A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation
A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation
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DOI：
10.1002/mbo3.1029
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发表时间：
2020-06-01
期刊：
影响因子：
3.4
通讯作者：
Lenz, Oliver
中科院分区：
文献类型：
--
作者：
Hartmann, Sven;Frielingsdorf, Stefan;Lenz, Oliver
[NiFe]-氢化酶催化氢分子可逆地转化为质子末端电子。该反应发生在位于二分氢化酶模块大亚基中的 NiFe(CN)(2)(CO) 辅因子处。相应的脱辅基蛋白通常带有 C 末端延伸，一旦成熟机制完成辅因子插入，该延伸就会被特定的内肽酶切割掉。该过程触发与氢化酶模块的小型电子转移亚基形成复合物，从而揭示出具有催化活性的酶。然而，C 端延伸在辅因子插入中的作用仍然难以捉摸。我们通过使用基因工程从真养产碱杆菌的膜结合[NiFe]-氢化酶(MBH)大亚基的apo形式中去除整个C末端延伸来解决这个问题。出乎意料的是，源自该预切割大亚基的MBH全酶靶向细胞质膜，赋予宿主菌株H-2依赖性生长，并且纯化的蛋白质表现出与天然MBH完全相同的催化活性。唯一的区别是细胞质膜中氢化酶的含量减少。这些结果表明，在 R 的情况下。 eutropaMBH，C 端延伸对于辅因子插入来说是可有可无的，并且似乎仅作为成熟促进剂发挥作用。
[NiFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)(2)(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo-protein carries usually a C-terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron-transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C-terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C-terminal extension from the apo-form of the large subunit of the membrane-bound [NiFe]-hydrogenase (MBH) fromRalstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H-2-dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of theR. eutrophaMBH, the C-terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.