Biochemical characterization and physiological properties of Escherichia coli UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase.
The UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.
Hervé
Mireille
M
CNRS Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, 91405 Orsay Cedex, France. mireille.herve@u-psud.fr
Boniface
Audrey
A
Gobec
Stanislav
S
Blanot
Didier
D
Mengin-Lecreulx
Dominique
D
D003960:Diaminopimelic Acid / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; D003960:Diaminopimelic Acid; D010453:Peptide Synthases