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.
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