Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine
Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine
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DOI：
10.1021/bi9705164
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发表时间：
1997-07-01
期刊：
影响因子：
2.9
通讯作者：
Matthews, RG
中科院分区：
文献类型：
--
作者：
Goulding, CW;Postigo, D;Matthews, RG
蛋氨酸合酶 (MetH) 催化甲基从结合的甲钴胺转移到高半胱氨酸，产生酶结合的钴胺素 (I) 和蛋氨酸。然后辅因子被甲基四氢叶酸重新甲基化。我们现在证明 MetH 能够催化游离钴 (I) 胺与甲基四氢叶酸的甲基化。 MetH 之前已被证明可以用游离甲钴胺作为甲基供体催化同型半胱氨酸的甲基化，在添加甲钴胺的一级反应中，我们已经使用均质酶证实了这一观察结果。缺乏全酶钴胺素结合区的截短多肽 MetH(2-649) 被过表达并纯化至同质。 MetH(2-649) 催化甲基四氢叶酸对游离钴 (I) 胺的甲基化，以及游离甲基钴胺对同型半胱氨酸的甲基化。此外，包含全酶残基2-353的蛋白质现已过表达并纯化至均质，并且该蛋白质催化从游离甲钴胺到高半胱氨酸的甲基转移，但不催化从甲基四氢叶酸到游离钴(I)胺的甲基转移。 MetH(2-649) 中的突变 Cys310Ala 和 Cys311Ala 完全消除了从外源甲钴胺到同型半胱氨酸的甲基转移，但不影响从甲基四氢叶酸到外源钴胺素的甲基转移，这与 MetH 的模块化结构一致。我们推断 MetH 是包含四个独立区域的模块蛋白：同型半胱氨酸结合区（残基 2-353）、甲基四氢叶酸结合区（残基 354-649）、负责结合钴胺素辅基的区域（残基 650-896）和 AdoMet 结合结构域（残基 897-1227）。
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).