The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum
The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum
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DOI：
10.1074/jbc.m807175200
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发表时间：
2008-12-26
影响因子：
4.8
通讯作者：
Gil, Jose A.
中科院分区：
文献类型：
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作者：
Fiuza, Maria;Canova, Marc J.;Gil, Jose A.
Mur连接酶在细菌细胞壁肽聚糖的生物合成中起着至关重要的作用，因此代表了设计新型抗菌药物的有吸引力的靶点。这些酶催化肽聚糖双糖肽单体单元的肽段的逐步形成。MurC负责将第一个残基（l -丙氨酸）添加到核苷酸前体UDP-MurNAc上。丝氨酸/苏氨酸蛋白激酶对蛋白质的磷酸化已成为原核生物调控的主要生理机制。本文研究了谷氨酸棒状杆菌MurC活性调控的磷酸化依赖机制。我们发现MurC在体外被PknA蛋白激酶磷酸化。磷氨基酸含量分析表明，磷酸化只发生在苏氨酸残基上。通过质谱分析鉴定了6个磷酸化受体残基，我们证实了对丙氨酸残基的诱变完全消除了pna依赖性的MurC磷酸化。体外和体内连接酶活性分析表明，这些苏氨酸残基突变后，MurC的催化活性受损。进一步的体外实验显示，与未磷酸化的蛋白相比，murc磷酸化的异构体的活性严重降低。据我们所知，这是第一次在体外证明MurC连接酶磷酸化。磷酸化与MurC酶活性降低相关的发现可能对肽聚糖生物合成的调节产生重大影响。
The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.