Purification and biochemical characterization of Mur ligases from Staphylococcus aureus.

Abstract 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.