Binding of substrate to N5-methyl-tetrahydropteroyltriglutamate-homocysteine transmethylase

Whitfield, Carolyn D.; Weissbach, Herbert

doi:10.1016/0006-291x(68)90412-9

Cited by 47 publications

(38 citation statements)

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“…Transcription of metE is highest when cells are grown aerobically in the absence of added B 12 and methionine (3) and can constitute 3-5% of the total cellular protein (4,5).…”

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Synergistic, Random Sequential Binding of Substrates in Cobalamin-Independent Methionine Synthase

2006

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Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH 3 -H 4 folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H 4 folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE·CH 3 -H 4 folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH 3 -H 4 folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed onpathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions. Methionine synthases catalyze the methylation of homocysteine (Hcy)1 by methyltetrahydrofolate (CH 3 -H 4 folate), yielding methionine and tetrahydrofolate (H 4 folate). Escherichia coli contains two genes, metH and metE, specifying two different methionine synthase enzymes that are differentially expressed (1). The MetH protein is cobalamindependent methionine synthase, in which the cobalamin (B 12 ) cofactor serves as an intermediary in methyl transfer, accepting a methyl group from CH 3 -H 4 folate and donating it to Hcy to form methionine. MetH holoenzyme is only expressed under conditions where B 12 is present in the medium, although the apoenzyme may be constitutively expressed (2). Since E. coli does not synthesize cobalamin, such conditions are typically encountered during growth of cells in the mammalian gut or under experimental growth conditions where cobalamin has been added to the growth medium. The MetE protein is cobalamin-independent methionine synthase, which does not contain any organic cofactor. This enzyme is believed to catalyze direct transfer of the methyl group between CH 3 -H 4 folate and Hcy, requiring that both

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“…Transcription of metE is highest when cells are grown aerobically in the absence of added B 12 and methionine (3) and can constitute 3-5% of the total cellular protein (4,5).…”

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confidence: 99%

“…The triglutamate CH 3 -H 4 PteGlu 3 is typically used as the substrate for in vitro experiments, although the diglutamate substrate also exhibits nearly maximal activity (8). MetE catalysis is also stimulated by low concentrations of phosphate and magnesium (5). The two enzymes show no detectable sequence homology; however, they do exhibit some similarities.…”

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confidence: 99%

Synergistic, Random Sequential Binding of Substrates in Cobalamin-Independent Methionine Synthase

2006

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“…However, in vitro studies have shown them to be equally or more effective than monoglutamates as enzyme substrates. One of these enzymes, a B12-independent methionine synthetase, which is present in some micro-organisms (Taylor & Weissbach, 1973;Whitfield et al, 1970) but not in mammalian tissues (Cheng et al, 1975;Coward et al, 1975), either only functions with folylpolyglumate substrates or utilizes the monoglutamate derivative extremely poorly. In vivo studies have also shown that while the rate-limiting step in the utilization of pteroylpolyglutamates by L. casei is their transport into the cell, once taken up, they are more effectively utilized for growth than the monoglutamate derivatives (Shane & Stokstad, 1976).…”

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Section: Introductionmentioning

confidence: 99%

“…The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al, 1974 Coward et al, , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).To investigate which step in pteroylmonoglutamate metabolism might be rate-limiting, the metabolism of 5-[Me-14C]methyl-H,PteGlu and 5-methyl-H,[3H]PteGlu (for nomenclature, see Methods) were compared in order to assess the turnover of intracellular folate via various metabolic pathways. Although two mechanisms have been described for 5-methyl-H4PteGlu formation in bacteria involving the reduction of 5, I o-methylene-H,PteGlu or the direct transfer of a methyl group from trimethylsulphonium to H,PteGlu (Wagner et al, 1967), the only known route for the metabolism of methyltetrahydrofolates is via methionine synthesis (Taylor & Weissbach, 1973) but attempts to detect methionine synthetase activity in L. casei have proved unsuccessful (Galivan, 1971 ;Kisliuk, 1971).…”

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Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

Shane¹,

Stokstad²

1977

Journal of General Microbiology

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The metabolism of 5-[Me-14C]methyltetrahydrofolate in Lactobacillus casei proceeded oxidatively with incorporation of label into purine and thymidylate derivatives. No labelled methionine was formed.(1)-5-Methyltetrahydrofolate, the natural isomer, was not a substrate for the L. casei folylpoly-y-glutamate synthetase although the unnatural (d)-isomer was slowly metabolized to the diglutamate form. I N T R O D U C T I O NWe have previously shown that pteroylpolyglutamates, once transported, are better growth promoters of Lactobacillus casei than pteroylmonoglutamates (Shane & Stokstad, I 976). The rate-limiting step in the utilization of pteroylpolyglutamates by this organism is transport into the cell, while intracellular metabolism, and not transport, is rate-limiting with pteroylmonoglutamates. In vitro studies have also shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (Burton & Metzenberg, 1975;Cheng, Shane & Stokstad, 1975;Coward et al., 1974 Coward et al., , 1975Curthoys & Rabinowitz, 1972;Kisliuk, Gaumont & Baugh, 1974;Powers & Snell, 1976;Salem, Pattison & Foster, 1972;Whitfield, Steers & Weissbach, 1970).To investigate which step in pteroylmonoglutamate metabolism might be rate-limiting, the metabolism of 5-[Me-14C]methyl-H,PteGlu and 5-methyl-H,[3H]PteGlu (for nomenclature, see Methods) were compared in order to assess the turnover of intracellular folate via various metabolic pathways. Although two mechanisms have been described for 5-methyl-H4PteGlu formation in bacteria involving the reduction of 5, I o-methylene-H,PteGlu or the direct transfer of a methyl group from trimethylsulphonium to H,PteGlu (Wagner et al., 1967), the only known route for the metabolism of methyltetrahydrofolates is via methionine synthesis (Taylor & Weissbach, 1973) but attempts to detect methionine synthetase activity in L. casei have proved unsuccessful (Galivan, 1971 ;Kisliuk, 1971). METHODSNomenclature. The abbreviations used are : PteGlu, pteroylglutamic acid, folic acid; PteGlu,, pteroylmonoto pteroyloligo-y-L-glutamic acid, n indicating the number of glutamic; acid residues; H,PteGlu,, 5,6,7,8-tetrahydropteroylmono-to ~,6,~,8-tetrahydropteroyloligo-y-~-glutamic acid. The symbols ( I ) and (d) are used to denote the natural and unnatural diastereoisomers of H,PteGlu,, respectively, due to the asymmetric centre at the C-6 position, and do not indicate optical activity.

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Metabolic Engineering for Biocatalyst Robustness to Organic Inhibitors

Royce

Jarboe

2018

Bioprocessing Technology for Production of Biopharmaceuticals and Bioproducts

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Microbial production of biorenewable fuels and chemicals is often limited by inhibition of the biocatalyst, either by increasing concentrations of the product compound or by contaminant compounds in the biomass-derived sugars. This inhibition can interfere with economically viable production. Here we discuss typical mechanisms of inhibition and methods for improving biocatalyst robustness. Inhibition often takes the form of inhibition of enzyme activity, depletion of cofactor pools, and membrane damage; methods are discussed for mitigating each of these types of inhibition. Various evolutionary schemes have been developed and implemented on a variety of inhibitory compounds, including butanol, acetic acid, furfural, and ethanol. Reverse engineering of these improved strains can provide insight into new metabolic engineering strategies.

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Binding of substrate to N5-methyl-tetrahydropteroyltriglutamate-homocysteine transmethylase

Cited by 47 publications

References 12 publications

Synergistic, Random Sequential Binding of Substrates in Cobalamin-Independent Methionine Synthase

Synergistic, Random Sequential Binding of Substrates in Cobalamin-Independent Methionine Synthase

Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

Metabolic Engineering for Biocatalyst Robustness to Organic Inhibitors

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