探究卤醇脱卤酶催化合成手性β-取代醇相关机制及其改造研究

卤醇脱卤酶在合成手性药物中间体β-取代醇领域应用潜能巨大。但由于缺乏对该酶相关规律性的深入了解，在很大程度上限制其广泛应用。本项目结合卤醇脱卤酶研究现状和申请者自身前期研究结果，拟从酶结构信息与底物结构多样性的双重视角，系统阐明卤醇脱卤酶在催化合成手性β-取代醇反应中亲核试剂多样性机制和对映体选择性规律，以实现该酶在催化手性药物中间体(R)-4-氰基-3-羟基丁酸乙酯合成中高效改造为具体实例。采用Alanine扫描技术和高效的CASTing方法构建多级突变体；综合考虑结构多样性及取代剂的空间位阻和电子效应构建多样性丰富的底物文库；建立高通量酶活检测系统；获取多维酶催化活性数据集；使用改进的双聚类分析手段并结合同源建模和分子柔性对接技术，充分挖掘数据的酶结构信息，并提炼其规律性指导具体应用。本研究实现了从全局方向把握底物结构特征与酶催化特性之间的关系，为高效合成手性药物中间体奠定坚实基础。

Halohydrin dehalogenases displays a great potential in the synthesis of chiral building blocks β-substituted alcohols for pharmaceuticals. However,the catalytic properties of the enzyme has not been investigated in details, which largely limits its industrial application. To address this issue, the substrate promiscuity and enantioselectivity of halohydrin dehalogenase will be fully explored by focusing on both enzyme structure and the substrate diversity simultanousely based on the previously studies. The research outcome will be subsequently applied to guide the enzyme modification for its ability to efficiently synthesize of ethyl (R)-4-cyano-3-hydroxybutyrate, the compound which is the regulated starting material for the production of the cholesterol-lowering drug atorvastatin (Lipitor). In the project, to fully understand the enzyme functionality, multi-level libraries will be constructed by using alanine scaning technique in conjunction with CASTing method; a diversity-rich epoxide library will be constructed by taking into account of both the type of epoxide and the structure diversity of its side-chain; high throughput activity assay will be estilished and optimized. The structure information related to a certain enzyme catalytic property will be revealed through data set mining by using an improved biclustering method in conjuction with homologous modelling and molecular docking. The studies will greatly facilitate not only the rational design of halohydrin dehalogenase,but also the enzyme application in the field of chiral drug synthesis.