反Prelog规则羰基还原酶立体选择性识别分子机制研究

R-alcohols are the important chiral blocks for organic synthesis. can selectively catalyze prochiral ketones to R-alcohols. The processes and molecular mechanisms of selective recognition between enzyme and substrate are crucial for the improvement properties of anti-Prelog carbonyl reductases. However, currently it lacks in the insightful information and acknowledges about the enzyme and substrate interaction and the molecular mechanism of selective recognition, which makes it very difficult to carry out the rational design to improve the properties of enzymes. In this project, anti-Prelog carbonyl reductases were selected to investigate the interaction between enzymes and substrates, dynamic structure changes and selective recognition processes by using the molecular dynamics and quantum mechanics. The molecular mechanisms of selective recognition between enzyme and substrate will be clarified and resolved in the theoretical and experimental levels based on the thermodymics and kinetics of biocatalysis with the help of modern instrumental analysis techniques. The novel anti-Prelog carbonyl reductases with excellent activities and selectivities will be finally obtained. This project will provide new techniques and theories for the improvements and application of anti-Prelog carbonyl reductases.

R-手性醇是一类重要的有机合成手性砌块。反Prelog规则羰基还原酶可将潜手性酮催化合成R-手性醇，该酶在R-手性醇合成中具有很好的发展潜力和应用前景。反Prelog规则羰基还原酶与底物的选择性识别过程和分子机制，对该酶性质的提高至关重要，但目前缺乏对反Prelog规则羰基还原酶与底物相互作用和立体选择性识别分子机制的深入研究和解析，对该酶的理性分子改造困难。本项目拟以反Prelog规则羰基还原酶为研究对象，采用分子动力学和量子力学方法研究酶和底物间相互作用、动态结构变化和立体选择性识别过程；利用催化过程动力学和热力学原理，借助现代仪器分析技术手段，在理论和实验水平上揭示和阐明反Prelog规则羰基还原酶和底物相互作用和立体选择性识别的分子机制；在此基础上构建高活力、高立体选择性的反Prelog规则羰基还原酶。项目将为反Prelog规则羰基还原酶的改造和应用提供新的技术方法和理论。

R-手性醇是一类重要的有机合成手性砌块。反Prelog规则羰基还原酶可将潜手性酮催化合成R-手性醇，在R-手性醇合成中具有很好的发展潜力和应用前景。本项目围绕反Prelog规则羰基还原酶的立体选择性机制研究，展开了以下工作：.1）运用基因组狩猎与数据挖掘技术，筛选获得49株高活性羰基还原酶。以羰基还原酶RtSCR9出发，利用随机突变和定点饱和突变技术对酶分子进行改造，获得活力提高的突变体RtSCR9-Ile144Lys，比酶活提高4.00倍，催化效率kcat/Km提高了3.15倍。.2）运用结构模拟、分子对接与定点突变等技术，揭示反Prelog规则羰基还原酶序列、结构与功能的关系，初步阐明了底物口袋的疏水性、NADPH结合域及潜手性酮底物在底物口袋的定位与构象等关键因素对反Prelog规则羰基还原酶催化活性与立体选择性的影响机制。.3）利用反Prelog规则羰基还原酶催化制备系列手性醇，实现了(2S,3R)-2-苯甲酰氨甲基-3-羟基丁酸甲酯、(R)-4-氯-3-羟基丁酸乙酯、(S)-N,N-双甲基-3-羟基-3-(2-噻吩基)丙酰胺、(3R,5S)-6-氯-3,5-二羟基己酸叔丁酯和(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮等手性醇药物中间体的高效生物制备。