First Step in Catalysis of the Radical S -Adenosylmethionine Methylthiotransferase MiaB Yields an Intermediate with a [3Fe-4S] 0 -Like Auxiliary Cluster
First Step in Catalysis of the Radical S -Adenosylmethionine Methylthiotransferase MiaB Yields an Intermediate with a [3Fe-4S] 0 -Like Auxiliary Cluster
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自由基 S-腺苷甲硫氨酸甲硫基转移酶 MiaB 催化的第一步产生具有 [3Fe-4S] 0 样辅助簇的中间体
DOI：
10.1021/jacs.9b11093
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发表时间：
2020
影响因子：
15
通讯作者：
Krebs, Carsten
中科院分区：
文献类型：
--
作者：
Zhang, Bo;Arcinas, Arthur J.;Radle, Matthew I.;Silakov, Alexey;Booker, Squire J.;Krebs, Carsten
在生物合成超修饰tRNA核苷酸2-甲基硫- n6 -（异戊烯基）腺苷（ms2i6A）的最后一步中，MiaB酶催化甲基硫（- SCH3）基团在n6 -（异戊烯基）腺苷（i6A）的C2位置上的附着。MiaB属于自由基-腺苷蛋氨酸（SAM）超家族的扩展酶亚群，除了所有家族成员都需要的[4Fe-4S]簇外，还含有一个或多个辅助的[4Fe-4S]簇，用于SAM的还原裂解，以提供常见的5 ' -脱氧腺苷5 ' -自由基（5 ' -dA•）中间体。虽然自由基SAM簇在生成5 ' -dA•中的作用已经被很好地理解，但对MiaB催化至关重要的辅助簇的详细作用仍不清楚。有人提出，辅助团簇可以作为外源衍生硫的配位位点，用于附着在底物上，或者团簇本身提供硫原子并在转换过程中牺牲。在这项工作中，我们报告了光谱和生化证据，证明在催化的甲基化步骤中，拟杆菌（bacteroides thetaiotaomicron, Bt） MiaB中的辅助[4Fe-4S]2+簇转化为[3Fe-4S]0样簇。Mössbauer对MiaB [3Fe-4S]0类星团的表征显示，与其他表征良好的立方体[3Fe-4S]0星团相比，它们具有不同寻常的光谱特性。具体来说，混合价段的铁位点不具有相同的Mössbauer参数。我们的研究结果支持辅助[4Fe-4S]簇在催化过程中是直接硫源的机制。
The enzyme MiaB catalyzes the attachment of a methylthio (−SCH3) group at the C2 position ofN6-(isopentenyl)adenosine (i6A) in the final step of the biosynthesis of the hypermodified tRNA nucleotide 2-methythio-N6-(isopentenyl)adenosine (ms2i6A). MiaB belongs to the expanding subgroup of enzymes of the radicalS-adenosylmethionine (SAM) superfamily that harbor one or more auxiliary [4Fe-4S] clusters in addition to the [4Fe-4S] cluster that all family members require for the reductive cleavage of SAM to afford the common 5′-deoxyadenosyl 5′-radical (5′-dA•) intermediate. While the role of the radical SAM cluster in generating the 5′-dA•is well understood, the detailed role of the auxiliary cluster, which is essential for MiaB catalysis, remains unclear. It has been proposed that the auxiliary cluster may serve as a coordination site for exogenously derived sulfur destined for attachment to the substrate or that the cluster itself provides the sulfur atom and is sacrificed during turnover. In this work, we report spectroscopic and biochemical evidence that the auxiliary [4Fe–4S]2+cluster inBacteroides thetaiotaomicron(Bt) MiaB is converted to a [3Fe-4S]0-like cluster during the methylation step of catalysis. Mössbauer characterization of the MiaB [3Fe-4S]0-like cluster revealed unusual spectroscopic properties compared to those of other well-characterized cuboidal [3Fe-4S]0clusters. Specifically, the Fe sites of the mixed-valent moiety do not have identical Mössbauer parameters. Our results support a mechanism where the auxiliary [4Fe-4S] cluster is the direct sulfur source during catalysis.