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Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

ABSTRACT: LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.

SUBMITTER: Lee CJ

PROVIDER: S-EPMC3149848 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

Lee Chul-Jin CJ Liang Xiaofei X Chen Xin X Zeng Daina D Joo Sang Hoon SH Chung Hak Suk HS Barb Adam W AW Swanson Shauna M SM Nicholas Robert A RA Li Yaoxian Y Toone Eric J EJ Raetz Christian R H CR Zhou Pei P

Chemistry & biology 20101216 1

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC or ...[more]

PMID: 21167751

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Project description:Tight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging of Klebsiella pneumoniae in increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations in fabZ and lpxC occurring together (encoding either FabZR121L/LpxCV37G or FabZF51L/LpxCV37G). K. pneumoniae mutants having only LpxCV37G or LpxCV37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37G accumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37G indicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37G was overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.

Dataset Information

Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

Publications

Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

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