一种新型杆状细菌形态分裂相关基因yhdE的结构及功能机制研究

杆状细菌中mre操纵子与细胞的形态、极性、分裂以及染色体分离等生物功能有着重要的关系。 其中的yhdE基因的生物功能还没得到深入地研究。yhdE基因具有明显的保守性,存在于细菌及一些哺乳动物基因组中。以往的研究结果显示枯草芽孢杆菌中yhdE基因可能与调控细胞的形态及分裂有关。申请人现有研究进展第一次发现yhdE是一个核苷酸水解酶，本项目将立足于申请人的研究进展，完成对yhdE核苷酸水解酶的酶动力学研究；通过解析YhdE单体的结构以及YhdE与UTP或TTP复合物的结构，并且进行结构比对；对活性中心中参与底物识别以及催化作用的关键氨基酸残基进行分析，辅以理论计算模拟,来阐明YhdE底物专一性机制，推测可能的催化机理。 同时运用投射电镜等物理化学方法对yhdE基因敲除，突变和过度表达情况下细胞的形态进行观察，并确定蛋白在细胞中定位。从而了解yhdE酶调控TTP/UTP对细胞形态影响机制。

In rode shape bacteria, such as E. coli or B. subtilis, mre operon is widely known as the cell sh ape operon and has also been found to be involved in DNA separation and cell division. However so far the determinants and the mechanisms that make the cell form a rod shape are still mystereis to scientists. The functionally unknown gene, yhdE, locates in the mre operon and is highly conserved across many organisms ranging from bacteria to mammals. To discover what role yhdE may play in this prominent operon, we performed extensive functional analyses on YhdE and found that it is a novel nucleotide phosphatase. Amongst various nucleotides, TTP was the preferred substrate of YhdE, followed by UTP. Glu33 of YhdE was identified as a critical catalytic residue: when mutated to alanine, the YhdE TTPase activity was abolished. This discovery may open insight into further investigations of this important gene and its roles in cell growth, division and rod shape formation. In this project, by using crystal structure of YhdE and its complex with TTP, we will study the enzymatic dynamics of YhdE nucleotidase, and further investigate the key residues at its active site and catalytic mechanisms of substrate specificity with computational dynamics and simulation methods. Moreover we will use physical and chemical techniques, such as cryo EM to study the effect of knockout, mutations or overexpression of yhdE on cell morphology, DNA segregation, cell division and cell growth, and set up the relationship between these cell functions with the size manipulation of the intracellular nucleotide pool.