Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide
Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide
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通过 C 端肽的切割来确定模块化膜相关 [NiFe]-氢化酶的合成和组装的协调
DOI：
10.1128/jb.00437-15
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发表时间：
2015
影响因子：
3.2
通讯作者：
Sawers
中科院分区：
文献类型：
--
作者：
Thomas;Sawers
在大肠杆菌的[NiFe]-氢化酶2（Hyd-2）的生物合成过程中，从催化大亚基前体pro-HybC切割15个氨基酸的C-末端肽。该肽仅在Hyp辅助蛋白机械的NiFe（CN）2CO辅因子插入完成后才被去除，这表明它在酶成熟过程中具有调节功能。我们在这里表明，inhypmutants未能合成和插入的NiFe辅因子，因此保留的肽，达特（双精氨酸translocon）信号肽的小亚基HybO没有被删除，亚基被降解。在缺乏大亚基的突变体中，达特信号肽也没有从前HybO中去除，表明成熟的大亚基必须主动接合小亚基以引发达特转运。我们验证了建议的调控作用的C-末端肽在控制酶的组装，通过遗传将其从前体的HybC，这使得组装和Tat依赖的膜协会的HybC-HybO异源二聚体缺乏NiFe（CN）2CO辅因子。最后，从Pro-HyaB，Hyd-1的大亚基，到HybC的C-末端肽的遗传转移并不影响其对辅助蛋白HybG，HypC paralysis，或特定蛋白酶HybD的依赖。这表明C-末端肽本身不是与Hyp机制相互作用所必需的，而是表明肽在维持适合于辅因子插入的蛋白质构象中的作用。总之，我们的研究结果表明，C-末端肽的催化亚基控制的Hyd-2的生物合成，装配，和膜协会。重要的是[NiFe]-氢化酶是多亚基酶的催化亚基含有NiFe（CN）2CO辅因子。以前的研究结果表明，合成和插入的辅因子的Hyp辅助蛋白后，这个大亚基改变构象后，从其C末端的短肽的蛋白水解去除。我们表明，这种肽的去除是必要的，允许裂解的达特信号肽的小亚基与伴随的异二聚体的膜协会发生。从大亚基的C-末端肽的遗传去除允许与小亚基和无NiFe辅因子酶的Tat依赖性膜插入的生产性相互作用。氢化酶之间的C-末端肽交换的基础上的结果表明，这种肽通过构象开关管酶组装。
During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN)2CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that inhypmutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN)2CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptideper seis not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2.IMPORTANCE[NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN)2CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch.
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