This project will support structural and mechanistic studies of enzymes in the isoprenoid biosynthetic pathway and the development of screening procedures for drug candidates that inhibit enzymes in the methylerythritol phosphate pathway to isopentenyl and dimethylallyl diphosphate. Enzymes to be studied include some of the proteins in the methylerythritol phosphate pathway (methylerythritol phosphate synthase, the transporter/kinase involved in uptake of methylerythritol, the enzyme that converts hydroxydimethylallyl diphosphate to isopentenyl diphosphate and dimethylallyl diphosphate), the type I and type II isopentenyl diphosphate isomerases, and the enzymes that catalyze cyclopropanation building reactions (squalene synthase, phytoene synthase, and chrysanthemyl diphosphate synthase). The proposed work has impact in two health-related areas of national concern. High serum cholesterol is a major determinant in coronary heart disease, the leading cause of death in the U.S. Although statins now on the market are effective, some individuals experience severe side effects and the drugs are not effective for individuals with the most severe homozygote form of familial hypercholesterolemia. The cyclopropanation of one molecule of farnesyl diphosphate by another catalyzed by squalene synthase is the first pathway specific reaction in cholesterol biosynthesis. Squalene synthase is an attractive target for drug development as an alternative to the statins by directly targeting the sterol branch of the pathway. The methylerythritol phosphate pathway is the exclusive route for isoprenoid biosynthesis in many bacteria and is orthogonal to the mevalonate pathway for isoprenoid biosynthesis in humans. Since isoprenoid biosynthesis is essential for bacterial growth, development of compounds that inhibit enzymes in the methylerythritol phosphate pathway are attractive drug candidates as antibacterial compounds with low mammalian toxicity. This project will employ a combination of genetic, molecular biological, enzymological, structural, and synthetic tools to achieve the goals stated above. Recombinant versions of all of the enzymes studied, both wild type and mutant, will be overproduced in bacteria. Mechanistic work will focus on the chemical mechanisms of the enzyme catalyzed reactions and those structural features of the protein important for catalysis. Structural work will feature X-ray studies conducted in collaboration and NMR studies performed on site. Salmonella typhimurium will be the vehicle used to construct strains suitable to screen compounds as inhibitors of enzymes in the methylerythritol phosphate pathway, including enzymes from difficult to handle pathogens. ? ?
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