O-demethylase from Acetobacterium dehalogenans -: Cloning, sequencing, and active expression of the gene encoding the corrinoid protein
O-demethylase from Acetobacterium dehalogenans -: Cloning, sequencing, and active expression of the gene encoding the corrinoid protein
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DOI：
10.1046/j.1432-1327.1998.2570515.x
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发表时间：
1998-10-01
期刊：
影响因子：
--
通讯作者：
Diekert, G
中科院分区：
文献类型：
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作者：
Kaufmann, F;Wohlfarth, G;Diekert, G
来自严格厌氧的同型产乙酸菌脱卤醋酸杆菌的醚裂解O-脱甲基酶催化4-羟基-3-甲氧基苯甲酸酯（香草酸）甲基转移为四氢叶酸。在第一步中，香草酸：类可啉蛋白甲基转移酶（甲基转移酶I）介导25-kDa类可啉蛋白的甲基化，辅因子还原为cob（I）丙氨酸。然后通过甲基可啉蛋白的作用将甲基转移到四氢叶酸：四氢叶酸甲基转移酶（甲基转移酶II）。使用来自corrinoid蛋白和香草酸：corrinoid蛋白甲基转移酶（甲基转移酶I）的氨基末端序列的引物，通过PCR扩增723 bp的片段，其中包含编码corrinoid蛋白的O-脱甲基酶的基因odmA。在odmA的下游，鉴定了编码甲基转移酶I的odmB基因的部分。从odmA推导的氨基酸序列显示约60%的相似性钴胺素结合结构域的蛋氨酸合酶从大肠杆菌（MetH）和corrinoid蛋白质的甲基转移酶系统参与从甲醇和甲胺产甲烷。该序列含有DXHXXG共有序列，该序列通常用于将类咕啉辅因子的二甲基苯并咪唑碱基置换为蛋白质中的组氨酸。odmA在E.大肠杆菌产生无色、氧不敏感的脱辅基蛋白，其能够结合1mol钴胺素或甲基钴胺素/mol蛋白。这两种重组形式的蛋白质在整体O-脱甲基反应中均具有活性。OdmA与羟钴胺素重构并被柠檬酸钛（III）还原为钴（I）维生素形式，通过甲基转移酶I在不可逆反应中被香草酸甲基化。携带OdmA的甲钴胺作为甲基转移酶II甲基化四氢叶酸的甲基供体。发现该反应是可逆的，因为甲基转移酶II也催化含有OdmA的cob（I）alamin与甲基四氢叶酸的甲基化。
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.