Molecular Microbiology (1991) 5(7), 1593-1597
ADONIS 0950382X9100175N
MicroReview Regulation of methionine synthesis in Escherichia coii H. Weissbach')' and N. Brot Roche Research Center, Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USA.
Summary The biosynthesis of methionine in Escherichia coli is under compiex reguiation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyimethionine which functions as a corepressor for the MetJ protein. Recentiy, a new reguiatory locus, metR, has been identified. The MetR protein is required for both mefE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metaboiic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metEgene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group. Introduction The genes involved in methionine biosynthesis in Escherichia coli are scattered throughout the chromosome and are referred to as the 'met reguion' (Bachman and Low, 1980). The cloning of many of these genes has made it possible to elucidate the mechanisms that are involved in the complex regulation of this pathway. As shown in Fig. 1, the biosynthesis of methionine involves the convergence of two metabolic pathways, one leading to the formation of homocysteine, and the other to the methyl donor, N^-CH3-H4-folate. The final methyl transfer to homocysteine can be carried out by either of two methyltransferases, i.e. the non-BT2 enzyme coded for by the metE gene or the B,2 methyitransferase {the product of the metH gene). A major metabolic product of methionine is S-adenosylmethionine (AdoMet), which is not only Received 1 February, 1991, "For correspondence. Tel. (201) 235 3376; Fax (201) 235 6578.
an important methyl donor in biological systems but is, as will be described later, a key component in the regulation of the methionine biosynthetic pathway. There are three distinct types of regulation of methionine biosynthesis in E. coli These include repression of the mef genes when the organism is grown in the presence of methionine, activation of specific met gene expression mediated by the MetR protein, and repression of specitic genes by vitamin B12. Table 1 lists the regulatory genes that are involved in methionine synthesis and their gene products. The regulation by methionine of its own synthesis involves two genes, metJ and metK. The metJ gene codes for a repressor protein that regulates ail of the met genes with the exception of metH. MetJ mutants constitutively express high levels of the methionine biosynthetic enzymes. MetK mutants also show a similar phenotype. This gene codes for AdoMet synthetase, the enzyme responsible for the synthesis of AdoMet, a corepressor for the MetJ repressor protein. The metR ger\e product is a transactivator of metE and metH expression but also autoregulates its own synthesis. Finally, the metH gene product (B,2 methyitransferase) is invoived in the repression of the metE and, to a lesser degree, metF expression. Each of these modes of regulation is discussed below.
Role of the mefJand metKgenes in methionine regulation As noted above, the expression of all of the met genes, with the exception of metH, is repressed when E. coli is grown in the presence of high levels of methionine (Cohn et ai, 1953; Wijesundera and Woods, 1953; Cohen and Jacob, 1953; Rowberg, 1965; Patte et ai, 1967). Two regulatory mutants, meU and metK (Table ^), were initially isolated that showed constitutive expression of the mef genes even in the presence of methionine (Holloway ef ai, 1970; Su and Greene, 1971; Kung et ai, 1972; Greene ef ai. 1970; 1973). It is now well established from in vitro experiments that the MetJ protein requires AdoMet as a corepressor for maximum activity (Shoeman etai. 1985a; Saint-Girons etai, 1986). This explains the observation that both genes are essential for reguiation of the methionine biosynthetic pathway in vivo.
1594
H Weissbach and N. Brot Homoserine
(F)
Homocysteine + N^-methyl-H4-foIate (H) Methionine
S-Adenosy I melhionine (AdoMet} R g . 1. The biosynthesis of methior)ine in E. coli. Letters in parentheses represent specific methionine {med genes.
The availability of a highly defined DNA-dependent coupled protein synthesis system (Kung et ai, 1977; Zaruckl-Schutz et ai, 1979) as well as a simplified gene expression system based on the formation of the first dior tripeptide of the gene product (Robakis et ai, 1981; Cenatiempo etai. 1982; Robakis etai. 1983), facilitated in vitro studies on the roie of the MetJ protein in expression of the met genes. The initial studies used a plasmid carrying ttie metF gene as template, and a simplified dipeptide system to examine the role of the purified MetJ protein (Smith etai, 1985) and AdoMet in me/Fexpression {Shoeman etai, 1985b). In this assay the synthesis of the AZ-terminal dipeptide of the metF gene product. fMet-Ser, was measured. The MetJ protein alone significantly repressed expression of the mefF gene v^rhen concentrations above 2 MQ pe"" incubation were used. However, in the presence of AdoMet, only one tenth of the amount of MetJ protein was required to obtain comparable inhibition. AdoMet gave a maximum response at between 5 x 10"^ M and 5 x 10"^ M. In other experiments. a highly defined in vitro system was used to study met gene expression in which the protein product was determined (Shoeman et ai. 1985a). The results clearly showed that the MetJ protein and AdoMet inhibited the in vitro synthesis of the MetF, MetB. and MetL proteins. The roie of AdoMel in the regulation of met gene expression has been further examined in our laboratory and by others (Shoeman etai. 1985b; Saint-Girons etai, 1986). Although AdoMet is well established as a biological methyiating agent, there is no evidence that it methyiates either the met gene regulatory region or the MetJ protein. Under the conditions used, the binding of AdoMet to MetJ was quite weak and a stable complex could not be isolated (Shoeman et ai. 1985b). although equilibrium dialysis studies have shown complex formation with an apparent dissociation constant of 200 M ^
(Saint-Girons etai, 1986). Methionine. S-adenosylhomocysteine or adenosine cculd not replace AdoMet in this reaction {Shoeman et ai, 1985b). It was concluded that AdoMet functions as a corepressor for the MetJ repressor protein by enhancing binding of the MetJ protein to the promoter region of the met genes by a factor of 10 or more. The regions on the me/gene promoters at which MetJ protein interacts were initially identified by DNase footprinting analysis. Subsequent examination of the DNA sequence of several met promoters by Saint-Girons and coworkers (Saint-Girons etai, 1984; 1983) led this group to the identification of an eight-base consensus sequence, AGACGTCT, that is now referred to as a 'met box". The met genes generally have at least two to five contiguous met boxes (a met box being defined as an eight-base sequence with at least 50% homology to the consensus sequence) and. where DNase footprinting studies have been performed, these regions are protected by the MetJ protein. The E. coli metH gene, whose expression is not directly regulated by methionine, does not contain a series of met boxes (Banerjee et ai, 1989; Oidetai, 1990; Marconi e/a/., 1991). Figure 2 summarizes the role of the metJ and metK in regulation of the met regulon. As indicated in the figure, metK codes for the enzyme that catalyses the formation of AdoMet. The bacterial cell represses the expression of the me/genes not by sensing the high level of methionine directly, but by means of the resulting increased formation of AdoMet, which can interact with the MetJ repressor protein. The MetJ protein (M, = 11996) functions as a dimer and binds to an eight-base-pair sequence (met tx}x) that is present in multiple contiguous copies in the promoter region of the met genes {Saint-Girons ef ai, 1984). AdoMet enhances this interaction by a factor of 10. In a series of elegant studies, the three-dimensional crystal structure of the MetJ protein has been determined in the presence or absence of the corepressor, AdoMet (Rafferty etai, 1988; 1989). The MetJ protein appears to be a dimer consisting of two highly intertwined monomers. Unlike other prokaryotic transcriptional factors, the DNA'binding region of MetJ does not contain a Table 1. Genes Involved m methionine regulation Regulatory gene
Gene product
Genes affected
Methionine
meU melK
Repressor AdoMet Synthelase
All mer genes except rrwtH
metR gene produa
metR
Activator Repressor
rrwtE. metH metR
Vitamin B,2
metH
Repressor (B,? mettiyltransferasef-'
metE. metF
Regulator
Methionine synthesis helix-turn-helix motif. It was suggested (Rafferty et al., 1989) that the MetJ protein recognizes the DNA target through two symmetry-related alpha-helices or a pair of beta-strands. The binding of AdoMet to MetJ does not result in a significant change in dimer conformation {Rafferty et ai, 1989). In detailed experiments with natural and synthetic operator sites, it was concluded that the MetJ protein dimers bind to operator sequences (mef boxes) in tandem arrays and that repression also depends on co-operative protein-protein contacts along the tandem array {Davidson and Saint Girons, 1989; Phillips e/a/., 1989).
Methionine + ATP
1595
metJ gene
MetJ protein (dimer)
Regulation by MetR of nwtEand mefHgenes The expression of the metE gene that codes for the nonBi2 methyitransferase {see Fig. 1) is unique in that it is regulated in three ways: (i) by excess methionine (mediated by the MetJ protein and AdoMet described above), (ii) by vitamin B,2, and (iii) by a newly identified protein, MetR. In our initial studies on expression of the E. coli metE gene we were puzzled by our inability to obtain good expression in vitro under conditions in which other cloned met genes were readily expressed (Chu et ai, 1985). This was especially surprising since it is known that this protein can represent close to 5% of the soluble protein of E. coli under derepressed conditions {Whitfield etai, 1970) The metEgene on plasmid pRSE562 (Cai et ai, 1989a) was functional, since transformation of a metE strain with the plasmid yielded wild-type levels of MetE activity which could be repressed by both methionine and vitamin B12. One possible explanation of our in vitro results was that a factor required for metE expression was lacking in the cell-free system. A major finding that related to our studies was the discovery by Stauffer and coworkers {Urbanowski etai. 1987) of a new mef regulatory locus, termed metR. MetR mutants were devoid of MetE activity and had only 20% of the wild-type MetH activity. Their results indicated that the mefHgene coded for a transactivator of both mefF and mefH expression. It was also reported that metR was located close to meff and that the metR gene product from Salmonella typhimurium was a protein of 31 kDa {Urbanowski ef ai, 1987). Sequence analysis in our laboratory showed that the plasmid containing the E. coli metE gene that we used as template also contained metR, which codes for a protein of 34 kDa (Maxon et ai, 1989). The metR gene Is adjacent to metEbut is transcribed in the opposite direction. In order to study the role of the MetR protein, the mefRgene was subcloned into a high-expression vector to overproduce the MetR protein, which was then purified to near homogeneity by ion-exchange chromatography {Maxon et ai. 1989). The purified protein (>95% pure) migrated
AGACGTCT
ATG
Met Box Fig. 2. Role of the met/and metK genes in repression of the methionine reQulon.
as a 34 kDa species. Addition of the purified MetR protein to in vitro incubations containing plasmids carrying the metE or metH genes as template resulted in a large stimulation of the synthesis of both gene products (Maxon et ai, 1989; Cai et ai, 1989a). However, more extensive studies on the MetR requirement showed that the amount of MetR needed to obtain maximum mef£ expression was 10-fold higher than that required for mefH expression (Cai ef ai. 1989a). Since other genetic and in vivo data implicated homocysteine in MetR regulation (Urbanowski and Stauffer, 1987; 1989), the effect of homocysteine was tested in the in vitro gene expression system (Cai ef ai., 1989b). It was shown that homocysteine had different effects on metE and metH repression. It markedly enhanced the MetR stimulation of metE synthesis, but had no effect, or caused only slight inhibition of MetRstimulated me/H expression in vitro. It was also noted that in these experiments the MetR protein autoregulates its own synthesis, and that this reaction is stimulated by homocysteine {Cai etai, 1989b). Footprinting studies and primer extension analysis have identified the MetJ- and MetR-binding regions, as well as the start of transcription for both the E. coli metR and metE genes in the mefH-meffl intergenic region {Cai Bt al., 1989a). The sequence of the metE-meiR intergenic region is shown in Fig. 3. The start of transcription for the metE gene is noted as +1, and is 169 bases upstream from the start of translation of metE. Two transcription start sites (RPi. RP2) were identified for metR at
1596
H. Weissbach and N. Brot R g , 3. Sequence of the metE-metR irWetgenic region The start of mefE transcnption is designated +1. Translation start sites are underlined and met boxes are overtined. The MetR-binding region is shown as a hatched area, and the MetJ-binding regions determined by (ootprinting experiments (Cai etai. 1989a) are shown as open boxes RP, and RP^ represent transcription start sites for the metR gene (from Cat etal. 1969a) that is transcribed in the opposite direction trom metE.
mefR TTCGAICAIS
AAAGTCCtTC
ACTrCS6IAT
GAATAATTTG
CGCTTGAGGA
ATATACAGTA
ACCGCCA;
ATGGATGTGT
AAACATCTGG
ACGGCTAAAA
MDIJ Hind ing
TCCfrTCGTCT
TTTAAATTTA
TGGTGCGTTG
GCTGCGTTTC
TCCACCCCGG
TCACTTACTT
CAGTAAGCTC
CCGGGGAIGA
ATAAACTTGC
CGCCTTCCCT
MrU Hi
metE AAATTCAAAA
TCCIkTAGGAT
TTACATATAA
TTAGAGGAAG
^-
AAAAAATGACAATA
.1J7
positions -29 and -47 from the start of mefEtranscription. Two MetJ-binding regions were observed, noted in Fig. 3 as open boxes. One, a high-affinity region, is located at positions - 8 to +27 and includes four met boxes (overlined). The other, a iow-aff inity region, is at positions +102 to +137. nearly encompassing three mef boxes. A singie MetR-binding region was detected at position -49 to -72. and extends into the coding region of the metR gene. The complete sequence of the S. typhimurium metR gene showed that it codes for a protein of -31 kDa, although in sodium dodecyi sulphate/polyacryiamide gei electrophoresis (SDS-PAGE) it migrated as a 34 kDa protein (Urbanowski et ai. 1987). Complete sequence analysis of the E. coli metR gene showed it to t>e a larger proteinof 35628 Da (Maxon Gf a/., 1990). The insertion of a single G residue at position 853 in the E. coli gene can explain the difference between the E. coli and S. typhimurium meff? sequences (Maxon etai, 1990). The MetR protein appears to be a member of a iarge family of bacteriai activator proteins, including LysR, lieY, and CysB, etc. that have a predicted helix-turn-helix DNA binding motif near the amino terminus (Henikoff ef ai, 1988). It was also noted (Maxon et ai. 1990) that the MetR protein contains a typical leucine zipper motif, i.e. four leucine resides spaced seven amino acids apart In the predicted heiicai region of the protein. Structures of this type were first detected in eukaryotic DNA-binding proteins (Landschuitz ef ai. 1988), and ieucine zipper motifs have now been observed in other prokaryotic proteins, including an E. coA sigma factor, o ^ (Sasse-Dwight and Gralia, 1990). replication proteins from bacterial plasmids (Giraldo ef ai, 1989; de la Campa ef ai. 1990). and the iactose repressor (Chakerian et ai, 1991). As with other eukaryotic leucine zipper proteins, the MetR protein
is found as a dimer and mutations in fhe leucine zipper region can inactivate the MetR protein (Maxon ef ai, 1990). Of speciai interest were the results of studies in which mefflwas truncated (Maxon etai. 1990). Removal of 43% of the carboxyl end of the protein had little effect on MetE and MetH activity relative to the wild-type protein. Removal of 62% of the protein (by restriction of the gene with Oral) resulted in about a 50% loss of MetE activity and a 10-20% loss of MetH activity. Additional truncation of the gene with Mlu\ to give a protein containing oniy 27% of the wiid-type MetR ied to complete loss of metE activity but no further toss of metH activity. One likely explanation for these results is that the region of the protein encoded by the DNA twtween the Oral and MliA sites is required for homocysteine activation. Loss of this region would be expected to affect metE expression but not mefH expression. After further restriction of the gene using Rsa\, the truncated protein was completely inactive for both mefH and mefH expression. On the basis of these resuits. a schematic representation of the putative active domains on the MetR protein is shown in Fig. 4. The Nterminal leucine zipper region between amino acids 19 and 40 is pictured as being necessary for dimer formation whereas the region {Rsa\-*Mlu\) adjacent to the leucine zipper is thought to be involved in DNA binding. The homocysteine-binding site is very iikely to be encoded by the nucieotide DNA between the Oral and Mlu\ sites on the gene.
Regulation by vitamin B^j When E. co//is grown in the presence of vitamin B,2 there is almost complete repression of metE expression and partial repression of mefF expression (Kung et ai, 1972;
Rsal
Mlul
BgUI
Oral
DNA Dimerization Amtno Acid
•/' 19
40
122
*
•/' 317
Hg. 4. Proposod functtonai regions of the MetR protein based on site-directed mutagenesis and truncation studies (Maxon etal.. 1989).
Methionine synthesis Milner et ai, 1969). The molecular basis of this regulation remains unclear, although genetic and biochemical studies have provided considerable information about this process. There is strong evidence that the MetH holoenzyme (B.,2 methyitransferase) is involved in the regulation (Milner et ai. 1969; Kung et ai. 1972): (i) MetH mutants do not show vitamin B,2 repression of metE expression, and (ii) only those B12 derivatives that form active holoenzyme in vivo are active in regulating metE expression. Other experiments have also implicated the metFgene, directly or indirectly, in this process {Mulligan etai, 1982; Wu and Urbanowski, 1989). The MetH holoenzyme is rather unusual since it not only has well-documented catalytic activity, but also appears to function as a regulatory protein. One can speculate that MetH serves to regulate metE expression in one of three ways. The MetH holoenzyme could (i) bind to the metE promoter and interfere with MetR binding, (ii) inactivate the homocysteine site on the MetR protein by methyiating it, or (iii) lower the Intraceiiular level of homocysteine (by converting it to methionine) so that MetR protein could not be activated. To date, there is no evidence to show that MetH holoenzyme binds to the metE promoter or prevents MetR binding to this promoter (our unpublished data). However, one difficulty with respect to these experiments is that the MetH holoenzyme as isolated may not be the functional form for regulation. For example, the purified MetH holoenzyme is known to be inactive as a catalyst (Weissbach and Taylor, 1970). Activation involves reduction of the enzyme-bound cobamide followed by formation of a methyl-Bi2 enzyme. It is possible that the active form of the enzyme involved in binding to the promoter is either the reduced or methylated species. Other experiments are in progress in this laboratory to determine whether the MetH holoenzyme can inactivate the MetR protein either directly or indirectly by significantly reducing the levels of homocysteine in the cell. Acknowledgements The authors would like to acknowledge major contributions of several colleagues who have worked on this problem over the years, including Robert Shoeman, Mary Maxon, Xiao-Yan Cai, Betty Redfield, John Wigboldus, Richard Marconi and Tim Coleman.
References Bachman, B.J., and Low, K.B. (1980) Microbiol Rev 44:1-56. Banerjee, R.V. etal. [A 989) J Biol Chem 264: 13885-13895. Cai, X.-Y. ef ai (1989a) Proc NatI Acad Sci USA 86: 44074411. Cai, X.-Y. etai (1989b) Biochem Biophys Res Commun 163:
79-83.
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Cenatiempo, V. etai (1982) Arch Biochem Biophys 218: 572578. Chakerian, A.E. et ai (1991) J Biol Chem 266:1371-1374. Chu, J. ef a/. (1985) Arch Biochem Biophys 239: 4 6 7 ^ 7 4 . Cohen, G.N., and Jacob, F. (1953) CR Hedb Seances Acad Sci Paris 248: 3490-3492. Cohn, M. ef ai (1953) CR Hebd Seances Acad Sci Paris 236: 746-748. Davidson, B.E., and Saint Girons, I. (1989) Mol Microbiol 2:
1639-1648. de la Campa, A.G. etai (1990) J Mo/S/o/213: 247-262. Giraldo, R. etal. (1989) NatureZ42: 866. Greene, R.C. etai (1970) Biochem Biophys Res Commun 38:
1120-1126. Greene, R.C. etai (1973) jeacter/o/115: 57-67. Henikoff, S. et ai (1988) Proc NatI Acad Sci USA 85: 6602-
6606. Holloway, C.T. etai 0970) JBacteriohOA: 734-747. Kung, H.-F. etai (1972) Arch Biochem Biophys 150: 23-31. Kung, H.-F. etai {W77) J Biol Chem 252: 6889-6894. Landschuitz, W.H. etai (1988) Science240:1759-1764. Marconi, R. etai (1990). Biochem BiophysHes Commun 175:
1057-1063. Maxon, M.E. etai (1989) Proc NatI Acad Sci USA 86: 85-S9. Maxon, M.E. et ai (1990) Proc NatI Acad Sci USA 87: 7076-
7079. Milner. L. etai (1969) Arch Biochem Biophys ^33:413-419. Mulligan, J.T. era/. (1982) JSacter/o/151: 609-619. Old, I.G. e/a/. (1990) Gene 87:15-21. Patte, J.C. ef ai (1967) Biochim Biophys Acta 136: 245-257. Phillips, S.E.V. etai (1989) Wafure341: 711-715. Rafferty, J.B. etai (1988) J Mol 6/0/200: 217-219. Rafferty, J.B. etal. (1989) Nafure341: 705-710. Robakis, N. etal. (1983) Meth Fnzymo/101: 690-706. Robakis, N. ef ai (1981) Proc NatI Acad Sci USA 78: 4 2 6 1 -
4264. Rowberg, R.J. (1965) Nature206: 962. Saint-Girons, I. etal. (1983). Wuc/-4c/ds Res 11: 6723-6732. Saint-Girons, I. etai (^984) J Biol Chem 259:14282-14285. Saint-Girons, I. etai (1986) JS/o/Chem261:10936-10940. Sasse-Dwight, S., and Gralia, J.D. (1990) Ce//62: 945-954. Shoeman, R. ef al. (1985a) Biochem Biophys Res Commun
133:731-739. Shoeman, R. etai {1985b) Proc NatI Acad Sci USA 82: 3eO^3605. Smith, A.A. ef ai (1985) Proc NatI Acad Sci USA 82: 61046108. Su, C.-H., and Greene, R.C. (1971) Proc NatI Acad Sci USA 68:367-371. Urbanowski. M.L., and Stauffer, G.V. (1987) J Bacteriol 169: 5841-5844. Urbanowski, M.L., and Stauffer, G.V. (1989) J Bacteriol 171:
3277-3281. Urbanowski, M.L etal. (1987) JSacfeno/169:1391-1397. Weissbach, H. and Taylor, R.T. (1970) WfHor 28:415-440. Whitfield, C D . et ai (1970) J Biol Chem 245: 390-401. Wijesundera, S., and Woods, D.D. (1953) S/oc/jem J 5 5 : viii. Wu, W.-F., and Urbanowski, M.L. {1989) Abstract of the 80th Annual Meeting of the American Society for Microbiology. Washington, D.C: American Society for Microbiology, p. 171. Zarucki-Schuiz, T. et ai {1979) Proc NatI Acad Sci USA 76:
6115-6119.
ADONIS 0950382X9100175N
MicroReview Regulation of methionine synthesis in Escherichia coii H. Weissbach')' and N. Brot Roche Research Center, Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USA.
Summary The biosynthesis of methionine in Escherichia coli is under compiex reguiation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyimethionine which functions as a corepressor for the MetJ protein. Recentiy, a new reguiatory locus, metR, has been identified. The MetR protein is required for both mefE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metaboiic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metEgene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group. Introduction The genes involved in methionine biosynthesis in Escherichia coli are scattered throughout the chromosome and are referred to as the 'met reguion' (Bachman and Low, 1980). The cloning of many of these genes has made it possible to elucidate the mechanisms that are involved in the complex regulation of this pathway. As shown in Fig. 1, the biosynthesis of methionine involves the convergence of two metabolic pathways, one leading to the formation of homocysteine, and the other to the methyl donor, N^-CH3-H4-folate. The final methyl transfer to homocysteine can be carried out by either of two methyltransferases, i.e. the non-BT2 enzyme coded for by the metE gene or the B,2 methyitransferase {the product of the metH gene). A major metabolic product of methionine is S-adenosylmethionine (AdoMet), which is not only Received 1 February, 1991, "For correspondence. Tel. (201) 235 3376; Fax (201) 235 6578.
an important methyl donor in biological systems but is, as will be described later, a key component in the regulation of the methionine biosynthetic pathway. There are three distinct types of regulation of methionine biosynthesis in E. coli These include repression of the mef genes when the organism is grown in the presence of methionine, activation of specific met gene expression mediated by the MetR protein, and repression of specitic genes by vitamin B12. Table 1 lists the regulatory genes that are involved in methionine synthesis and their gene products. The regulation by methionine of its own synthesis involves two genes, metJ and metK. The metJ gene codes for a repressor protein that regulates ail of the met genes with the exception of metH. MetJ mutants constitutively express high levels of the methionine biosynthetic enzymes. MetK mutants also show a similar phenotype. This gene codes for AdoMet synthetase, the enzyme responsible for the synthesis of AdoMet, a corepressor for the MetJ repressor protein. The metR ger\e product is a transactivator of metE and metH expression but also autoregulates its own synthesis. Finally, the metH gene product (B,2 methyitransferase) is invoived in the repression of the metE and, to a lesser degree, metF expression. Each of these modes of regulation is discussed below.
Role of the mefJand metKgenes in methionine regulation As noted above, the expression of all of the met genes, with the exception of metH, is repressed when E. coli is grown in the presence of high levels of methionine (Cohn et ai, 1953; Wijesundera and Woods, 1953; Cohen and Jacob, 1953; Rowberg, 1965; Patte et ai, 1967). Two regulatory mutants, meU and metK (Table ^), were initially isolated that showed constitutive expression of the mef genes even in the presence of methionine (Holloway ef ai, 1970; Su and Greene, 1971; Kung et ai, 1972; Greene ef ai. 1970; 1973). It is now well established from in vitro experiments that the MetJ protein requires AdoMet as a corepressor for maximum activity (Shoeman etai. 1985a; Saint-Girons etai, 1986). This explains the observation that both genes are essential for reguiation of the methionine biosynthetic pathway in vivo.
1594
H Weissbach and N. Brot Homoserine
(F)
Homocysteine + N^-methyl-H4-foIate (H) Methionine
S-Adenosy I melhionine (AdoMet} R g . 1. The biosynthesis of methior)ine in E. coli. Letters in parentheses represent specific methionine {med genes.
The availability of a highly defined DNA-dependent coupled protein synthesis system (Kung et ai, 1977; Zaruckl-Schutz et ai, 1979) as well as a simplified gene expression system based on the formation of the first dior tripeptide of the gene product (Robakis et ai, 1981; Cenatiempo etai. 1982; Robakis etai. 1983), facilitated in vitro studies on the roie of the MetJ protein in expression of the met genes. The initial studies used a plasmid carrying ttie metF gene as template, and a simplified dipeptide system to examine the role of the purified MetJ protein (Smith etai, 1985) and AdoMet in me/Fexpression {Shoeman etai, 1985b). In this assay the synthesis of the AZ-terminal dipeptide of the metF gene product. fMet-Ser, was measured. The MetJ protein alone significantly repressed expression of the mefF gene v^rhen concentrations above 2 MQ pe"" incubation were used. However, in the presence of AdoMet, only one tenth of the amount of MetJ protein was required to obtain comparable inhibition. AdoMet gave a maximum response at between 5 x 10"^ M and 5 x 10"^ M. In other experiments. a highly defined in vitro system was used to study met gene expression in which the protein product was determined (Shoeman et ai. 1985a). The results clearly showed that the MetJ protein and AdoMet inhibited the in vitro synthesis of the MetF, MetB. and MetL proteins. The roie of AdoMel in the regulation of met gene expression has been further examined in our laboratory and by others (Shoeman etai. 1985b; Saint-Girons etai, 1986). Although AdoMet is well established as a biological methyiating agent, there is no evidence that it methyiates either the met gene regulatory region or the MetJ protein. Under the conditions used, the binding of AdoMet to MetJ was quite weak and a stable complex could not be isolated (Shoeman et ai. 1985b). although equilibrium dialysis studies have shown complex formation with an apparent dissociation constant of 200 M ^
(Saint-Girons etai, 1986). Methionine. S-adenosylhomocysteine or adenosine cculd not replace AdoMet in this reaction {Shoeman et ai, 1985b). It was concluded that AdoMet functions as a corepressor for the MetJ repressor protein by enhancing binding of the MetJ protein to the promoter region of the met genes by a factor of 10 or more. The regions on the me/gene promoters at which MetJ protein interacts were initially identified by DNase footprinting analysis. Subsequent examination of the DNA sequence of several met promoters by Saint-Girons and coworkers (Saint-Girons etai, 1984; 1983) led this group to the identification of an eight-base consensus sequence, AGACGTCT, that is now referred to as a 'met box". The met genes generally have at least two to five contiguous met boxes (a met box being defined as an eight-base sequence with at least 50% homology to the consensus sequence) and. where DNase footprinting studies have been performed, these regions are protected by the MetJ protein. The E. coli metH gene, whose expression is not directly regulated by methionine, does not contain a series of met boxes (Banerjee et ai, 1989; Oidetai, 1990; Marconi e/a/., 1991). Figure 2 summarizes the role of the metJ and metK in regulation of the met regulon. As indicated in the figure, metK codes for the enzyme that catalyses the formation of AdoMet. The bacterial cell represses the expression of the me/genes not by sensing the high level of methionine directly, but by means of the resulting increased formation of AdoMet, which can interact with the MetJ repressor protein. The MetJ protein (M, = 11996) functions as a dimer and binds to an eight-base-pair sequence (met tx}x) that is present in multiple contiguous copies in the promoter region of the met genes {Saint-Girons ef ai, 1984). AdoMet enhances this interaction by a factor of 10. In a series of elegant studies, the three-dimensional crystal structure of the MetJ protein has been determined in the presence or absence of the corepressor, AdoMet (Rafferty etai, 1988; 1989). The MetJ protein appears to be a dimer consisting of two highly intertwined monomers. Unlike other prokaryotic transcriptional factors, the DNA'binding region of MetJ does not contain a Table 1. Genes Involved m methionine regulation Regulatory gene
Gene product
Genes affected
Methionine
meU melK
Repressor AdoMet Synthelase
All mer genes except rrwtH
metR gene produa
metR
Activator Repressor
rrwtE. metH metR
Vitamin B,2
metH
Repressor (B,? mettiyltransferasef-'
metE. metF
Regulator
Methionine synthesis helix-turn-helix motif. It was suggested (Rafferty et al., 1989) that the MetJ protein recognizes the DNA target through two symmetry-related alpha-helices or a pair of beta-strands. The binding of AdoMet to MetJ does not result in a significant change in dimer conformation {Rafferty et ai, 1989). In detailed experiments with natural and synthetic operator sites, it was concluded that the MetJ protein dimers bind to operator sequences (mef boxes) in tandem arrays and that repression also depends on co-operative protein-protein contacts along the tandem array {Davidson and Saint Girons, 1989; Phillips e/a/., 1989).
Methionine + ATP
1595
metJ gene
MetJ protein (dimer)
Regulation by MetR of nwtEand mefHgenes The expression of the metE gene that codes for the nonBi2 methyitransferase {see Fig. 1) is unique in that it is regulated in three ways: (i) by excess methionine (mediated by the MetJ protein and AdoMet described above), (ii) by vitamin B,2, and (iii) by a newly identified protein, MetR. In our initial studies on expression of the E. coli metE gene we were puzzled by our inability to obtain good expression in vitro under conditions in which other cloned met genes were readily expressed (Chu et ai, 1985). This was especially surprising since it is known that this protein can represent close to 5% of the soluble protein of E. coli under derepressed conditions {Whitfield etai, 1970) The metEgene on plasmid pRSE562 (Cai et ai, 1989a) was functional, since transformation of a metE strain with the plasmid yielded wild-type levels of MetE activity which could be repressed by both methionine and vitamin B12. One possible explanation of our in vitro results was that a factor required for metE expression was lacking in the cell-free system. A major finding that related to our studies was the discovery by Stauffer and coworkers {Urbanowski etai. 1987) of a new mef regulatory locus, termed metR. MetR mutants were devoid of MetE activity and had only 20% of the wild-type MetH activity. Their results indicated that the mefHgene coded for a transactivator of both mefF and mefH expression. It was also reported that metR was located close to meff and that the metR gene product from Salmonella typhimurium was a protein of 31 kDa {Urbanowski ef ai, 1987). Sequence analysis in our laboratory showed that the plasmid containing the E. coli metE gene that we used as template also contained metR, which codes for a protein of 34 kDa (Maxon et ai, 1989). The metR gene Is adjacent to metEbut is transcribed in the opposite direction. In order to study the role of the MetR protein, the mefRgene was subcloned into a high-expression vector to overproduce the MetR protein, which was then purified to near homogeneity by ion-exchange chromatography {Maxon et ai. 1989). The purified protein (>95% pure) migrated
AGACGTCT
ATG
Met Box Fig. 2. Role of the met/and metK genes in repression of the methionine reQulon.
as a 34 kDa species. Addition of the purified MetR protein to in vitro incubations containing plasmids carrying the metE or metH genes as template resulted in a large stimulation of the synthesis of both gene products (Maxon et ai, 1989; Cai et ai, 1989a). However, more extensive studies on the MetR requirement showed that the amount of MetR needed to obtain maximum mef£ expression was 10-fold higher than that required for mefH expression (Cai ef ai. 1989a). Since other genetic and in vivo data implicated homocysteine in MetR regulation (Urbanowski and Stauffer, 1987; 1989), the effect of homocysteine was tested in the in vitro gene expression system (Cai ef ai., 1989b). It was shown that homocysteine had different effects on metE and metH repression. It markedly enhanced the MetR stimulation of metE synthesis, but had no effect, or caused only slight inhibition of MetRstimulated me/H expression in vitro. It was also noted that in these experiments the MetR protein autoregulates its own synthesis, and that this reaction is stimulated by homocysteine {Cai etai, 1989b). Footprinting studies and primer extension analysis have identified the MetJ- and MetR-binding regions, as well as the start of transcription for both the E. coli metR and metE genes in the mefH-meffl intergenic region {Cai Bt al., 1989a). The sequence of the metE-meiR intergenic region is shown in Fig. 3. The start of transcription for the metE gene is noted as +1, and is 169 bases upstream from the start of translation of metE. Two transcription start sites (RPi. RP2) were identified for metR at
1596
H. Weissbach and N. Brot R g , 3. Sequence of the metE-metR irWetgenic region The start of mefE transcnption is designated +1. Translation start sites are underlined and met boxes are overtined. The MetR-binding region is shown as a hatched area, and the MetJ-binding regions determined by (ootprinting experiments (Cai etai. 1989a) are shown as open boxes RP, and RP^ represent transcription start sites for the metR gene (from Cat etal. 1969a) that is transcribed in the opposite direction trom metE.
mefR TTCGAICAIS
AAAGTCCtTC
ACTrCS6IAT
GAATAATTTG
CGCTTGAGGA
ATATACAGTA
ACCGCCA;
ATGGATGTGT
AAACATCTGG
ACGGCTAAAA
MDIJ Hind ing
TCCfrTCGTCT
TTTAAATTTA
TGGTGCGTTG
GCTGCGTTTC
TCCACCCCGG
TCACTTACTT
CAGTAAGCTC
CCGGGGAIGA
ATAAACTTGC
CGCCTTCCCT
MrU Hi
metE AAATTCAAAA
TCCIkTAGGAT
TTACATATAA
TTAGAGGAAG
^-
AAAAAATGACAATA
.1J7
positions -29 and -47 from the start of mefEtranscription. Two MetJ-binding regions were observed, noted in Fig. 3 as open boxes. One, a high-affinity region, is located at positions - 8 to +27 and includes four met boxes (overlined). The other, a iow-aff inity region, is at positions +102 to +137. nearly encompassing three mef boxes. A singie MetR-binding region was detected at position -49 to -72. and extends into the coding region of the metR gene. The complete sequence of the S. typhimurium metR gene showed that it codes for a protein of -31 kDa, although in sodium dodecyi sulphate/polyacryiamide gei electrophoresis (SDS-PAGE) it migrated as a 34 kDa protein (Urbanowski et ai. 1987). Complete sequence analysis of the E. coli metR gene showed it to t>e a larger proteinof 35628 Da (Maxon Gf a/., 1990). The insertion of a single G residue at position 853 in the E. coli gene can explain the difference between the E. coli and S. typhimurium meff? sequences (Maxon etai, 1990). The MetR protein appears to be a member of a iarge family of bacteriai activator proteins, including LysR, lieY, and CysB, etc. that have a predicted helix-turn-helix DNA binding motif near the amino terminus (Henikoff ef ai, 1988). It was also noted (Maxon et ai. 1990) that the MetR protein contains a typical leucine zipper motif, i.e. four leucine resides spaced seven amino acids apart In the predicted heiicai region of the protein. Structures of this type were first detected in eukaryotic DNA-binding proteins (Landschuitz ef ai. 1988), and ieucine zipper motifs have now been observed in other prokaryotic proteins, including an E. coA sigma factor, o ^ (Sasse-Dwight and Gralia, 1990). replication proteins from bacterial plasmids (Giraldo ef ai, 1989; de la Campa ef ai. 1990). and the iactose repressor (Chakerian et ai, 1991). As with other eukaryotic leucine zipper proteins, the MetR protein
is found as a dimer and mutations in fhe leucine zipper region can inactivate the MetR protein (Maxon ef ai, 1990). Of speciai interest were the results of studies in which mefflwas truncated (Maxon etai. 1990). Removal of 43% of the carboxyl end of the protein had little effect on MetE and MetH activity relative to the wild-type protein. Removal of 62% of the protein (by restriction of the gene with Oral) resulted in about a 50% loss of MetE activity and a 10-20% loss of MetH activity. Additional truncation of the gene with Mlu\ to give a protein containing oniy 27% of the wiid-type MetR ied to complete loss of metE activity but no further toss of metH activity. One likely explanation for these results is that the region of the protein encoded by the DNA twtween the Oral and MliA sites is required for homocysteine activation. Loss of this region would be expected to affect metE expression but not mefH expression. After further restriction of the gene using Rsa\, the truncated protein was completely inactive for both mefH and mefH expression. On the basis of these resuits. a schematic representation of the putative active domains on the MetR protein is shown in Fig. 4. The Nterminal leucine zipper region between amino acids 19 and 40 is pictured as being necessary for dimer formation whereas the region {Rsa\-*Mlu\) adjacent to the leucine zipper is thought to be involved in DNA binding. The homocysteine-binding site is very iikely to be encoded by the nucieotide DNA between the Oral and Mlu\ sites on the gene.
Regulation by vitamin B^j When E. co//is grown in the presence of vitamin B,2 there is almost complete repression of metE expression and partial repression of mefF expression (Kung et ai, 1972;
Rsal
Mlul
BgUI
Oral
DNA Dimerization Amtno Acid
•/' 19
40
122
*
•/' 317
Hg. 4. Proposod functtonai regions of the MetR protein based on site-directed mutagenesis and truncation studies (Maxon etal.. 1989).
Methionine synthesis Milner et ai, 1969). The molecular basis of this regulation remains unclear, although genetic and biochemical studies have provided considerable information about this process. There is strong evidence that the MetH holoenzyme (B.,2 methyitransferase) is involved in the regulation (Milner et ai. 1969; Kung et ai. 1972): (i) MetH mutants do not show vitamin B,2 repression of metE expression, and (ii) only those B12 derivatives that form active holoenzyme in vivo are active in regulating metE expression. Other experiments have also implicated the metFgene, directly or indirectly, in this process {Mulligan etai, 1982; Wu and Urbanowski, 1989). The MetH holoenzyme is rather unusual since it not only has well-documented catalytic activity, but also appears to function as a regulatory protein. One can speculate that MetH serves to regulate metE expression in one of three ways. The MetH holoenzyme could (i) bind to the metE promoter and interfere with MetR binding, (ii) inactivate the homocysteine site on the MetR protein by methyiating it, or (iii) lower the Intraceiiular level of homocysteine (by converting it to methionine) so that MetR protein could not be activated. To date, there is no evidence to show that MetH holoenzyme binds to the metE promoter or prevents MetR binding to this promoter (our unpublished data). However, one difficulty with respect to these experiments is that the MetH holoenzyme as isolated may not be the functional form for regulation. For example, the purified MetH holoenzyme is known to be inactive as a catalyst (Weissbach and Taylor, 1970). Activation involves reduction of the enzyme-bound cobamide followed by formation of a methyl-Bi2 enzyme. It is possible that the active form of the enzyme involved in binding to the promoter is either the reduced or methylated species. Other experiments are in progress in this laboratory to determine whether the MetH holoenzyme can inactivate the MetR protein either directly or indirectly by significantly reducing the levels of homocysteine in the cell. Acknowledgements The authors would like to acknowledge major contributions of several colleagues who have worked on this problem over the years, including Robert Shoeman, Mary Maxon, Xiao-Yan Cai, Betty Redfield, John Wigboldus, Richard Marconi and Tim Coleman.
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