Publications

• UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme.

  Jackman J.E., Raetz C.R., Fierke C.A.

  The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. ... >> More

  The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity. << Less

  Biochemistry 38:1902-1911(1999) [PubMed] [EuropePMC]

• Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis.

  Whittington D.A., Rusche K.M., Shin H., Fierke C.A., Christianson D.W.

  The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committ ... >> More

  The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committed step in lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal structure of LpxC from Aquifex aeolicus, which reveals a new alpha+beta fold reflecting primordial gene duplication and fusion, as well as a new zinc-binding motif. The catalytic zinc ion resides at the base of an active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty acid. This tunnel accounts for the specificity of LpxC toward substrates and inhibitors bearing appropriately positioned 3-O-fatty acid substituents. Notably, simple inhibitors designed to target interactions in the hydrophobic tunnel bind with micromolar affinity, thereby representing a step toward the structure-based design of a potent, broad-spectrum antibacterial drug. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:8146-8150(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid a biosynthesis.

  Sorensen P.G., Lutkenhaus J., Young K., Eveland S.S., Anderson M.S., Raetz C.R.

  The first enzyme of lipid A assembly in Escherichia coli is an acyltransferase that attaches an R-3-hydroxymyristoyl moiety to UDP-GlcNAc at the GlcNAc 3-OH. This reaction is reversible and thermodynamically unfavorable. The subsequent deacetylation of the product, UDP-3-O-[R-3-hydroxymyristoyl]-G ... >> More

  The first enzyme of lipid A assembly in Escherichia coli is an acyltransferase that attaches an R-3-hydroxymyristoyl moiety to UDP-GlcNAc at the GlcNAc 3-OH. This reaction is reversible and thermodynamically unfavorable. The subsequent deacetylation of the product, UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc, is therefore the first committed step of lipid A biosynthesis. We now demonstrate that inhibition of either the acyltransferase or the deacetylase in living cells results in a 5-10-fold increase in the specific activity of the deacetylase in extracts prepared from such cells. Five other enzymes of the lipid A pathway are not affected. The elevated specific activity of deacetylase observed in extracts of lipid A-depleted cells is not accompanied by a significant change in the Km for the substrate, but is mainly an effect on Vmax. Western blots demonstrate that more deacetylase protein is indeed made. However, deacetylase messenger RNA levels are not significantly altered. Inhibition of lipid A biosynthesis must either stimulate the translation of available mRNA or slow the turnover of pre-existing deacetylase. In contrast, inhibition of 3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis has no effect on deacetylase specific activity. The underacylated lipid A-like disaccharide precursors that accumulate during inhibition of Kdo formation may be sufficient to exert normal feedback control. << Less

  J. Biol. Chem. 271:25898-25905(1996) [PubMed] [EuropePMC]

• The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

  Young K., Silver L.L., Bramhill D., Cameron P., Eveland S.S., Raetz C.R., Hyland S.A., Anderson M.S.

  The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in U ... >> More

  The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene. << Less

  J. Biol. Chem. 270:30384-30391(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase functions through a general acid-base catalyst pair mechanism.

  Hernick M., Gennadios H.A., Whittington D.A., Rusche K.M., Christianson D.W., Fierke C.A.

  UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of L ... >> More

  UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of k(cat)/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4+/-0.1 and 9.1+/-0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile. << Less

  J. Biol. Chem. 280:16969-16978(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.