Evolutionary coupling-inspired engineering of alcohol dehydrogenase reveals the influence of distant sites on its catalytic efficiency for stereospecific synthesis of chiral alcohols,Computational and Structural Biotechnology Journal

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Evolutionary coupling-inspired engineering of alcohol dehydrogenase reveals the influence of distant sites on its catalytic efficiency for stereospecific synthesis of chiral alcohols
Computational and Structural Biotechnology Journal ( IF 4.4 ) Pub Date : 2021-10-26 , DOI: 10.1016/j.csbj.2021.10.031
Jie Gu ₁ , Byu Ri Sim ₂ , Jiarui Li ₁ , Yangqing Yu ₁ , Lei Qin ₁ , Lunjie Wu ₁ , Yu Shen ₁ , Yao Nie _{1,

3} , Yi-Lei Zhao ₂ , Yan Xu _{1,

4}

Affiliation

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24–7.81 fold increase in value compared with that of the wild type, when reacted with 8 aromatic ketones or -ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.

中文翻译：

醇脱氢酶的进化耦合工程揭示了远距离位点对其手性醇立体定向合成催化效率的影响

醇脱氢酶（ADH）因其能够以高立体选择性催化合成重要的手性醇药物中间体而备受关注。 ADH 蛋白质工程工作通常集中于重塑底物结合袋。然而，尽管潜在的分子机制仍不清楚，但口袋外的遥远位点也可能影响其活性。当前的研究旨在将进化耦合启发的工程应用于 ADH CpRCR 并识别潜在的突变位点。通过保守性分析、系统发育分析和残基分布分析，证实共同进化热点Leu34和Leu137在自然选择的压力下高度进化，并可能与蛋白质的催化功能有关。因此，选择远离活性中心的Leu34和Leu137进行突变。当与 8 种芳香酮或酮酯反应时，生成的 CpRCR-L34A 和 CpRCR-L137V 变体表现出高立体选择性，与野生型相比，其值增加了 1.24-7.81 倍。相应的计算研究表明，L34和L137可能将蛋白质结构的变构波动从远端突变位点延伸到活性位点。此外，L34 和 L137 突变以多种方式改变了预反应状态，即氢化物相对于目标羰基的位置。这些发现提供了对酶催化机制的见解，并从位点相互作用网络的角度促进其调节。

更新日期：2021-10-26

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