This proposal will support genetic, structural, and mechanistic studies of enzymes in the isoprenoid biosynthetic pathway. The specific targets are isopentenyl diphosphate isomerase, squalene synthase, phytoene synthase, chrysanthemyl diphosphate synthase, and enzymes in the non-mevalonate pathway. Isopentenyl diphosphate isomerase and squalene synthase catalyze key reactions in cholesterol biosynthesis. Phytoene synthase and chrysanthemyl diphosphate synthase, although not directly involved in sterol metabolism, catalyze reactions closely related to those seen for squalene synthase. The proposed work impacts to two areas of national health. High serum cholesterol is a major factor in the progression of coronary heart disease, the leading cause of death in the United States. While major therapeutic breakthroughs have been achieved with inhibitors of hydroxymethylglutaryl-CoA reductase, the drugs sometimes produce side effects attributed to inhibition of essential non-sterol branches of the isoprenoid pathway. Targets for inhibition of cholesterol synthesis downstream from hydroxymethylglutaryl-CoA reductase are being actively investigated. The increasing resistance of pathogenic bacteria toward the available arsenal of drugs is of growing national concern. In mammals, the fundamental 5-carbon building blocks for isoprenoid biosynthesis are synthesized by the mevalonate pathway, while the non-mevalonate pathway is the exclusive source of these compounds in many pathogenic bacteria such as E. coli, salmonella, M. tuberculosis. Since isoprenoid compounds are required for viability, the possibility exists for developing a novel class of potent, highly selective antibacterial agents based on inhibitors of the non-mevalonate pathway. This project seeks to establish the reactions and identify the enzymes in the non-mevalonate pathway; to determine the chemical mechanisms for enzymes in cholesterol metabolism and, eventually, for those in the non- mevalonate pathway; to obtain multi-milligram quantities of enzymes through recombinant DNA techniques; to obtain 3- dimensional structures for isoprenoid enzymes; and to determine how the enzymes catalyze their respective reactions. Specific goals for the next period include (1) locate and characterize genes and enzymes in the non-mevalonate pathway, (2) establish the chemical mechanisms for isomerization of isopentenyl diphosphate to dimethylallyl diphosphate and the conversion of farnesyl diphosphate to squalene, (3) locate of crucial catalytic site residues by phylogenetic correlations, covalent modification, and site-directed mutagenesis; and (4) determine 3-dimensional Structures of isoprenoid enzymes through collaborative studies.
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