丝/苏氨酸蛋白激酶对结核分枝杆菌磷酸葡糖胺变位酶Tb_GlmM磷酸化修饰反应的分子调控机制研究

Phosphoglucosamine mutase (GlmM) is responsible for the formation of UDP-N-acetylglucosamine, an important precursor component of cell wall structure, in the human pathogenic bacterium Mycobacterium tuberculosis. Mycobacterium tuberculosis GlmM (Tb_GlmM) is essential for mycobacteria viability, and exists only in prokaryotes, which makes Tb_GlmM an ideal target for development of new anti-tuberculosis drugs. Our recent studies indicated that Tb_GlmM was phosphorylated in vivo, and the phosphorylation state of Tb_GlmM was crucial for maintaining the fully catalytic ability of enzyme. Site-directed mutagenesis of Tb_GlmM showed that a reversible phosphorylation in Ser102 played a pivotal role in regulating Tb_GlmM catalytic ability. At the molecular level, the reversible phosphorylation in Ser102 was tightly regulated by various serine/threonine protein kinases (STPKs), however, the exact regulatory mechanism was unclear. The aim of this project is to further clarify the molecular mechanism that is critical for regulating Tb_GlmM phosphorylation in a physiological manner. The research includes following contents: To identify the specific STPKs which are responsible for Tb_GlmM phosphorylation in vivo and illustrate its possible effects on Tb_GlmM phosphorylation; To identify all the phosphorylation sites on Tb_GlmM and further clarify the effects of each phosphorylation site on regulate Tb_GlmM phosphorylation state and enzyme activity; To determine the phenotypic properties in Mycobacterium tuberculosis H37Ra strains with different Tb_GlmM phosphorylation states. According to the analysis results, the molecular mechanism of Tb_GlmM phosphorylation which regulated by STPKs will be revealed. The study will provide the more evidence for theory on enzyme modification in prokaryote, and also provide a new way for development of Tb_GlmM enzyme inhibitors, as new antituberculosis drugs.

结核分枝杆菌磷酸葡糖胺变位酶Tb_GlmM是参与细胞壁合成的关键酶，为结核分枝杆菌生长所必需，是理想的抗结核药物作用新靶标。本课题组前期研究发现Tb_GlmM第102位保守丝氨酸残基的磷酸化修饰是酶活化的关键。进一步分析提示该保守丝氨酸的磷酸化受到多种丝/苏氨酸蛋白激酶STPKs的调控，但具体机制不明。因此本课题将重点探讨STPKs对Tb_GlmM磷酸化反应的调控，具体内容包括：从蛋白水平确定生理状态下作用于Tb_GlmM的靶STPKs及其作用效应；从分子水平鉴定STPKs作用位点及各磷酸化位点对Tb_GlmM磷酸化状态及酶活性的影响；从菌体水平观察Tb_GlmM不同磷酸化位点的磷酸化状态对细菌生长的影响。通过以上结果系统阐明生理状态下STPKs调控Tb_GlmM磷酸化反应的分子机制。本课题研究结果不仅丰富了原核生物酶修饰与调控的理论知识，也为GlmM抑制剂的定向设计研发提供了新方向。