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 reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction 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 specific activities under standard assay conditions. The decrease in activity corresponds to decreased k(cat)/K(m,UDP-GlcNAc) values for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in k(cat)/K(m,UDP-GlcNAc) mainly reflects an increased K(m,UDP-GlcNAc). His(125) of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate.
Wyckoff
T J
TJ
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
C R
CR
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008050:Lipid A / Q000096:biosynthesis; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D008050:Lipid A; D006639:Histidine; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase