ENZYME INTERMEDIATE STRUCTURES BY NMR

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This renewal proposal requests funds to continue our studies ont he structure-function relationships for the enzyme 5-enolpyruvylshikimate-3- phosphate (EPSP) synthase, while developing further a new method for the structural characterization of enzymatic reactions in general by time- resolved solid-state NMR spectroscopy. The project will be extended to include uridine diphosphate N-acetyl-glucosamine enolpyruvyl transferase (UDP-NAG EPT) and 3-deoxy-D-manno-2-octulosonate-8-phosphate (KD08P) synthase. The research program is designed to determine, using nuclear magnetic resonance (NMR) spectroscopy, the structure of the enzyme-bound intermediates of three enolpyruvyl transfer enzymes; EPSP synthase; UDP- NAG EPT and KD08P synthase. EPSP synthase catalyzes the condensation of shikimate-3-phosphate and phosphoenolpyruvate, and the product, EPSP, is a key intermediate in the biosynthesis of aromatic amino acids. UDP-NAG EPT is a key enzyme in bacterial cell wall biosynthesis, and KD08P synthase is involved in bacterial lipopolysaccharide biosynthesis. The specific aims of this renewal proposal are to: (1) carry out site-directed mutagenesis studies on specific active site residues of EPSP synthase, (2) carry out time-resolved solid-state REDOR NMR measurements on EPSP synthase, measuring longer distances than was originally proposed in GM43215, and (3) extend this approach to two other enolpyruvyl transferase enzymes, UDP-NAG EPT and KD08P synthase, as time permits. We believe that the consequences of these studies are particularly interesting and exciting, not just for extending our understanding of the structure-function relationship of EPSP synthase and related enolpyruvyl transferase enzymes, but also for developing methodologies that can provide detailed time-resolved structural information on enzymatic reactions in general, which even sophisticated techniques like Laue X-ray diffraction have difficulty obtaining. The long-term goal of our research is to collaborate with a Laue X-ray crystallographer, whose structural information of the protein as whole will be crucial, and generate a "move" of the molecular details of an enzyme in action. This might enable the rational of antibacterial agents in the future.