Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.
Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.
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DOI：
10.1021/acs.biochem.6b00670
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发表时间：
2016-10-04
期刊：
影响因子：
2.9
通讯作者：
Elliott SJ
中科院分区：
文献类型：
--
作者：
Maiocco SJ;Arcinas AJ;Landgraf BJ;Lee KH;Booker SJ;Elliott SJ
甲硫基转移酶 (MTTases) 代表 S-腺苷甲硫氨酸 (AdoMet) 自由基超家族酶的一个亚家族，可催化甲基硫醚 (-SCH3) 部分在未活化的碳中心上的附着。这些酶含有两个 [4Fe-4S] 簇，其中一个参与 AdoMet 的还原断裂，生成 5'-脱氧腺苷 5'-自由基，另一个被称为辅助簇，被认为在构建甲硫基并将其附着到底物上发挥核心作用。由于对 AdoMet 自由基超家族中结合辅因子的氧化还原特性知之甚少，我们使用蛋白质电化学方法平行检测了两种 MTTase：MiaB 和 RimO。我们解析了每个 [4Fe-4S] 簇的氧化还原电位，表明辅助簇的电位高于 AdoMet 结合簇的电位，并证明在任一酶与 AdoMet 一起孵育时，会出现该酶独特的低电位状态。我们的结果与辅助簇在底物甲硫基化过程中瞬时甲基化的机制一致。
The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5′-deoxyadenosyl 5′-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation.