Cloning, expression and characterization of the Streptococcus pyogenes murE gene encoding a UDP-MurNAc-l-alanyl-d-glutamate: l-lysine ligase

The l-lysine-adding enzyme encoded by the murE gene catalyzes the ATP-dependent formation of UDP-MurNAc-l-alanyl-d-glutamyl-l-lysine (UDP-MurNAc-tripeptide). MurE has been cloned from Streptococcus pyogenes (Spy) and expressed as a glutathione-S-transferase (GST)/polyhistidine (His12) fusion in Escherichia coli (Eco). Initial velocity studies show that the fusion enzyme has values of kcat=9s−1 and of Km (ATP) = 125μM, Km (l-lysine) = 122μM and Km (UDP-MurNAc-dipeptide) = 20.5μM, at 23°C. Spy murE is expressed using a new plasmid developed for the expression of GST-HIS12 tagged proteins in Eco. Identification and purification of the UDP-MurNAc-l-alanyl-d-glutamyl-l-lysine product of the GST-HIS12-Spy MurE enzyme is also described. Surprisingly, this product is a substrate for Eco MurF, an enzyme that normally handles a UDP-MurNAc-tripeptide that contains a diaminopimelic acid residue instead of l-lysine.