小亚基对核酮糖-1,5-二磷酸羧化/加氧酶催化功能的影响

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.

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。

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。由于Rubisco8个大亚基rbcL和8个小亚基rbcS的十六聚体复杂结构，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，通过提高Rubisco固碳能力获得的突变体及杂合rbcS设计，研究探索rbcS影响Rubisco固定CO2催化功能的作用机理。本项目课题研究基本按照计划执行完成，研究结果将加深对Rubisco小亚基rbcS与功能关系的理解，为改造和优化复杂结构的Rubisco提供可借鉴思路和技术途径。.本课题成功构建了Rubisco对CO2催化效率的高活性筛选体系，以及针对Rubisco选择性的筛选体系，并获得了Rubisco高活性突变体；又鉴于Rubisco固碳功能或是加氧活性的酶学性质研究办法复杂不易实现，本课题设计了一种in vivo的Rubisco功能评价办法，用乳酸产量表征Rubisco的固碳能力，为作为重要固碳元件的Rubisco应用研究提供技术途径；依据以上获得的Rubisco高活性突变体rbcS，以及不同植物来源Rubisco的rbcS序列，设计杂合rbcS，证明了伴侣蛋白rbcX对植物源rbcS在E. coli中表达的重要作用，以及从大小亚基相互作用的角度分析研究，探索rbcS对Rubisco催化功能的作用机理。.由该项目支持，在投SCI论文2篇，培养硕士研究生1人。.