[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.
[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.
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DOI：
10.1021/bi501205e
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发表时间：
2015-03-10
期刊：
影响因子：
2.9
通讯作者：
Broderick JB
中科院分区：
文献类型：
--
作者：
Betz JN;Boswell NW;Fugate CJ;Holliday GL;Akiva E;Scott AG;Babbitt PC;Peters JW;Shepard EM;Broderick JB
HydE和HydG是[FeFe]-氢化酶（HydA）成熟所需的自由基s -腺苷蛋氨酸酶，并产生其独特的催化簇特征的非蛋白有机配体。HydA的催化簇（h簇）是典型的[4Fe-4S]立方烷与2fe亚簇的桥接，该亚簇包含两个一氧化碳、三个氰化物和一个桥接二硫甲胺作为配体。虽然最近的研究揭示了HydG生物合成双原子配体的性质，但对HydE的功能知之甚少。在此，我们提供了生物化学、光谱、生物信息学和分子建模数据，这些数据共同绘制了活性位点，并为海德在h簇生物合成中的作用提供了重要的见解。电子顺磁共振和紫外可见光谱研究表明，重组的HydE结合了两个[4Fe-4S]簇，并与s -腺苷- l-蛋氨酸结合。D2O中的氘加入到s -腺苷- l-蛋氨酸裂解产物5 ' -脱氧腺苷中，再加上分子对接实验表明，HydE底物含有一个硫醇官能团。这些信息，连同HydE序列相似性和基因组背景网络，使我们能够重新定义HydE的假定机制，远离biob类硫插入化学；这些数据共同表明，h簇二硫甲基胺桥中的硫原子的来源可能来自海德的含硫醇底物。
HydE and HydG are radical S-adenosylmethionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the non-protein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxide, three cyanide, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatics, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-L-methionine. Incorporation of deuterium from D2O into 5’-deoxyadenosine, the cleavage product of S-adenosyl-L-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, have allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster are likely derived from HydE’s thiol containing substrate.