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.