The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in $Corynebacterium\ glutamicum$
Résumé
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.
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