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。

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，为理想的抗结核药物作用靶标。磷酸化是生物体最重要的蛋白共价修饰方式，是蛋白的功能“开关”，干预蛋白磷酸化过程已成为药物研发的重要策略。研究发现Tb_GlmM存在磷酸化修饰，且磷酸化修饰是维持酶活性的关键因素，因此，本课题对Tb_GlmM蛋白磷酸化机制进行深入探讨。特异性磷酸丝/苏氨酸抗体检测发现，Tb_GlmM同时存在丝氨酸和苏氨酸残基的磷酸化修饰，但质谱检测未鉴定出Tb_GlmM磷酸化位点，考虑可能由于Tb_GlmM蛋白整体磷酸化水平较低，且不同生理状态下Tb_GlmM磷酸化水平及位点不同导致。序列比对发现Tb_GlmM第102位丝氨酸残基(Ser102)高度保守，该位点突变导致酶活性完全丧失。体外激酶反应发现Tb_GlmM可发生自我磷酸化，Ser102位点突变后，蛋白磷酸化水平无明显改变，但自我磷酸化现象消失，证明Tb_GlmM中Ser102的磷酸化修饰反应为自我磷酸化，Ser102的自我磷酸化是维持酶活性的关键。对Tb_GlmM截短蛋白分析发现，蛋白C端79个氨基酸残基可能参与催化Ser102的自磷酸化反应。Ser102自我磷酸化机制的阐明为Tb_GlmM抑制剂的定向设计研发提供了理论基础。Tb_GlmM中存在多个磷酸化位点，除Ser102，其他位点磷酸化均需激酶参与，文献报道丝/苏氨酸蛋白激酶(STPKs)可能参与GlmM的初始磷酸化过程。为寻找催化Tb_GlmM磷酸化发生的激酶，我们成功表达了11种结核分枝杆菌STPKs的胞内结构域，并验证了激酶活性。然而利用Phos-tag凝胶、ADP-Glo Kinase Assay、Pull-down多种分析方法，均未发现STPKs与Tb_GlmM之间发生相互作用，提示在结核分枝杆菌中Tb_GlmM的初始磷酸化由其他激酶催化，仍需进一步寻找可能的相关激酶。