PLP依赖的II-氨基酸脱羧酶家族祖先基因序列重构及其趋异进化机制研究

项目名称： PLP依赖的II-氨基酸脱羧酶家族祖先基因序列重构及其趋异进化机制研究

项目编号： No.31470793

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 黄俊

作者单位： 浙江科技学院

项目金额： 80万元

中文摘要： 本研究拟以II-氨基酸脱羧酶家族为研究对象，运用分子生物学、生物信息学和计算生物学的方法，对该酶家族进行氨基酸多序列比对、建立分子系统发育树以及重建进化树中特定节点的祖先序列，依据得到的酶蛋白序列合成相应的基因并通过实验鉴定活性。根据酶学性质表征、结构数据、分子对接和分子动力学模拟数据，比较不同节点祖先酶的突变位置、催化多功能性和稳定性之间的差异，分析氨基酸位点与酶结构、酶催化活性/催化多功能性之间的内在联系。利用通才的祖先酶为亲本酶，通过定向进化等获得高酶活力、热稳定性的谷氨酸脱羧酶突变体，并确定由特定节点的祖先酶成为专一性谷氨酸脱羧酶的可能分子进化途径。此研究不仅为具有stem-cell-like的祖先酶构建提供理论指导和平台技术，从而为理解酶趋异进化的过程和机制提供最直接的科学证据，而且为针对目标底物或活性定制专才的新酶提供新的改造方法和新的性能。

中文关键词： II-氨基酸脱羧酶家族；谷氨酸脱羧酶；计算分子进化；催化多功能性

英文摘要： In this study,molecular biology, bioinformatics and computational biology methods are applied to ancestral genetic sequence resurrection and divergent evolution analysis of group II amino acid decarboxylase family. The multiple amino acid sequences of the family are aligned, and the molecular phylogenetic tree is established. The ancestral amino acid sequences of specific nodes in the evolutionary tree are resurrected by molecular evolution algorithm. The corresponding gene sequences are synthesized according to the calculated ancestral protein amino acid sequences, and ancestral protein activity are characterized by experiments. Based on the data of enzymatic properties characterization, structure and bioinformatics (molecular docking and molecular dynamics simulation) data, mutation position, catalytic promiscuity and stability of the enzymes are compared in different node positions. The interactions among amino acid sites and enzyme structure, enzyme catalytic activity/promiscuity are analyzed. The glutamate decarboxylase mutants with high enzyme activity and thermal stability are obtained using the generalist ancestors as parent protein, and the possible molecular evolution path from the ancestor to specific glutamate decarboxylase is characterized. This study not only provides theoretical guidance and platform technology to resurrect the stem-cell-like ancestral enzyme in order to understand the enzyme divergent evolution process and mechanism, but also provide new methods and new function for tailoring the new specialist enzyme for the target substrate or specific activity.

英文关键词： group II amino acid decarboxylase family;glutamate decarboxylase;computational molecular evolution;catalytic promiscuity