一种新型去糖基酶的催化机理及其关键催化结构域的鉴定

There are many natural products which have novel structures and significant pharmacological activities existed in nature as aglycones. However, difficulties in preparing the aglycone moieties greatly limited related drug research & development progress. The enzymes having the ability to remove the glycosyl group are all obtained from nature. Rational design and development of such enzymes is still in serious lack of the theoretical basis. To solve this issue, a novel enzyme Xyl_S, which was previously isolated and characterized by the applicant, is used as the research object. This work aimed at in-depth research into its catalytic mechanism and structure-function relationship from both small and macromolecular levels: from small molecular level, characterization of the sugar moiety configuration changes, and inhibition studies will be performed in order to reveal its catalytic mechanism; from macromolecular level, protein recombination and enzymatic assays will be performed to identify its key catalytic domain, then, systematic characterization of the catalytic properties of the recombinants to further study its structure-function relationship; finally, verification studies might be performed by site-directed mutagenesis and in silico modeling. The research work performed here will provide solid experimental foundations and theoretical basis for final elucidation of the structure-function relationship of Xyl_S, and provide guidance for the development of the glycosyl removing enzymes which have high activity and selectivity, and excellent stability.

自然界中很多糖苷化合物的苷元都具有重要药理活性，但苷元的制备困难极大地限制了基于该类化合物的创新型药物研发。目前已有的去糖基酶都是从自然界中筛选得到的，其活力和选择性难以满足需求，而对该类酶的理性设计和制备仍严重缺乏理论依据。为解决这一问题，本项目拟以前期工作中开发出的新型去糖基酶Xyl_S为研究对象，分别从小分子和大分子两个水平上对其催化机理以及结构-功能关系进行深入研究：小分子水平上，通过产物糖分子鉴定以及抑制动力学研究揭示该酶的催化机理；大分子水平上，通过重组表达结合酶活性监测对其关键催化结构域进行鉴定，并在此基础上，对不同重组体进行酶学表征，深入探讨其结构-功能关系；最后，以定点突变和计算机建模法进一步验证。本工作的进行，不仅为最终揭示Xyl_S酶的结构-功能关系提供扎实的实验基础和理论依据，还为具高活力、高选择性和良好稳定性的理想糖基去除酶的开发提供指导。

自然界中很多具有重要药理活性、结构新颖的天然产物都是以糖苷的形式存在的，苷元母核往往是新药开发的基础。对于一些大位阻、强疏水性的糖苷化合物，找到能够去除其糖基、制备相应苷元的酶，仍极具挑战性。本项目根据大位阻、强疏水性糖苷化合物去糖基酶设计开发中存在的共性问题，对一种新型去糖基酶Xyl_S，分别从小分子和大分子两个水平上对其催化机理和关键催化结构域进行了鉴定：小分子水平上，通过产物糖分子鉴定以及抑制动力学研究揭示该酶的催化机理，结果表明，目标酶可能是LPMO酶，即该酶催化糖基去除，是通过氧化还原反应进行，反应过程中需要氧气的参与，且产物分子中有过氧化氢的产生；大分子水平上，通过天然来源酶分子进行底物谱分析和蛋白质组学分析发现，其关键催化亚基在临近种属中都是存在的，且具有很强的保守性。对该菌株细胞壁表面的超分子结构进行了基于电子显微镜的分析，在水不溶性底物的诱导下，细胞壁表面出现了15-20nm左右的凸起，该凸起具有同水不溶性底物如纤维素的强结合能力，极大地有利于细胞降解该底物作为能量。本工作的进行，不仅初步揭示Xyl_S酶的催化机理，为结构-功能关系研究提供扎实的实验基础和理论依据，还将为具备高活力、高选择性和良好稳定性的理想糖基去除酶的理性设计与开发提供了指导。