Lipopolysaccharide (LPS) is the major lipid component of the outermost monolayer of most gram-negative bacteria. This monolayer forms a diffusion barrier to hydrophobic and large hydrophilic agents to their periplasmic and cytosolic targets. The first step of LPS biosynthesis in E. coil is catalyzed by UDP-GIcNAc O-acyltransferase (lpxA). This enzyme catalyzes transfer of (R)-3-hydroxymyristic acid from the Acyl Carrier Protein (ACP) to UDP-GlcNAc. Cell lines with antibiotic supersusceptible phenotypes have been identified which are defective in lpxA as well as other early steps in LPS biosynthesis, establishing the validity of the LPS biosynthetic pathway and this enzyme in particular as targets for inhibitor design.
The specific aims of this proposal are: 1. To solve the x-ray crystal structure of the apoenzyme. 2. To determine binding constants of sugar-nucleotide and acyl-ACP ligands to the enzyme. 3. To solve the x-ray crystal structure of enzyme/sugar-nucleotide complexes. 4. To crystallize tight binding enzyme/acyl-ACP complexes. Good, well-ordered single crystals of the apoenzyme are available. These crystals diffract to 2.5 Angstroms resolution and will support an x-ray crystal structure investigation. Fatty acyl-ACP substrate analogs will be prepared and the binding constants to the enzyme of these analogs and sugar nucleotides will be determined. This binding data will be used to investigate the substrate specificity due to fatty acyl chain length and acyl chain substituents as well as that due to the ACP polypeptide. Sugar nucleotides which form tight complexes with the enzyme will be investigated by soaking apoenzyme crystals in solutions of these compounds and determining their crystal structures. These results will be used to determine the interactions of the enzyme with its sugar nucleotide substrate. Tight binding acyl-ACP complexes identified in the binding studies will be used in crystallization experiments with the goal of preparing enzyme/acyl-ACP co-crystals. The information gained from the crystal structures of the apoenzyme and enzyme-ligand forms of the enzyme will provide a detailed description of the active site for use in the design of inhibitors directed against gram-negative pathogens.
