Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is a glucosamine-based phospholipid that constitutes the outer monolayer of the outer membrane of most Gram-negative bacteria. Also known as endotoxin, lipid A is the active component of LPS that stimulates the immune system and causes lifethreatening Gram-negative septic shock, a severe condition characterized by disseminated intra-vascular coagulation and multiple organ failure. Lipid A biosynthesis is an essential pathway that is conserved in virtually all Gram-negative organisms. The committed step of lipid A biosynthesis is catalyzed by UDP-3-O- (acyl)-N-acetylglucosamine deacetylase (LpxC). LpxC belongs to a novel family of zinc-dependent metalloamidases and shares no sequence homology with any known mammalian proteins. Hence, it is an excellent target for the design of novel antibiotics. Indeed, inhibition of LpxC causes rapid bacterial death and cures mice infected with a lethal intraperitoneal dose of Escherichia coil (E. coh). However, potent inhibitors against the LpxC from E. coli are relatively inactive against divergent LpxCs from other Gramnegative bacteria, particularly, those from Aquifex aeolicus and Pseudomonas aeruginosa. Although LpxCs have been the subject of extensive biochemical studies and pharmacological screenings, the unusual inhibitor specificity and the lack of structural information on LpxCs and their complexes, either with substrates or inhibitors, hinder further mechanistic studies on LpxCs and the optimization of their inhibitors. The overall goal of this proposal is to reveal the largely unknown molecular mechanism underlying LpxC catalysis in lipid A biosynthesis and to provide a structural basis to rationalize the specificity of LpxCs from different Gram-negative species. These studies should also facilitate the development of novel antibiotics targeting LpxC. In the proposed work, the specific aims are: 1) determining the solution structure of the LpxC from Aquifex aeolicus (AaLpxC); 2) determining the solution structure of the AaLpxC/TU-514 inhibitor complex and characterizing the interaction between AaLpxC and its substrate; 3) determining the solution structure of the LpxC from E. coil (EcLpxC); 4) characterizing the interactions between EcLpxC and various inhibitors using structural and biochemical approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI055588-04
Application #
6986164
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Korpela, Jukka K
Project Start
2003-06-15
Project End
2007-11-30
Budget Start
2005-12-01
Budget End
2007-11-30
Support Year
4
Fiscal Year
2006
Total Cost
$300,762
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Titecat, Marie; Liang, Xiaofei; Lee, Chul-Jin et al. (2016) High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors. J Antimicrob Chemother 71:2874-82
Liang, Xiaofei; Gopalaswamy, Ramesh; Navas 3rd, Frank et al. (2016) A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058. J Org Chem 81:4393-8
Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin et al. (2016) Drug design from the cryptic inhibitor envelope. Nat Commun 7:10638
Martin, Jeffrey W; Zhou, Pei; Donald, Bruce R (2015) Systematic solution to homo-oligomeric structures determined by NMR. Proteins 83:651-61
Sampson, Timothy R; Napier, Brooke A; Schroeder, Max R et al. (2014) A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion. Proc Natl Acad Sci U S A 111:11163-8
Lee, Chul-Jin; Liang, Xiaofei; Gopalaswamy, Ramesh et al. (2014) Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC. ACS Chem Biol 9:237-46
Wang, Su; Zhou, Pei (2014) Sparsely-sampled, high-resolution 4-D omit spectra for detection and assignment of intermolecular NOEs of protein complexes. J Biomol NMR 59:51-6
Masoudi, Ali; Raetz, Christian R H; Zhou, Pei et al. (2014) Chasing acyl carrier protein through a catalytic cycle of lipid A production. Nature 505:422-6
Emptage, Ryan P; Tonthat, Nam K; York, John D et al. (2014) Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4'-kinase LpxK. J Biol Chem 289:24059-68
Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A et al. (2013) Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis. Science 341:1012-1016

Showing the most recent 10 out of 36 publications