The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase - Chemical modification and site-directed mutagenesis

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the r eversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl ca rrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of li pid A biosynthesis. Lipid A is required for the growth and virulence of mos t Gram-negative bacteria, making its biosynthetic enzymes intriguing target s for the development of new antibacterial agents. LpxA is a member of a la rge family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and argin ine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in c onjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specif ic activities under standard assay conditions. The decrease in activity cor responds to decreased k(cat)/K-m,K-UDP-GlcNAc values for all the mutants. W ith the exception of H125A, in which no activity is seen under any assay co ndition, the decrease in k(cat)/K-m,K-UDP-GlcNAc mainly reflects an increas ed K-m,K-UDP-GlcNAc. His(125) of E. coli LpxA may therefore function as a c atalytic: residue, possibly as a general base. LpxA does not catalyze measu rable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3- O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mecha nism with an acyl-enzyme intermediate.