Engineering bi-functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration
Engineering bi-functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration
复制标题
通过肽接头介导的融合工程设计甲酸脱氢酶和亮氨酸脱氢酶的双功能酶复合物以加速辅因子再生
DOI：
10.1002/elsc.201600232
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发表时间：
2017
影响因子：
2.7
通讯作者：
Fang BS
中科院分区：
文献类型：
--
作者：
Zhang Yonghui;Wang Yali;Wang Shizhen;Fang Baishan;Fang Baishan;Fang Baishan;Wang SZ;Fang BS
本研究报道了肽连接剂在构建双功能甲酸脱氢酶（FDH）和亮氨酸脱氢酶（LeuDH）酶复合物中的应用，用于高效的辅助因子再生和L -叔亮氨酸（L - tle）生物转化。7种FDH - LeuDH融合酶分别具有不同的肽连接体，且均具有亲本酶活性。利用I - TASSER服务器预测了LeuDH - FDH和FDH - LeuDH模型的三维结构，研究了FDH和LeuDH的结合顺序。酶学表征表明，刚性肽连接物的插入比柔性肽连接物具有更好的活性和热稳定性。选择合适的柔性肽连接体的融合酶复合物的产率比自由酶混合物提高了1.2倍。此外，FDH和LeuDH的结构分析表明，N‐、C‐末端结构域的二级结构及其与功能域的相对位置也与融合酶配合物的催化性能有很大关系。结果表明，刚性肽连接体可以保证肽段的独立折叠和酶结构的稳定，而柔性肽连接体则可能使酶段靠近，从而获得更好的辅因子通道。
This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.