PMID:9201956
| Citation |
Goulding, CW, Postigo, D and Matthews, RG (1997) Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine. Biochemistry 36:8082-91 |
|---|---|
| Abstract |
Methionine synthase (MetH) catalyzes the transfer of a methyl group from bound methylcobalamin to homocysteine, yielding enzyme-bound cob(I)alamin and methionine. The cofactor is then remethylated by methyltetrahydrofolate. We now demonstrate that MetH is able to catalyze methylation of free cob(I)alamin with methyltetrahydrofolate. MetH had previously been shown to catalyze methylation of homocysteine with free methylcobalamin as the methyl donor, in a reaction that is first-order in added methylcobalamin, and we have confirmed this observation using homogenous enzyme. A truncated polypeptide lacking the cobalamin-binding region of the holoenzyme, MetH(2-649), was overexpressed and purified to homogeneity. MetH(2-649) catalyzes the methylation of free cob(I)alamin by methyltetrahydrofolate and the methylation of homocysteine by free methylcobalamin. Furthermore, a protein comprising residues 2-353 of the holoenzyme has now been overexpressed and purified to homogeneity, and this protein catalyzes methyl transfer from free methylcobalamin to homocysteine but not from methyltetrahydrofolate to free cob(I)alamin. The mutations Cys310Ala and Cys311Ala in MetH(2-649) completely abolish methyl transfer from exogenous methylcobalamin to homocysteine but do not affect methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, consistent with a modular construction for MetH. We infer that MetH is a modular protein comprising four separate regions: a homocysteine binding region (residues 2-353), a methyltetrahydrofolate binding region (residues 354-649), a region responsible for binding the cobalamin prosthetic group (residues 650-896), and an AdoMet-binding domain (residues 897-1227). |
| Links |
PubMed Online version:10.1021/bi9705164 |
| Keywords |
5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/metabolism; Bacterial Proteins/metabolism; Binding Sites; Escherichia coli/enzymology; Escherichia coli/metabolism; Homocysteine/metabolism; Methylation; Protein Binding; S-Adenosylmethionine/metabolism; Tetrahydrofolates/metabolism; Vitamin B 12/analogs & derivatives; Vitamin B 12/metabolism |
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