The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum

Luís M. Mateos,Michel Becchi,Marc J. Canova,Dominique Mengin-Lecreulx,Maria Fiuza,Michal Letek,Delphine Patin,Virginie Molle,José A. Gil,Isabelle Zanella-Cléon

doi:10.1074/jbc.m807175200

Abstract

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.

Highlights

The biggest challenges for modern biomedical research is the continuous development of new antimicrobial drugs targeting bacterial essential mechanisms such as cell division or peptidoglycan (PG)4 biosynthesis [1]
MurNAc:L-alanine ligase (MurC) Is a Substrate of the C. glutamicum PknA—The peptide moiety of the PG monomer unit is assembled stepwise in the cytoplasm by the successive actions of four Mur ligases designated as MurC, MurD, MurE, and MurF
Based on protein sequence alignments and on the three-dimensional structure of the E. coli MurC protein [26], these three domains in the C. glutamicum MurC may extend between Met1–Gly118, Ser119–Arg334, and Arg335–Asn486, respectively (Figs. 1 and 4)

Summary

EXPERIMENTAL PROCEDURES

The murC gene was amplified by PCR using C. glutamicum ATCC 13869 genomic DNA as a template and the primers pair murC1/ murC2 (Table 2), containing NdeI and NheI restriction sites, respectively. E. coli BL21(DE3)Star cells transformed with this construction were used for expression and purification of His6-tagged MurC, as previously described [21]. Purification of the soluble His6-tagged MurC protein from C. glutamicum was performed as described previously [21]. Plasmids pGEXA, pGEXB, pGEXL, and pTEVGfull (Table 1) were used for the expression and purification in E. coli of the four recombinant STPKs from C. glutamicum as previously described [18]. Analysis of the Phosphoamino Acid Content of Proteins— MurC sample (5 ␮g) phosphorylated in vitro by the GST-tagged PknA, and unreacted [␥-33P]ATP were separated by one-dimensional gel electrophoresis and electroblotted onto an Immobilon polyvinylidene difluoride membrane.

Genotype or description

This work

RESULTS AND DISCUSSION

Phosphorylated tryptic and chymotryptic peptide sequence