小亚基对核酮糖-1,5-二磷酸羧化/加氧酶催化功能的影响

项目名称： 小亚基对核酮糖-1,5-二磷酸羧化/加氧酶催化功能的影响

项目编号： No.21506245

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 柳国霞

作者单位： 中国科学院微生物研究所

项目金额： 21万元

中文摘要： 核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）是光合生物固定CO2的关键酶和限速酶，提高其对CO2催化效率及CO2/O2选择性，对提高光合生物固碳效率有重要意义。目前对Rubisco大亚基rbcL的改造结果不理想，而小亚基rbcS如何影响其催化活性未有报道。本项目以rbcS为研究对象，旨在解析rbcS影响Rubisco催化功能的作用机理。针对rbcS定向进化，筛选两个性能分别提高的突变体，分析确立对其活性关键作用的rbcS突变位点；从大小亚基相互作用界面、底物CO2吸附结合能力、产物3PGA逃逸通道层面出发，理性设计以上位点阐明rbcS作用机理；依据此机理，结合不同性能Rubisco的rbcS序列结构分析结果，合成非天然rbcS，使其组装的Rubisco两个性能同时提高，验证此机理的正确性。研究结果有助于深入理解Rubisco结构-功能关系，为其他酶分子的小亚基功能性提供可借鉴思路。

中文关键词： 核酮糖-1；5-二磷酸羧化/加氧酶；小亚基；CO2催化效率；CO2/O2选择性；突变位点

英文摘要： Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key and rate-limiting enzyme for CO2 fixation in photosynthetic organisms. The enhancement of CO2 catalytic efficiency and CO2/O2 specificity significantly improves the efficiency of photosynthetic carbon fixation. The results of large subunit rbcL study of Rubisco are not ideal, otherwise, small subunit rbcS has been reported to effect on Rubisco catalytic activity without clear mechanism. In view of this problem, the project will study and clarify the magical mechanism. Our work will focus on rbcS, directed evolution is firstly executed to obtain mutants with screening which exhibit better CO2 catalytic efficiency or selectivity, respectively, and find out rbcS mutations which have the key role in Rubisco activity. To respect the interface of rbcL and rbcS, binding ability of substrate CO2 and escape passage of product 3PGA, the mechanism of rbcS effect on Rubisco catalytic function will be established with rational design rbcS mutation sites found above. According to the proposed mechanism, the non-natural rbcS will be designed and synthesized after comparisons of sequence and structure of various properties rbcS in different species, the assembled super Rubisco will be originated to verify if our supposed mechanism correct. Furthermore, the results of the project will help us to deeply understand the relationship between the structure and function of Rubisco, still provide valuable ideas to other enzyme molecules in order to explore the small subunit functionality.

英文关键词： ribulose-1;5-bisphosphate carboxylase/ oxygenase;small subunit;CO2 catalytic efficiency;CO2/O2 specificity;mutation sites