Characterization of an O-Demethylase of Desulfitobacterium hafniense DCB-2
Characterization of an O-Demethylase of Desulfitobacterium hafniense DCB-2
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DOI：
10.1128/jb.00146-12
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发表时间：
2012-07-01
影响因子：
3.2
通讯作者：
Diekert, Gabriele
中科院分区：
文献类型：
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作者：
Studenik, Sandra;Vogel, Michaela;Diekert, Gabriele
除了产乙酸细菌，只有脱硫杆菌已被描述为利用和切割苯甲醚在缺氧条件下，然而，没有醚切割O-脱甲基酶从后者的生物体已被确定和调查至今。在这项研究中，操纵子编码的O-脱甲基酶组分的Desulfitobacterium longniense菌株DCB-2的基因克隆和异源表达在大肠杆菌。甲基转移酶I和II的特点。甲基转移酶I介导的醚裂解和转移的甲基基团的超还原corrinoid的corrinoid蛋白。脱硫杆菌甲基转移酶I有66%的同一性（80%的相似性）的香草酸脱甲基转移酶I（ODMB）的脱卤醋酸杆菌。底物谱也类似于后一种酶;然而，脱硫杆菌甲基转移酶I对愈创木酚表现出更高水平的活性，并使用氯甲烷作为底物。甲基转移酶II催化甲基基团从甲基化的类咕啉蛋白转移到四氢叶酸。它与A的甲基转移酶II也有很高的同源性（接近70%）。脱卤剂在E. colt作为无辅因子的脱辅基蛋白，其可以用羟钴胺素或甲钴胺素重构以在甲基转移酶I和II测定中起作用。六个COG 3894蛋白质，这被假定为激活酶介导的还原后，无意中氧化的corrinoid辅因子的corrinoid蛋白质的功能，进行了研究，就其能力，以减少重组重建corrinoid蛋白。在这六种蛋白质中，只有一种被发现催化还原的corrinoid蛋白。
Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (similar to 70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. colt as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.