Articles Molecular Recognition by Escherichia coli UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine Deacetylase Is Modulated by Bound Metal Ions † Marcy Hernick and Carol A. Fierke* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed August 10, 2006; ReVised Manuscript ReceiVed October 12, 2006 ABSTRACT: The metal-dependent enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacet- ylase (LpxC) catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP- 3-O-(R-3-hydroxymyristoyl)glucosamine and acetate. This is the committed step in the biosynthesis of lipid A, and for this reason, LpxC is a target for the development of antibiotics in the treatment of Gram- negative bacterial infections. Here we examine the importance of bound metal ion(s) and fatty acids for molecular recognition of ligands by LpxC. The K D product value increases >1000-fold with the loss of the hydroxymyristoyl moiety, indicating that the enhanced catalytic efficiency of substrates containing this acyl group is mainly due to increased binding affinity. New fluorescent binding assays for measuring the affinity of LpxC for fatty acids indicate that myristate binds to LpxC 10-fold less tightly than palmitate and that fatty acid affinity is only modestly dependent on pH. Furthermore, LpxC homologues from different species have similar affinities for fatty acids despite alterations in protein sequence. In contrast, the affinity of LpxC for both product and fatty acids is significantly influenced (e40-fold) by changes in the number and identity of metal ions bound to the LpxC active site. Therefore, interactions with these metal ions are critical for molecular recognition of ligands by LpxC and may mimic similar contacts with active site inhibitors. These data indicate that the potency of LpxC inhibitors in vitro can be altered by assay conditions used in screening and/or development of LpxC inhibitors and that the metal ion status of LpxC in vivo will likely influence the effectiveness of LpxC inhibitors as antibiotics. Pathogenic Gram-negative bacteria, including Pseudo- monas aeruginosa, Escherichia coli, Klebsiella sp., and Enterococcus sp., are responsible for approximately half of the serious human infections, and complications from Gram- negative sepsis account for ∼100000 deaths/year in the United States alone (1-3). Gram-negative bacteria such as P. aeruginosa are responsible for the chronic pulmonary infections associated with cystic fibrosis (CF) (4, 5), which is the leading cause of morbidity and mortality in CF patients. This finding highlights the need for more effective antibacte- rial agents for the treatment of Gram-negative bacterial infections. Treatment is complicated by the innate resistance of these pathogens, acquired multidrug resistance, and the limited number of effective antibiotics; therefore, new therapeutic alternatives, including drugs that act on new targets, are needed (6-8). The innate resistance of Gram-negative bacteria is largely attributed to the outer membrane surrounding these organisms (9, 10). Lipid A is the core of lipopolysaccharides (LPS) 1 that form the outer membranes of Gram-negative bacteria and is also the component of LPS responsible for stimulating the immune system in septic shock (11). Consequently, there is interest in the development of inhibitors of lipid A biosynthesis as both antibiotics and antiendotoxins (11-13). Recently, several Gram-negative bacteria have been identified for their potential as bioterror agents and are listed as NIAID category A and B priority pathogens (including Yersinia pestis, Francisella tularensis, Coxiella burnetti, Brucella sp., Burkholderia mallei, Rickettsia prowazekii, Salmonella sp., and Campylobacter jejuni), further increasing the urgency for developing new antibiotics effective against these organ- isms (14). † This work was supported by the National Institutes of Health (Grant GM40602 to C.A.F.) and the Cystic Fibrosis Foundation (Grant HERNIC05F0 to M.H.). * To whom correspondence should be addressed. Phone: (734) 936- 2678. Fax: (734) 647-4865. E-mail: fierke@umich.edu. 1 Abbreviations: LPS, lipopolysaccharide; LpxC, UDP-3-O-(R-3- hydroxymyristoyl)-N-acetylglucosamine deacetylase; UDPGlcNAc, uri- dine-5′-diphosphate N-acetylglucosamine; myrUDPGlcNAc, UDP-3- O-(R-3-hydroxymyristoyl)-N-acetylglucosamine; myrUDPGlcNH2, UDP- 3-O-(R-3-hydroxymyristoyl)glucosamine; EcLpxC, Escherichia coli LpxC; AaLpxc, Aquifex aeolicus LpxC; BSA, bovine serum albumin; TCEP, tris(carboxyethyl)phosphine; EGTA, ethylene glycol bis(2- aminoethyl ether)-N,N,N′,N′-tetraacetic acid; GABC, general acid-base catalyst; BODIPY 500/510 C 4,C9, 5-butyl-4,4-difluoro-4-bora-3a,4a- diaza-s-indacene-3-nonanoic acid; ADIFAB, acrylodated intestinal fatty acid binding protein. 14573 Biochemistry 2006, 45, 14573-14581 10.1021/bi061625y CCC: $33.50 © 2006 American Chemical Society Published on Web 11/17/2006