Mechanism and inhibition of LpxC: an essential zinc-dependent deacetylase of bacterial lipid A synthesis

Abstract

Multi-drug resistant (MDR), pathogenic Gram-negative bacteria pose a serious health threat, and novel antibiotic targets must be identified to combat MDR infections. One promising target is the zinc-dependent metalloamidase UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyzes the committed step of lipid A (endotoxin) biosynthesis. LpxC is an essential, single copy gene that is conserved in virtually all Gram-negative bacteria. LpxC structures, revealed by NMR and X-ray crystallography, demonstrate that LpxC adopts a novel 'beta-alpha-alpha-beta sandwich' fold and encapsulates the acyl chain of the substrate with a unique hydrophobic passage. Kinetic analysis revealed that LpxC functions by a general acid-base mechanism, with a glutamate serving as the general base. Many potent LpxC inhibitors have been identified, and most contain a hydroxamate group targeting the catalytic zinc ion. Although early LpxC-inhibitors were either narrow-spectrum antibiotics or broad-spectrum in vitro LpxC inhibitors with limited antibiotic properties, the recently discovered compound CHIR-090 is a powerful antibiotic that controls the growth of Escherichia coli and Pseudomonas aeruginosa, with an efficacy rivaling that of the FDA-approved antibiotic ciprofloxacin. CHIR-090 inhibits a wide range of LpxC enzymes with sub-nanomolar affinity in vitro, and is a two-step, slow, tight-binding inhibitor of Aquifex aeolicus and E. coli LpxC. The success of CHIR-090 suggests that potent LpxC-targeting antibiotics may be developed to control a broad range of Gram-negative bacteria.

Figure 1. The lipid A biosynthesis pathway

The biosynthesis of Kdo₂-Lipid A in E. coli requires nine enzymes, beginning with the LpxA-catalyzed acylation of UDP-GlcNAc. Because this reaction is thermodynamically unfavorable, the second reaction catalyzed by LpxC (deacetylation) is the committed step of lipid A biosynthesis.

Figure 2. The structure of LpxC

The three-dimensional structure of LpxC is characterized by a unique “β-α-α-β sandwich” fold. This structure was solved with one bound molecule of the substrate analog, TU-514, that mimics the hexose ring and acyl chain of the substrate. The α-helix of the hydrophobic binding passage encapsulates the acyl chain of TU-514 (pdb-1XXE; this figure was generated using Pymol).

Figure 3. Proposed LpxC mechanisms

Panel A. The LpxC catalytic mechanism proposed by McClerren and coworkers (2005). This proposal suggests that E78 abstracts a proton from zinc-bound water, thereby activating the water for attack on the carbonyl carbon. The proposal presented here suggests that H265 stabilizes the oxyanion intermediate, and E78 later donates a proton to the terminal amine. Panel B. An alternate hypothesis by Hernick and coworkers (2005) suggests that T191 stabilizes the oxyanion intermediate and H265 donates a proton to the liberated amine.

Figure 4. The chemical structures and antibiotic properties of LpxC inhibitors

Panel A. LpxC inhibitors are structurally diverse, though all shown here contain a hydroxamate moiety. The E. coli LpxC K_i for each compound is shown. Each compound, except TU-514, is an effective antibiotic. Panel B. A disc-diffusion assay with 10 µg of each compound applied to a E. coli W3110 bacterial lawn demonstrates that L-161,240 and CHIR-090 are effective antibiotics. Drug-resistant colonies are visible in the zone of L-161,240 growth control distal to ciprofloxacin, suggesting that resistance to this compound is readily selected. There are no resistant colonies visible in the zone of growth control for any other compound tested here.