Purification and biochemical characterization of Mur ligases from Staphylococcus aureus.
The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His(6)-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted L-Ala, L-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for L-Ala. S. aureus MurE was very specific for L-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and L-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (L-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and L-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.
Patin
Delphine
D
Univ Paris-Sud, Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, Orsay F-91405, France.
Boniface
Audrey
A
Kovač
Andreja
A
Hervé
Mireille
M
Dementin
Sébastien
S
Barreteau
Hélène
H
Mengin-Lecreulx
Dominique
D
Blanot
Didier
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003960:Diaminopimelic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D009842:Oligopeptides; D010457:Peptidoglycan; D011994:Recombinant Proteins; D012694:Serine; D003960:Diaminopimelic Acid; D000255:Adenosine Triphosphate; D008025:Ligases; D000409:Alanine; D005998:Glycine