两种甾体皂苷-糖苷酶的水解特异性与其酶蛋白质结构关系的研究

项目名称： 两种甾体皂苷-糖苷酶的水解特异性与其酶蛋白质结构关系的研究

项目编号： No.81202417

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 药物学、药理学

项目作者： 周文斌

作者单位： 中国人民解放军军事医学科学院

项目金额： 23万元

中文摘要： 甾体皂苷是一类重要的活性天然产物，其C-3位的糖链结构对其药理活性具有重要意义。前期发现了2种水解甾体皂苷α-1，2-鼠李糖或α-1，4-糖基的特异糖苷酶，并基本明确了位置选择性水解规律。本课题拟在以上工作基础上，利用已得到的两种酶蛋白的一级结构，通过同源模板蛋白模拟搭建两种糖苷酶的三维结构；嵌合薯蓣皂苷等底物分子，通过结合时的构象，确定影响两种酶底物空间结构及活性的关键氨基酸序列。克隆表达2种糖苷酶，并对关键氨基酸进行定点突变，测定酶活，以验证结构模拟结果的合理性，解释酶蛋白结构与糖基水解选择性间的关系。以上研究，不仅有助于进一步了解2种特异性甾体皂苷糖苷酶的空间位置选择性，及其与蛋白结构的关系，同时对普遍认识酶催化的本质及选择性的结构基础也具有重要意义。

中文关键词： 甾体皂苷；糖苷酶；特异性水解；三维结构；

英文摘要： The steroidal saponin is a kind of important natural products, and its C-3 suger chain structure takes great significance to its pharmacological activity. Two kinds of specific glycosidase, which can hydrolyses the steroidal saponins with α-1,4-glycose were observed in our previous study, and the law of their selective hydrolysis was elucidated basically. This study intends to established the three-dimensional structures of above glycosidase through the simulation technique of homologpus template protein base on the primary structures, and to determine the key amino acid sequences which influence the structure and activity of the two enzymes according to the conformation of the enzymes combined with the dioscin and the other substrate. This study also intends to clone the two enzymes, mutate the key amino acid, and assay the activity, so that to verify the rationality of protein simulation and explain the relationship between protein structure and selectivity of glycosyl hydrolysis. Not only this study will be helpful to further undersand the spatial selectivity of the two steroidal saponin glycosidase and their relationship with protein structure, but also it was very significant to understand the essence of enzyme catalysis and the structure basis of selectivity.

英文关键词： steroidal saponins；glycosidases；hydrolysis specificity；three dimensional structure；