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 muM, K-m (L-lysine) = 122 muM and K-m (UDP-MurNAc-dipeptide) = 20.5 muM, at 23degreesC. 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. (C) 2004 Published by Elsevier Inc.