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

Abstract 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 (His 12 ) fusion in Escherichia coli ( Eco ). Initial velocity studies show that the fusion enzyme has values of k cat =9 s −1 and of K m (ATP) = 125 μM, K m ( l -lysine) = 122 μM and K m (UDP-MurNAc-dipeptide) = 20.5 μM, at 23 °C. Spy murE is expressed using a new plasmid developed for the expression of GST-HIS 12 tagged proteins in Eco . Identification and purification of the UDP-MurNAc- l -alanyl- d -glutamyl- l -lysine product of the GST-HIS 12 - 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.