腈水解酶对映体选择性形成机制研究及其理性定制

The understanding of the relationship between protein structure and function is the prerequisites of enzyme designed making. The project is focus on the molecular mechanism of enzyme enantioselectivity. According to the recent literatures, nitrilase demonstrated great enantioselectivity to R-mandelonitrile, however its enantioselectivity mechanism has not been clarified. It hinders the rational design of nitrilase. In our previous study, a new nitrilase from Pseudomonas psychrotolerans, NIT9, was found can hydrolyze racemic mandelonitrile to S-mandelic acid with highly enantiomeric excess value. By combining random mutagenesis with site-directed mutagenesis, two enantiocomplementary nitrilase mutants, were created, which exhibited high S- and R-selectivity toward mandelonitrile, respectively. Besides, the second nitrilase crystal structure was obtained. In this study, two main research contents are conducted to investigate the influence of structure, energy and the profile of proton transfer on the nitrilase enantioselectivity. Firstly, the enantioselective mechanism will be elucidated by protein structure analysis, dynamic evaluation of binding conformation and QM/MM free energy calculation. Subsequently, the structure, energy and the profile of proton transfer of four nitrilases with distinct enantioselectivity will be compared to achieve a full-depth understanding of the enzyme enantioselective mechanism and the relationship of protein structure and function. Secondly, the rational design will be used to obtain nitrilase with with highly catalytic activity and S-selectivity and in turn to verify the above enantioselective mechanism. This study will provide new insight into the mechanism of enzyme enantioselectivity and at the same time lay the foundation for enzyme design.

酶分子“量身定制”时代的步伐取决于对酶结构-功能关系的深度揭示。本项目针对“酶分子对映体选择性是如何形成的”这一科学问题，以及现有腈水解酶对R-扁桃腈具有更高选择偏好性但其机制尚不明确的现象，基于申请人已经获得了目前国际最高S-扁桃腈选择性腈水解酶、国际第二个腈水解酶晶体结构及其对映体选择性骤异的突变体，拟采用动态结合构象分析和QM/MM结合自由能计算两者结合互补的方法，对比并解析具有不同对映体选择性的腈水解酶，从空间位阻、关键结构域、结合自由能和催化中心质子传递四个角度揭示腈水解酶对映体选择性的形成机制，并采用in silico的策略实现其理性定制，以期在酶对映体选择性机制方面获得新发现、提出新观点，突破S-扁桃腈选择性酶数量少、活性低、选择性差的瓶颈，同时基于已有基础有望实现“定制酶”对扁桃腈类似物的选择性系列催化，为酶分子“量身定制”提供新素材、揭示新机制、引入新方法。

课题组基于主持人前期研究成果，选择具有重要应用前景的具水合活性的腈水解酶ATCC43949以及来自 Bradyrhizobium japonicum USDA 110 的腈水解酶bll6402为研究对象，以苯乙腈以及不同位置取代的苯乙腈和外消旋异丁基丁二腈为底物，揭示了具水合活性腈水解酶不同突变热点对不同底物的化学选择性和立体选择性的影响，准确定位了腈水解酶化学选择性和立体选择性相关的氨基酸残基，揭示了腈水解酶立体选择性的机制。项目研究加深了腈水解酶的发展，为腈水解酶化学选择性以及立体选择性分子改造过程中建立小而精的突变库，较高优势突变率的先进酶分子改造技术奠定了基础，解决了立体选择性腈水解酶分子改造的问题，拓宽了腈水解酶理性定制的范围，为实现腈水解酶的高值化利用奠定了基础。

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