乳酸乳球菌NZ9000中YlbD的功能及催化机制研究

肽聚糖是细菌细胞壁的重要组成成分，在维持细胞形状、保持细胞完整性方面发挥了重要作用。UDP-MurNAc-三肽（UMT）合成酶基因murE是肽聚糖合成途径中高度保守的必需基因。然而，前期工作表明乳酸乳球菌murE的敲除不影响菌株的正常生长，初步验证发现，YlbD具有UMT合成酶功能且与肺炎链球菌MurTSp（MurT/GatD复合物，肽聚糖氨基转移酶）存在同源性。本项目拟首先确定乳酸乳球菌中YlbD的必需性，然后通过体外酶活、基因组水平改变启动子强弱等实验确证其UMT合成酶和肽聚糖氨基转移酶功能，并研究其菌种间保守性。同时，通过YlbD和MurE的酶活动力学、底物偏好性、与底物共结晶、关键催化位点分析等实验，揭示两者的UMT合成酶催化机制的异同。最终确定乳酸乳球菌中YlbD的功能并阐明其催化机制，为全面解析乳酸乳球菌细胞壁合成途径及相关催化机制提供帮助。

Peptidoglycan is an important component of bacterial cell wall, which plays a vital role in maintaining cell shape and integrity. MurE, UDP-MurNAc tripeptide (UMT) synthase, is essential and highly conserved in the peptidoglycan biosynthesis. However, previous work showed that the knockout of murE did not affect the growth of Lactococcus lactis. Meanwhile, it was found that a putative protein YlbD functioned as UMT synthase, and was homologous with MurTsp of Streptococcus pneumoniae (MurT/GatD complex, required for amidation of peptidoglycan). In this project, we aim to determine whether YlbD is essential in L. lactis firstly, and then identify the functions of catalyzing UMT synthesis and peptidoglycan amidation by enzymatic activity assay and changing the promoter strength at genome-level. And the function conservatism among species as UMT synthase will be studied. Besides, it is proposed to reveal the similarities and differences of the catalytic mechanism of UMT synthase between YlbD and MurE, via the studies on kinetics, substrate preference, cocrystallization with substrate and the conserved catalytic sites. Ultimately, we hope to identify the functions of the putative protein YlbD and the catalytic mechanism functioned as UMT synthase in L. lactis. Our results will be helpful on comprehensive understanding of cell wall biosynthesis and the involved catalytic mechanism of L. lactis.