Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.ATP依赖型Mur连接酶（Mur synthetases）在细胞壁肽聚糖（peptidoglycan, PG）的生物合成中发挥关键作用：它们以ATP水解为能量代价，催化关键氨基酸残基与茎肽的连接反应，因此成为抗菌药物研发的潜在靶点。本研究对分裂/细胞壁（dcw）操纵子进行了系统表征，并鉴定出一个可驱动Mur合成酶与ftsQ、ftsW等关键细胞分裂基因共转录的启动子。此外，本研究将此前针对结核分枝杆菌（Mycobacterium tuberculosis）MurE的研究拓展至该菌的MurC、MurD及MurF合成酶。对纯化重组酶的功能分析显示，二价阳离子的存在是其酶活发挥的绝对必要条件。本研究同时发现，高浓度的ATP与UDP糖底物会抑制所有Mur合成酶的酶活，这表明肽聚糖生物合成通路的胞质阶段受到严格的调控。与此前关于结核分枝杆菌MurD及棒状杆菌MurC合成酶通过磷酸化进行调控的研究结果一致，本研究发现所有Mur合成酶均能与丝氨酸/苏氨酸蛋白激酶（Ser/Thr protein kinases）PknA和PknB发生特异性相互作用。此外，本研究深入解析了所有Mur合成酶与参与细胞分裂及细胞壁肽聚糖生物合成的蛋白质之间的相互作用网络，以重新评估这些关键酶作为抗结核药物研发新型治疗靶点的重要价值。