Structural Insight into the Catalytic Mechanisms of an L-Sorbosone Dehydrogenase.
Structural Insight into the Catalytic Mechanisms of an L-Sorbosone Dehydrogenase.
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DOI：
10.1002/advs.202301955
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发表时间：
2023-10
期刊：
影响因子：
15.1
通讯作者：
Zhou, Jingwen
中科院分区：
文献类型：
--
作者：
Li, Dong;Deng, Zhiwei;Hou, Xiaodong;Qin, Zhijie;Wang, Xinglong;Yin, Dejing;Chen, Yue;Rao, Yijian;Chen, Jian;Zhou, Jingwen
L-山梨糖酮脱氢酶（SNDH）是参与2-酮-L-古洛糖酸生物合成的关键酶，2-酮-L-古洛糖酸是工业规模生产维生素C的直接前体。阐明其结构和催化机理是提高SNDH性能的关键。通过解析氧化葡糖醛酸WSH-004的SNDH晶体结构，发现Cys 295和催化Cys 296残基之间存在可逆的二硫键。它允许SNDH在氧化和还原状态之间切换，导致打开或关闭衬底口袋。此外，发现Cys 296影响NADP+与SNDH的结合姿势。结合体外生物化学和定点突变研究，提出了SNDH基于氧化还原的动态调控和催化机制。此外，通过延伸底物通道，获得了活性增强的突变体。本研究不仅阐明了脱氢酶的生理调控机制，而且为类似酶的工程改造提供了理论依据。基于对SNDH结构的分析，本研究提出了SNDH的氧化还原机制，即允许底物口袋打开或关闭，从而影响其活性。此外，通过对SNDH底物通道的分析，确定了底部隧道瓶颈区域，并通过释放该区域，获得了活性显著增强的突变体。
L‐Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2‐keto‐L‐gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH‐004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site‐directed mutagenesis studies, the redox‐based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes. Based on the analysis of SNDH structure, this study proposes a redox mechanism of SNDH, which allows the substrate pocket to be opened or closed, thereby affecting its activity. In addition, through the analysis of substrate channel of SNDH, the bottom tunnel bottleneck area is identified, and by releasing it, a mutant with significantly enhanced activity is obtained.
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