亚铁和α-酮戊二酸依赖的乙烯合成酶催化机制和分子改造研究

植物和与植物共生的微生物可以合成乙烯。微生物利用可再生的生物质废料或二氧化碳为碳源合成乙烯，可减轻对化石能源的依赖。丁香假单胞杆菌中的乙烯合成酶（EFE）是极有开发潜力的靶点，因为单个efe基因的表达就可以完成乙烯的合成。在氧气和亚铁结合状态下，EFE催化α-酮戊二酸氧化脱羧生成乙烯，但同时催化L-精氨酸的羟化，对L-精氨酸的依赖和消耗限制了生物乙烯的产量并提高了成本。虽然已有EFE晶体结构的报道，但对于α-酮戊二酸和L-精氨酸是如何结合EFE的分子机制仍不清楚。本项目拟综合应用蛋白质晶体学、生物化学和谱学分析的手段揭示EFE催化双反应的内在分子机制。我们将解析无氧条件下EFE结合底物的复合物晶体结构，阐释EFE结构和功能关系。在结构信息的指导下，结合理性设计和定向进化，制备和筛选出高乙烯的转化能力和低精氨酸羟化活性的突变体酶，推动乙烯生物合成的工业化应用。

Ethylene is produced naturally by plants and some microbes that live with plants. Bio-ethylene could supplement or replace fossil fuels-derived way in chemical industry, using renewable sources of carbon such as waste biomass (plant matter) and CO2. Ethylene-forming enzyme (EFE) is a promising biothechnology target because expression of a single efe gene is sufficient for ethylene production. EFE converts α-ketoglutarate into ethylene by the oxidative decarboxylation of α-ketoglutarate, however, it also catalyzes the C5 hydroxylation of L-arginine (L-Arg) which limits the yield and raises the cost of L-Arg dependent ethylene production. Although several crystal structures of EFE have been published, the molecular mechanism of how α-ketoglutarate and L-Arg binds to enzyme is still not clear. We aim to elucidate the catalytic mechanism by x-ray crystallography，biochemistry and Mössbauer spectroscopy. We plan to determine the crystal structures of EFE in complex with α-ketoglutarate, L-Arg, or both under anerobic conditions. Based on the structure information, the combinatorial active-site saturation test and iterative saturation mutagenesis will be applied to EFE engineering for obtaining engineered EFE with higher α-ketoglutarate conversion efficiency and lower L-Arg oxidation activity.