This project supports structural and mechanistic studies of enzymes in the methylerythritol phosphate (MEP) pathway to isoprenoid compounds found in bacteria and plasmodium from MEP synthase to isopen- tenyl diphosphate (IPP) isomerase. All but one of these enzymes were discovered during the past 10 years and an in-depth understanding of their mechanisms of action will provide important support for drug development. This project incorporates a combination of genetic, molecular biological, enzymological, structural, and synthetic organic tools to determine the chemical mechanisms of the enzymatic reactions and what features in the catalytic site are important for catalysis. Clones of the enzymes have been constructed previously in the Pi's laboratory and will be used to produce wild type and mutant proteins in E. coli. Compounds designed to test mechanistic hypotheses, to provide non-reactive analogues for X-ray studies, and to inhibit the enzymes will be synthesized and evaluated using techniques that were developed by the PI. Inhibitors and alternate substrates for the enzymes from MEP synthase to hydroxydimethylallyl diphosphate will be studied by time course experiments that bypass the need to synthesize intermediates that are difficult to obtain. Two convergently evolved versions of IPP will be studied. Enantiomerically pure irreversible inhibitors will be used to covalently modify essential active site residues of type I IPP isomerase stereoselectively using X-ray structures to determine the sites and stereochemistries of the modifications. The pKa's of essential active site residues will be determined by NMR titration experiments. Type II IPP isomerase is an unusual flavin-requiring enzyme that does not catalyze a net oxidation/reduction. The mechanism of the reaction and the role of the cofactor in catalysis will be studied through the use of mechanism-based inhibitors, UV and EPR studies of the cofactor, and X-ray analysis of the enzyme and enzyme-inhibitor complexes. The isoprenoid biosynthetic pathway provides small molecules for many different essential cellular functions and is found in all organisms. Human diseases directly related to the pathway or treatable by drugs that inhibit enzymes in the pathway include atherosclerosis leading to myocardial and cerebral infarction and gangrene, Paget's disease and, ras-related cancers. Unique features of the isoprenoid pathway in bacteria and plasmodium parasites present new opportunities for developing a new class of drugs to treat infections.
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