The isoprenoid biosynthetic pathway occurs in all organisms and synthesizes over 33,000 natural metabolites. There are eight branches of the pathway in humans for synthesis of modified tRNAs, sterols, dolochols, respiratory quinones, heme a, and three different groups of prenylated proteins. Human diseases directly linked to defects in isoprenoid metabolism include atherosclerosis, muscle disease, progressive renal failure, Ras-related cancers, Alzheimer's disease, Chediak-Higashi syndrome, and retinal degeneration. This proposal funds structural and mechanistic studies of prenyltransferases. The enzymes catalyze the major building reactions in the isoprenoid required to construct complex structures from five-carbon building blocks. Reactions at major branch points in the pathway are catalyzed by these enzymes. Their activities control the flux of intermediate metabolites into the various branches of the pathway. As a result, most prenyltransferases are highly regulated and, thus, are attractive targets for drug development. They catalyze the alkylation of electron-rich moieties by allylic diphosphates. The electron-rich acceptors being studied are carbon-carbon double bonds (farnesyl diphoshate synthase, undecaprenyl diphosphate synthase), aromatic rings (dimethyallyltryptophan synthase), hydroxyl groups (geranylgeranylglyceryl phosphate synthase), amino groups (dimethyallyl-tRNA synthase), and sulfhydryl groups (protein prenyltransferase). The long term goal of this project is to establish the mechanisms for binding and catalysis. This information will facilitate the development of new classes of inhibitors for the enzymes. The approach involves i) identification and characterization of genes for prenyltranferases, ii) construction of bacterial and fungal strains for mutagenesis and overproduction of the proteins, iii) steady-state and presteady-state kinetic studies, iv) synthesis and evaluation of alternate substrates and inhibitors, v) structural studies of wild type and mutant prenyltranferases, and vi) development of new tools to facilitate this work.
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