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.
|Steussy, C Nicklaus; Vartia, Anthony A; Burgner 2nd, John W et al. (2005) X-ray crystal structures of HMG-CoA synthase from Enterococcus faecalis and a complex with its second substrate/inhibitor acetoacetyl-CoA. Biochemistry 44:14256-67|
|Sutherlin, Autumn; Hedl, Matija; Sanchez-Neri, Barbara et al. (2002) Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate biosynthesis. J Bacteriol 184:4065-70|
|Hedl, Matija; Sutherlin, Autumn; Wilding, E Imogen et al. (2002) Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis. J Bacteriol 184:2116-22|
|Kim, D Y; Stauffacher, C V; Rodwell, V W (2000) Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase. Protein Sci 9:1226-34|
|Kim, D Y; Stauffacher, C V; Rodwell, V W (2000) Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation. Biochemistry 39:2269-75|
|Wilding, E I; Kim, D Y; Bryant, A P et al. (2000) Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus. J Bacteriol 182:5147-52|
|Bochar, D A; Tabernero, L; Stauffacher, C V et al. (1999) Aminoethylcysteine can replace the function of the essential active site lysine of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase. Biochemistry 38:8879-83|
|Bochar, D A; Stauffacher, C V; Rodwell, V W (1999) Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mol Genet Metab 66:122-7|
|Tabernero, L; Bochar, D A; Rodwell, V W et al. (1999) Substrate-induced closure of the flap domain in the ternary complex structures provides insights into the mechanism of catalysis by 3-hydroxy-3-methylglutaryl-CoA reductase. Proc Natl Acad Sci U S A 96:7167-71|
|Bochar, D A; Stauffacher, C V; Rodwell, V W (1999) Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase. Biochemistry 38:15848-52|
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