Fiuza Maria, Canova Marc J, Patin Delphine, Letek Michal, Zanella-Cléon Isabelle, Becchi Michel, Mateos Luís M, Mengin-Lecreulx Dominique, Molle Virginie, Gil José A
Departamento de Biología Molecular, Area de Microbiología, Facultad de Biología, Universidad de León, León 24071, Spain.
J Biol Chem. 2008 Dec 26;283(52):36553-63. doi: 10.1074/jbc.M807175200. Epub 2008 Oct 29.
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.
Mur连接酶在细菌细胞壁肽聚糖的生物合成中发挥着至关重要的作用，因此是新型抗菌药物设计的有吸引力的靶点。这些酶催化肽聚糖二糖肽单体单元肽部分的逐步形成。MurC负责将第一个残基（L-丙氨酸）添加到核苷酸前体UDP-MurNAc上。Ser/Thr蛋白激酶对蛋白质的磷酸化作用最近已成为原核生物中一种主要的生理调节机制。在此，研究了谷氨酸棒杆菌中MurC活性的磷酸化依赖性调节机制的假说。我们表明，MurC在体外被PknA蛋白激酶磷酸化。对磷酸氨基酸含量的分析表明，磷酸化仅发生在苏氨酸残基上。通过质谱分析鉴定出六个磷酸受体残基，并且我们证实将这些残基突变为丙氨酸残基完全消除了PknA依赖性的MurC磷酸化。体外和体内连接酶活性测定表明，这些苏氨酸残基发生突变后，MurC的催化活性受到损害。进一步的体外测定表明，与未磷酸化的蛋白相比，MurC磷酸化异构体的活性严重降低。据我们所知，这是首次在体外证明MurC连接酶的磷酸化。磷酸化与MurC酶活性降低相关的这一发现可能对肽聚糖生物合成的调节产生重大影响。
