LipA), and the activating enzymes of pyruvate formate lyase and anaerobic ribonucleotide reductase; however, the list of proteins within this family is rapidly expanding (257). Radical SAM enzymes all contain a [4Fe-4S] cluster, which upon reduction transfers an electron to the closely bound AdoMet molecule. This results in homolytic cleavage of the molecule, to generate methionine and the reactive adenosyl radical (Fig. 13B). The adenosyl radical serves to abstract a proton from the substrate to create a substrate radical, thereby initiating the chemical transformation unique to each enzyme. In general, AdoMet functions as a cosubstrate resulting in the production of 5'-deoxyadenosine and methionine. However, a few radical SAM enzymes have been found in other organisms that utilize AdoMet as a cofactor and regenerate AdoMet following each turnover.
AdoMet also provides an important component of the modified tRNA nucleoside queuosine (Fig. 13C) (130). In the penultimate step of de novo queuosine biosynthesis, S-adenosylmethionine:tRNA ribosyltransferase-isomerase (the queA gene product) transfers the ribosyl group of AdoMet to 7-(aminomethyl)-7-deazaguanosine (preQ1) modified tRNA, which is then rearranged to produce epoxyqueuosine (oQ). This unique reaction is the only known example where the ribosyl moiety of AdoMet is utilized. Adenine and methionine are also produced from the action of QueA. Further discussion of queuosine biosynthesis can be found in Chapter Transfer RNA Modification.
Aminobutyryl derivatives of AdoMet (obtained from the methionine portion of AdoMet) are also donated in a variety of cellular processes (Fig. 13D–F). In polyamine biosynthesis, an aminopropyl group obtained from decarboxylated AdoMet is transferred to putrescine, producing spermidine and methylthioadenosine (MTA, Fig. 13D). Further details concerning spermidine biosyntheses may be found in Chapter Biosynthesis of Arginine and Polyamines. The 3-amino-3-carboxylpropyl group of AdoMet is utilized for the production of 3-(3-amino-3-carboxypropyl)uridine (acp3U), a modification of phenylalanine tRNA (Fig. 13E) (196). And, in the second step of biotin biosynthesis, a PLP-dependent aminotransferase, DAPA synthase (the bioA gene product), transfers the amino group of AdoMet to 7-keto-8-amino pelargonic acid (KAPA) to generate 7,8-diaminopelargonic acid (DAPA, Fig. 13F). This is the only known reaction where AdoMet contributes its amino group to a transamination reaction.
MTA is generated as a byproduct of spermidine synthesis and as a consequence of acp3U tRNA modification (see Fig. 13). It is then cleaved by the pfs gene product (discussed below) to produce methylthioribose (MTR) and recover the adenine moiety. Many bacteria possess methionine salvage pathways that recycle MTR to regenerate methionine. This pathway was first fully characterized by Abeles and colleagues in Klebsiella pneumoniae, and has now been worked out in other organisms such as Bacillus subtilis and Pseudomonas aeruginosa (58, 116, 247, 249). However, E. coli and Salmonella do not appear to possess an analogous mechanism for methionine salvage. Instead, E. coli have been found to excrete MTR into the medium (244, 245). In organisms that salvage methionine, MTR is phosphorylated to MTR-1-phosphate by a kinase that is not present in E. coli and Salmonella, indicating that the pathway is not intact. This is consistent with the finding that a methionine auxotroph (metE) is unable to utilize MTA as a source of methionine (60). A discussion of additional experiments that suggest the absence of a methionine salvage pathway in E. coli may be found in the previous edition of this chapter (99).
S-Adenosylhomocysteine (AdoHcy) is produced as a consequence of the numerous AdoMet-dependent methyl transfer reactions that occur within the cell. In E. coli and Salmonella, this molecule is recycled in two discrete steps to complete the methyl cycle (Fig. 1). The pfs gene product catalyzes the hydrolysis of AdoHcy to yield S-ribosylhomocysteine (RibHcy) and adenine; RibHcy is further broken down by the luxS gene product to regenerate homocysteine (53). This is in contrast to mammalian systems where homocysteine is recovered from AdoHcy in a single step catalyzed by AdoHcy hydrolase.
Pfs (also called MTA/SAH nucleosidase or MTAN) (EC 3.2.2.9) catalyzes an irreversible cleavage of the N9-C1' glycosidic linkage of AdoHcy to produce adenine and RibHcy. This is the same enzyme that is responsible for the hydrolysis of 5'-methylthioadenosine (MTA) to generate adenine and 5'-methylthioribose (MTR, see above) (68, 74). Crystal structures of Pfs reveal a relatively open active site, which helps accommodate the bulkier tail of AdoHcy (156). MTA appears to be the better substrate with a Km that is 10-fold lower than that of AdoHcy (KmMTA = 0.43 μM versus KmAdoHcy = 4.3 μM) (54, 68). Nevertheless, both activities play important roles within the cell, since MTases are extremely sensitive to AdoHcy levels and MTA can inhibit polyamine synthesis (26, 28, 34, 57, 76, 262).
Recent biochemical studies have provided significant insight into the reactions catalyzed by Pfs. The active enzyme appears to be a dimer composed of identical 25-kDa subunits (156). Numerous crystal structures of the protein have been solved, including enzyme complexes that are believed to lie along the reaction coordinate (156, 157, 159, 160). Structural analyses in combination with kinetic isotope effect, steady-state kinetic, and mutational studies have led to a detailed description of the reaction mechanism (5, 158, 253). Upon substrate binding, the enzyme appears to undergo a conformational change that closes the active site and excludes extraneous water. The reaction then proceeds via the formation of a dissociative transition state possessing oxacarbenium ion character, which is attacked by an activated water in an SN1-type reaction to generate the two products. Based on these studies, transition state analogues have recently been generated and found to function as potent inhibitors of Pfs (160, 254). With dissociation constants in the femtomolar range, these molecules rank among the most effective noncovalent enzyme inhibitors known. This is particularly significant from a biological standpoint where Pfs is viewed to be an attractive target for broad-spectrum antibiotics.
The pfs gene is cotranscribed with btuF, which encodes a periplasmic B12-binding protein that together with the btuC and btuD gene products forms an ABC-type transporter for B12 uptake. While B12 is certainly important for the methyl cycle (as a cofactor for the B12-dependent methionine synthase, MetH), the significance of this association is unclear. There does not appear to be any evidence for involvement of Pfs in B12 transport.
E. coli lacking pfs were found to exhibit a severe growth defect (33). Cells grew slowly on LB medium, but were completely inhibited in minimal medium; addition of methionine only partially reversed this effect. Surprisingly, biotin was found to play a crucial role in abrogating the growth defect (33). While biotin supplementation alone does not completely restore normal growth, the pfs strain is phenotypically similar to cells that lack biotin synthase (the bioB gene product) (45). Biotin synthase is a radical SAM enzyme that utilizes a 5'-deoxyadenosyl radical (from AdoMet, see above) to produce biotin. 5'-Deoxyadenosine is generated as a consequence of this reaction and has been proposed to function as a potent inhibitor of biotin synthase (45, 201). Pfs was found to be able to utilize 5'-deoxyadenosine as a substrate, apparently degrading it both in vitro and in vivo (45). Hence, cells lacking Pfs may accumulate 5'-deoxyadenosine, which serves to inhibit biotin synthase and leads to the observed biotin auxotrophy. There are also indications that 5'-deoxyadenosine may similarly affect a second radical SAM enzyme known to be required for aerobic growth, lipoic acid synthase (the lipA gene product); supplementation of the pfs strain with lipoic acid in addition to biotin further counteracts the growth inhibition by 5'-deoxyadenosine (45). Thus, while MTA appears to be the preferred substrate of Pfs in vitro, the promiscuous specificity of the enzyme may allow a single nucleosidase to contribute to multiple important processes within the cell.
The recovery of homocysteine from RibHcy in E. coli was first described by Duerre and Miller in the 1960s (75, 76, 184). A "ribosylhomocysteinase" enzyme was reported to cleave the thioester linkage of RibHcy to generate homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD; see Fig. 1 and Fig. 14). However, further progress was not made until 2001 when the reaction was unexpectedly implicated in bacterial quorum sensing, thereby leading to an explosion of interest in this metabolic process (241, 304).
Quorum sensing is a means of cell-cell communication in which bacteria monitor their population density by measuring the concentration of small secreted signal molecules known as autoinducers. Based on this information, cells can alter their gene expression to better respond to changing environmental conditions. The autoinducer-2 (AI-2) system was first characterized in Vibrio harveyi, but production of AI-2 activity by the luxS gene product was discovered in a variety of bacteria, including E. coli and Salmonella (16, 265, 266). This suggested that AI-2 may provide a universal signal for interspecies communication, in contrast to other autoinduction systems, which appear to be specific for intraspecies signaling (309). Further work led to the surprising discovery that LuxS was the "ribosylhomocysteinase" enzyme previously described and that the DPD byproduct was the precursor to AI-2 (65, 179, 241, 250, 304, 316).
Several crystal structures of LuxS orthologues were solved before the reaction it catalyzed was even known (118, 164, 232). The enzyme is a homodimer with each active site positioned at the dimer interface. A divalent metal was discovered within the active site and found to play a critical role in catalysis. LuxS was initially crystallized with a zinc ion, however, further studies indicated that iron might be the metal utilized in vivo (320). Iron is found in the purified "native" enzyme and LuxS containing iron or cobalt is approximately 10-fold more active than the zinc-containing enzyme.
Recent studies have also provided significant insight into the kinetics and mechanism of LuxS (209, 222, 318, 319, 320). The enzyme catalyzes what is chemically an interesting reaction in an unprecedented manner. The metal ion appears to interact directly with the RibHcy substrate and participate in a series of acid-base-catalyzed proton transfers to generate 2- and 3-ketone intermediates, followed by a β-elimination reaction to yield the homocysteine and DPD products (Fig. 15). Homocysteine can then be remethylated by methionine synthase to produce methionine. However, DPD is quite unstable in solution and spontaneously cyclizes to form a series of interconverting furanones, including (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), which appears to function as the autoinducer for Salmonella (Fig. 14) (185). The AI-2 signal for V. harveyi is the boron adduct of the opposite THMF isomer (S-THMF), perhaps due to the abundance of boron in the marine niche of this organism (42). Thus the E. coli and V. harveyi autoinduction systems appear to be specific for distinct DPD derivatives within an ensemble of molecules that are in equilibrium with each other.
Quorum sensing has been implicated in numerous pathogenic processes; therefore, the regulation of LuxS and AI-2 synthesis is of considerable interest (142, 289). Instead of simply providing a density-dependent signal, AI-2 appears to communicate the metabolic state and growth potential of the cell (17, 265, 266). AI-2 levels have been shown to vary in response to growth phase, carbon source (due to catabolite repression), and stress conditions (66, 265, 292, 310). The concentration of extracellular AI-2 is closely correlated with pfs (which codes for the MTA/SAH nucleosidase) but not luxS expression, suggesting that AI-2 production is regulated at the level of LuxS substrate availability (17). No evidence has been found for regulation of either luxS or pfs transcription by AI-2 concentration. E. coli and Salmonella also contain systems responsible for uptake and degradation of AI-2 (267, 310). Hence, note that measurements of extracellular levels of AI-2 (which is derived from DPD) are not necessarily indicative of LuxS activity within the methyl cycle.
Cells lacking LuxS do not appear to have an obvious growth defect, suggesting that AdoMet recycling via the methyl cycle is not an essential process (260, 268, 293). (While there was no detectable difference in growth for Salmonella and E. coli W3110, a luxS mutant strain of enterohemorrhagic E. coli was reported to actually grow faster than the wild-type parent.) However, microarray studies indicate that a mutation in luxS impacts many genes (67, 260, 293). The pleiotropic nature of the luxS mutation makes it difficult to deconvolute the effects that are the result of AI-2-mediated signaling from those that are simply due to alterations in metabolic processes. In V. harveyi, the reaction catalyzed by LuxS clearly links the methyl cycle to AI-2-mediated communication; however, it has been argued that E. coli and Salmonella respond to DPD derivatives primarily as toxic metabolites rather than signaling molecules (293, 303, 304). Thus, additional studies will be necessary to completely decipher the role(s) that LuxS plays within the cell. Further discussion of LuxS in relation to bacterial quorum sensing may be found in Chapter Acidic and Alkaline Stress.
Given the many contributions of methionine to fundamental cellular processes, it may not be surprising that there appears to be a link between methionine availability and the cellular response to various stress conditions. Emerging evidence suggests that inimical processes may target the methionine biosynthetic pathway leading to a methionine limitation that can have profound cellular consequences.
The availability of methionine has long been known to be critical for bacteria experiencing heat-shock conditions. Early studies showed that at increased temperatures E. coli growing in minimal media adopt a significantly slower growth rate, which may be reversed by supplementation with exogenous methionine (226, 227). The methionine limitation was traced to a defect in the first enzyme of the biosynthetic pathway, homoserine transsuccinylase (HTS, the metA gene product) (228). HTS was found to be a thermally unstable enzyme that is reversibly inactivated at elevated temperatures. Its transcription is upregulated by heat shock in an rpoH-dependent manner, presumably to help compensate for decreased HTS activity (22, 229). However, while mRNA levels increase 40- to 50-fold, protein levels only rise approximately 2- to 5-fold due to energy-dependent proteolysis of HTS (23). At even higher temperatures (above 45°C), the protein appears to accumulate within insoluble aggregates and cells become methionine auxotrophs (107).
Cultures challenged by oxidative stress also appear to experience a growth limitation that depends on methionine levels. E. coli that are deficient for the manganese and iron superoxide dismutases (the sodA and sodB gene products, respectively) require the addition of methionine or cysteine for aerobic growth (20, 40). This phenotype was originally traced to leakage of sulfite through a damaged cellular envelope (19, 20). However, subsequent analyses have suggested that the bacterial membrane is largely unaffected by oxidative stress and therefore further investigations are warranted (128). In unrelated studies, 3'-phosphoadenylylsulfate (PAPS) reductase, an important enzyme in sulfate assimilation, was found to be oxidatively inactivated, and it is conceivable that this enzyme may be targeted in the sodA sodB strain (165).
Oxidative stress conditions have recently been shown to have a dramatic impact on the final step in methionine biosynthesis, that catalyzed by MetE (B12-independent methionine synthase, the metE gene product) (121). Biochemical studies indicate that MetE is inactivated by oxidation of a single cysteine residue (cysteine 645 in the E. coli enzyme), which lies strategically at the entrance to the active site. Formation of a mixed disulfide between cysteine 645 and glutathione leads to a conformational change and serves to inactivate the enzyme in a reversible manner. In E. coli challenged by oxidative stress, MetE was found to be exquisitely sensitive to oxidation (121, 163). Moreover, under these conditions, a methionine auxotrophy occurs concomitant with MetE oxidation (121). Thus in cells confronted with many types of oxidative stress, methionine appears to become limiting for growth as a result of oxidant-mediated inactivation of MetE.
MetE was also reported to be prone to aggregation in E. coli shifted to 45°C (188). However, the purified protein is stable at temperatures up to 55°C (121). Since aerobic heat shock may be coupled with oxidative stress, aggregation of MetE may not be an intrinsic function of temperature, but instead, a consequence of oxidative conditions in vivo. However, additional studies are necessary to fully investigate this phenomenon.
Oxidation of methionine itself also occurs during oxidative stress. To combat this damage, cells employ methionine sulfoxide reductases (298). MsrA and MsrB (the products of the msrA and msrB genes) function to reduce free methionine sulfoxide as well as oxidized protein methionyl residues. (The presence of these enzymes allows bacteria to utilize methionine sulfoxide as a limiting source of methionine.)
A methionine limitation also appears to be induced by weak organic acids, such as acetic acid. In E. coli, the source of this response was traced to the last step of methionine biosynthesis, the conversion of homocysteine to methionine (225). Treatment with weak acids appears to generate a level of oxidative stress, since many antioxidant and chaperone proteins are induced; therefore, it seems reasonable to expect that MetE could be inhibited under these conditions (6). However, overexpression of MetE was reportedly unable to correct the methionine deficiency (225). Nevertheless, inhibition of this step by acetate was found to result in intracellular accumulation of homocysteine. Such high concentrations of homocysteine were determined to be toxic due to inhibition of threonine deaminase, the enzyme that catalyzes the first step in isoleucine biosynthesis (225, 281). Therefore, upon acetate treatment cells are not only limited for methionine, but the resulting buildup of homocysteine also leads to inhibition of branched-chain amino acid biosynthesis. Supplementation with methionine alleviates the growth defect by supplying necessary methionine and repressing the methionine biosynthetic genes, thereby reducing homocysteine levels and relieving the inhibition of threonine deaminase. It seems physiologically sensible for cells to shut down branched-chain amino acid biosynthesis under conditions where methionine is limiting and translation is likely slowed considerably. In Salmonella, weak acids have also been reported to affect homoserine transsuccinylase (HTS), particularly in strains lacking polyphosphate kinase (217). Polyphosphate is proposed to act as a chemical chaperone for HTS, helping to refold it or increase the proteolysis of denatured protein that accumulates under stressful conditions.
Several reports have indicated that the methionine biosynthetic pathway is also impacted by treatment with S-nitrosoglutathione (GSNO) and related compounds (64, 81, 180). The effect seems to be centered around homocysteine, which has been shown to readily react with GSNO. Homocysteine levels are suggested to be a factor in helping to protect against GSNO toxicity and modulate the cellular response. Alternate explanations have been proposed to explain the protective effects of homocysteine, but their relative importance is not yet clear.
Not unexpectedly, sulfur-limiting conditions have recently been demonstrated to have a major impact on methionine biosynthesis (108, 109). For E. coli growing with a poor source of sulfur, expression of almost all of the methionine biosynthetic genes (metA, metB, metC, metF, metK, and metL) is increased, except metE, whose expression is severely decreased. It is reasoned that sulfur limitation leads to lower AdoMet levels, which causes derepression of the met genes resulting in the observed increase in their expression. However, metE expression requires MetR, which utilizes homocysteine as its coactivator. Depletion of homocysteine when sulfur is limiting prevents activation of metE expression, thereby overriding MetJ/AdoMet derepression, leading to a decrease in metE transcription. The utility of the MetR subcircuit makes sense in the case of sulfur limitation, where some level of oxidative stress appears to be induced. Increased synthesis of MetE (a large protein that is inactivated by oxidative stress) would be wasteful. When homocysteine levels rise indicating conditions are more favorable, MetE may be expressed and methionine biosynthesis can resume.
Several of the methionine biosynthetic proteins have been found to require cellular chaperones for production of the active enzymes. E. coli depleted of GroEL (by shifting a temperature-sensitive groEL strain to the nonpermissive temperature), greatly overexpress cystathionine β-lyase (CBL, the metC gene product) and MetE (122). In fact, MetE becomes the most abundant protein synthesized, even though cells are growing in rich media and are not lacking methionine. Subsequent studies identified MetE, methylenetetrahydrofolate reductase (MTHFR, the metF gene product), and S-adenosylmethionine synthetase (MAT, the metK gene product) as in vivo substrates of GroEL (123). Moreover, folding of MTHFR and MAT has been shown to absolutely require GroEL (149). Hence, upon depletion of GroEL, misfolded MAT may be expected to accumulate. Lack of AdoMet, the MetJ corepressor, should serve to derepress the met genes, leading to the observed overexpression of MetE and CBL. Additional studies suggest that MetE, MTHFR, and MetH (B12-dependent methionine synthase, the metH gene product) may interact with the DnaK and ClpB chaperones, while MetE and MetH also appear to associate with trigger factor (69, 188, 312). Thus conditions that place pressure on cellular chaperones and folding pathways could severely impact the methionine biosynthetic pathway.
Modulation of methionine levels in response to stressful conditions further increases the complexity of its regulation (see previous section on regulation of methionine biosynthesis). However, such a mechanism provides the cell with the means to control its response at multiple levels. The availability of methionine and one-carbon units should influence the rate of protein synthesis, since formylated methionine is required for initiation of translation (94). Limiting methionine under adverse conditions may slow protein translation (see, for example, references 205 and 227), preventing rapid growth under dangerous conditions and allowing the cell to focus its energy towards combating the crisis at hand. Moreover, it seems likely that autoinducer-2 (AI-2) production is affected by methionine availability (since the precursor for AI-2 synthesis, AdoMet, is directly derived from methionine), which could provide an efficient means of communicating the metabolic state of the cell under hostile circumstances. Thus an emerging understanding of the impact that stressful environments have on methionine biosynthesis further delineates the central role of this basic metabolic pathway in the lifestyle of E. coli and Salmonella.
We thank R. Pejchal for providing Fig. 7 and 8.
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Module3.6.1.7
