Kaufmann F, Wohlfarth G, Diekert G
Institut für Mikrobiologie der Universität Stuttgart, Germany.
Eur J Biochem. 1998 Oct 15;257(2):515-21. doi: 10.1046/j.1432-1327.1998.2570515.x.
The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II also catalysed the methylation of cob(I)alamin containing OdmA with methyltetrahydrofolate.
来自严格厌氧的同型产乙酸菌脱卤产乙酸杆菌的醚裂解O - 脱甲基酶催化4 - 羟基 - 3 - 甲氧基苯甲酸（香草酸）向四氢叶酸的甲基转移。第一步，香草酸：类咕啉蛋白甲基转移酶（甲基转移酶I）介导25 kDa类咕啉蛋白的甲基化，此时辅因子还原为钴胺素（I）。然后，甲基通过甲基类咕啉蛋白：四氢叶酸甲基转移酶（甲基转移酶II）的作用转移到四氢叶酸上。利用从类咕啉蛋白和香草酸：类咕啉蛋白甲基转移酶（甲基转移酶I）的氨基末端序列衍生的引物，通过PCR扩增出一个723 bp的片段，该片段包含编码O - 脱甲基酶类咕啉蛋白的odmA基因。在odmA的下游，鉴定出了编码甲基转移酶I的odmB基因的一部分。从odmA推导的氨基酸序列与大肠杆菌甲硫氨酸合酶（MetH）的钴胺素结合结构域以及参与甲醇和甲胺产甲烷作用的甲基转移酶系统的类咕啉蛋白显示出约60%的相似性。该序列包含典型的DXHXXG共有序列，用于蛋白质中的组氨酸取代类咕啉辅因子的二甲基苯并咪唑碱基。odmA在大肠杆菌中的异源表达产生了一种无色、对氧不敏感的脱辅基蛋白，该蛋白能够结合1 mol钴胺素或甲基钴胺素/mol蛋白。这两种重组形式的蛋白在整个O - 脱甲基反应中均具有活性。用羟基钴胺素重构并用柠檬酸钛（III）还原为钴胺素（I）形式的OdmA在不可逆反应中被甲基转移酶I用香草酸甲基化。携带OdmA的甲基钴胺素作为甲基供体，用于甲基转移酶II对四氢叶酸的甲基化。发现该反应是可逆的，因为甲基转移酶II也催化含OdmA的钴胺素（I）与甲基四氢叶酸的甲基化反应。
