中国科学院天津工业生物技术研究所的朱敦明研究团队，通过重塑结合位点使腈水解酶催化的二腈不对称水解反应发生手性翻转。 该项研究成果发表在2020年11月3日的《德国应用化学》上。

在这项研究中，通过采用“镜像”策略，即对底物结合口袋进行对称性分析，研究人员在来自集胞藻PCC6803的腈水解酶中，发现了两个关键的影响3-异丁基戊二腈水解手性选择性的氨基酸残基，即W170和V198。将这两个氨基酸残基互换导致手性选择性发生翻转（由S, 90% ee变为R, 47% ee）。通过常规的点饱和突变和组合突变，研究人员进一步重塑了底物结合口袋，获得了具有更高活性和手性选择性（R, 99% ee）的突变体E8。该突变型的酶被用于制备光学纯的(R)-3-异丁基-4-氰基丁酸，并在一系列3-取代的戊二腈水解反应中表现出类似的手性选择性翻转。

这项研究为翻转腈水解酶及其他酶催化含两个相同反应基团的前手性底物的不对称反应的光学选择性提供了一个通用的策略，在不对称合成中这种策略是急需的。

据了解，腈水解酶催化的前手性二腈的不对称水解是一种有吸引力的合成手性氰基羧酸——一种用于合成制药工业中重要的γ-氨基酸的前体——的方法。然而，已经测试过的腈水解酶在催化3-烷基或芳基取代的戊二腈时主要生成(S)-构象的氰基羧酸。

附：英文原文

Title: Inverting the enantiopreference of nitrilase‐catalyzed desymmetric hydrolysis of prochiral dinitriles by reshaping the binding pocket with a “mirror‐image” strategy

Author: Shanshan Yu, Jinlong Li, Peiyuan Yao, Jinhui Feng, Yunfeng Cui, Jianjiong Li, Xiangtao Liu, Qiaqing Wu, Jianping Lin, Dunming Zhu

Issue&Volume:03 November 2020

Abstract: Nitrilase‐catalyzed desymmetric hydrolysis of prochiral dinitriles is an attractive method for the synthesis of chiral cyanocarboxylic acids, the precursors to pharmaceutically important γ‐amino acids. However, the tested nitrilases towards 3‐alkyl‐ or aryl substituted glutaronitriles mostly generate ( S  )‐configurated cyanocarboxylic acids. Herein a “mirror‐image” strategy, i.e. symmetry analysis of substrate‐binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis  sp. PCC6803 towards 3‐isobutyl glutaronitrile ( 1a  ). Exchange of these two residues resulted in the enantiopreference inversion ( S  , 90% ee  to R  , 47% ee  ). By further reshaping the substrate‐binding pocket via routine site‐saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99% ee  , R  ) was obtained. The mutant enzyme was applied in the preparation of optically pure ( R  )‐3‐isobutyl‐4‐cyanobutanoic acid (( R  )‐ 2a  ), and showed similar stereopreference inversion towards a series of 3‐substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other classes of enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups, that are often desirable in asymmetric synthesis.

DOI: 10.1002/anie.202012243

Source: https://onlinelibrary.wiley.com/doi/10.1002/anie.202012243