The long-term goal of this proposal is to map the catalytic site of HMG-CoA reductase (HMGR) and to relate the secondary-tertiary structure of eukaryotic HMGRs to that of P. mevalonii HMGR and to other 2- and 4-electron NAD+-dependent oxidoreductases.
The Specific Aims i nclude; 1) Identify amino acids of both P. mevalonii and hamster HMGR which function in catalysis, substrate recognition, or maintaining conformation. 2) Optimize production of the catalytic domain of hamster HMGR and initiate its crystallization. 3) Generate mutant proteins, initially of P. mevalonii HMGR, and ultimately of the catalytic domain of hamster HMGR, to facilitate solution of the three-dimensional structure of both proteins. HMGR is the target enzyme for chemotherapy of hypercholesterolemias by drugs such as Lovastatin R. and knowledge derived from this project will impact on the rational design of drugs that inhibit HMG-CoA reductase for use in individuals with elevated blood cholesterol, a large and prognostically significant fraction of the United States population. Amino acids which function in catalysis, in substrate recognition, or in maintaining a native structure will be identified. Techniques to be employed include site-directed mutagenesis, overexpression and purification of mutant enzymes, characterization of enzymic and physical properties, and determination of three-dimensional structures by X-ray crystallography. Residues to be mutated will initially be selected based on inferred roles in catalysis or substrate binding and their conservation across species. A high resolution three-dimensional structure for Pseudomonas mevalonii HMGR, when available, will guide selection of additional residues to mutate.
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