蓝细菌a-酮戊二酸脱羧酶的结构-功能关系、催化机制及其催化不对称碳-碳键形成研究

蓝细菌a-酮戊二酸脱羧酶是蓝细菌三羧酸循环中一个关键酶，然而目前还没有对其结构-功能关系及催化机制的研究报道。研究其结构-功能关系及催化机制对于深入理解蓝细菌的三羧酸循环和拓宽a-酮酸脱羧酶家族的催化底物多样性都具有重要意义。. 本项目将以聚球藻PCC7002中A2770蛋白为研究对象，通过分子同源建模及蛋白质结晶、靶点氨基酸定点突变、酶动力学分析、氢核磁共振、圆二色谱及质谱等技术手段阐明蓝细菌a-酮戊二酸脱羧酶的结构-功能关系及催化机制，在此基础上开展a-酮戊二酸脱羧酶不对称催化合成手性2-羟基酮研究，进一步拓宽a-酮酸脱羧酶家族的催化底物多样性，同时也为通过基因工程手段改造蓝细菌合成醇类燃料奠定基础。

Cyanobacterial a-ketoglutarate decarboxylase is a key enzyme in cyanobacterial three carboxylic acid cycle. However, these is no research report about the structure-function relationships and catalytic mechanism of cyanobacterial a-ketoglutarate decarboxylase. It is of great significance for in-depth understanding of cyanobacterial three carboxylic acid cycle and exploring the catalytic substrate diversity of a-keto acid decarboxylases family to study the structure-function relationships and catalytic mechanism of cyanobacterial a-ketoglutarate decarboxylase.. The A2770 gene of Synechococcus PCC7002 encoded an a-ketoglutarate decarboxylase. In this project the structure-function relationships and catalytic mechanism of A2770 protein will be elucidated by molecular homology modeling and protein crystallization, site-directed mutagenesis, enzyme kinetic analysis, 1H NMR, time-resolved circular dichroism spectroscopy and mass spectrometry techniques. Then the application of A2770 protein to catalyze the asymmetric synthesis of chiral 2-hydroxy ketone will be explored. Based on the aforementioned results the catalytic substrate diversity of a-keto acid decarboxylases will be further expanded, and also these results will lay the foundation for constructing genetically engineered cyanobacteria to biosynthesize alcohol fuels.