Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology , Jiangnan University , Wuxi , 214122 Jiangsu , China.
State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis , Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai 200032 , China.
J Am Chem Soc. 2018 Oct 3;140(39):12645-12654. doi: 10.1021/jacs.8b08640. Epub 2018 Sep 24.
Diaryl ketones are important building blocks for synthesizing pharmaceuticals and are generally regarded as "difficult-to-reduce" ketones due to the large steric hindrance of their two bulky aromatic side chains. Alcohol dehydrogenase from Kluyveromyces polyspora ( KpADH) has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate (4-chlorophenyl)(pyridine-2-yl)ketone (CPMK) with a moderate R-selectivity of 82% ee. To modulate the stereoselectivity of KpADH, a "polarity scanning" strategy was proposed, in which six key residues inside and at the entrance of the substrate binding pocket were identified. After iterative combinatorial mutagenesis, variants Mu-R2 and Mu-S5 with enhanced (99.2% ee, R) and inverted (97.8% ee, S) stereoselectivity were obtained. The crystal structures of KpADH and two mutants in complex with NADPH were resolved to elucidate the evolution of enantioselective inversion. Based on MD simulation, Mu-R2-CPMK and Mu-S5-CPMK were more favorable in the formation of prereaction states. Interestingly, a quadrilateral plane formed by α-carbons of four residues (N136, V161, C237, and G214) was identified at the entrance of the substrate binding pocket of Mu-S5; this plane acts as a "polar gate" for substrates. Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents, the pro- S orientation of CPMK is defined when it passes through the "polar gate" in Mu-S5, whereas the similar plane in wild-type is blocked by several aromatic residues. Our result paves the way for engineering stereocomplementary ADH toward bulky diaryl ketones and provides structural insight into the mechanism of stereoselective inversion.
二芳基酮是合成药物的重要构建模块，由于其两个庞大的芳基侧链的大位阻，通常被认为是“难还原”的酮。由于 Kluyveromyces polyspora（Kp）醇脱氢酶（ADH）对二芳基酮底物（4-氯苯基）（吡啶-2-基）酮（CPMK）具有较高的转化率，并且具有 82%ee 的中等 R-选择性，因此被鉴定为一种强大的生物催化剂。为了调节 KpADH 的立体选择性，提出了“极性扫描”策略，其中确定了底物结合口袋内部和入口处的六个关键残基。经过反复组合诱变，获得了具有增强（99.2%ee，R）和反转（97.8%ee，S）立体选择性的变体 Mu-R2 和 Mu-S5。解析了 KpADH 及其与 NADPH 复合物的两种突变体的晶体结构，以阐明对映选择性反转的演变。基于 MD 模拟，Mu-R2-CPMK 和 Mu-S5-CPMK 更有利于预反应状态的形成。有趣的是，在底物结合口袋的入口处鉴定到由四个残基（N136、V161、C237 和 G214）的α-碳原子形成的四边形平面；这个平面充当底物的“极性门”。由于氯苯基和吡啶取代基之间的电荷特性差异，当 CPMK 通过 Mu-S5 中的“极性门”时，其呈现出 pro-S 取向，而在野生型中类似的平面则被几个芳基残基所阻挡。我们的结果为工程化针对大位阻二芳基酮的立体互补 ADH 铺平了道路，并为立体选择性反转的机制提供了结构见解。
