Kinetic Analysis of the Zinc-Dependent Deacetylase in the Lipid A Biosynthetic Pathway ² Amanda L. McClerren, Pei Zhou, Ziqiang Guan, Christian R. H. Raetz, and Johannes Rudolph* Department of Biochemistry, Duke UniVersity Medical Center, P.O. Box 3813, Durham, North Carolina 27710 ReceiVed September 16, 2004; ReVised Manuscript ReceiVed October 19, 2004 ABSTRACT: The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2′′-position of UDP-3- O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus,a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k cat /K M and k cat for the deacetylation of the substrate UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK 1 ) was fitted to 6.1 ( 0.2 for k cat and 5.7 ( 0.2 for k cat /K M . The descending basic limb (pK 2 ) was fitted to 8.0 ( 0.2 for k cat and 8.4 ( 0.2 for k cat /K M . The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water. Lipopolysaccharide (LPS) 1 is a unique glycolipid found at the cell surface of most Gram-negative bacteria, including Escherichia coli and Salmonella typhimurium. LPS is an important immunogenic determinant of Gram-negative bac- terial infections and provides a formidable barrier to many potentially bactericidal compounds and environmental haz- ards. Lipid A is the hydrophobic anchor moiety of LPS, securing it to the outer surface of the outer membrane. Lipid A is a hexaacylated (1′-6)-linked disaccharide of glu- cosamine that is glycosylated with two 3-deoxy-D-manno- octulosonic acid (Kdo) sugars at position 6′ to comprise the minimal structure of LPS required for growth in most Gram- negative bacteria (1). The biosynthetic pathway of lipid A is conserved and involves nine essential enzymes. The second of these is LpxC, a deacetylase that removes acetate from the nitrogen at the 2′′-position of UDP-3-O-acyl-N-acetylglucosamine. Because the first step of biosynthesis is thermodynamically unfavorable, deacetylation by LpxC is the first committed step of the pathway, making LpxC an attractive target for antibiotic design. LpxC, like other zinc-dependent hydrolases, can be effectively inhibited by hydroxamate-containing compounds (2-5). One of these is TU-514, a substrate- analogue inhibitor of LpxC that binds to the active site Zn 2+ . TU-514 is the only compound known to inhibit a broad range of LpxC orthologues, showing potency against LpxC from both E. coli and Aquifex aeolicus, the latter orthologue having only 32% sequence identity with the one from E. coli (3). However, TU-514 lacks efficacy in vivo, most likely because it is excluded by the cell envelope. Progress in the develop- ment of more effective inhibitors of this important target may be facilitated by a better understanding of the catalytic mechanism of LpxC. Recently, the structure of LpxC from A. aeolicus was determined by NMR (6) and X-ray crystallography (7). The protein consists of two domains with similar folds, each containing a layer of R-helices packing against a primary -sheet. LpxC contains a novel Zn 2+ -binding motif, HKXXD, with Zn 2+ coordination by His74, His226, and Asp230 (Figure 1). A water molecule serves as the fourth ligand in the free enzyme but can be replaced by the carbonyl oxygen of the hydroxamate in the inhibitor-bound structure. Con- served Lys227 could function to stabilize the transition state or organize the active site. Conserved residues Glu73 and His253 are also located in the active site and have been shown to coordinate a second inhibitory Zn 2+ ion present in the crystal structure. Additionally, these two residues may be in position to hydrogen bond to the substrate or zinc- bound water molecule. Asp234 is also conserved and is located one helical turn away from the Zn 2+ ligand Asp230, within H-bonding distance to His253 (Figure 1). Although LpxC shows no amino acid sequence homology with other known zinc-dependent hydrolases and has a ² This research was supported by NIH Grant GM-51310 to C.R.H.R. and NIH AI-055588 to P.Z. The mass spectrometry facility in the Department of Biochemistry of the Duke University Medical Center and Z.G. were supported by the LIPID MAPS Large Scale Collaborative Grant number GM069338 from NIH. * To whom correspondence should be addressed. Phone: (919) 668- 6188. Fax: (919) 613-8642. E-mail: rudolph@biochem.duke.edu. 1 Abbreviations: LPS, lipopolysaccharide; UDP-GlcNAc, UDP-N- acetylglucosamine; UDP-3-O-acyl-GlcNAc, UDP-3-O-acyl-N-acetyl- glucosamine; BSA, bovine serum albumin. 1106 Biochemistry 2005, 44, 1106-1113 10.1021/bi048001h CCC: $30.25 © 2005 American Chemical Society Published on Web 01/07/2005