Broad, long-term Objectives. To elucidate, at a molecular level: l) the mechanism of catalysis, 2) specificity determinants for substrates and coenzyme, and 3) why phosphorylation down regulates activity of the 4- electron oxidoreductase HMG-CoA reductase.
Specific Aims. 1) Identify active site residues which function in catalysis, substrate recognition, or maintaining conformation. 2) Provide physical evidence in support of the P.I. 's proposed novel mechanism for regulation of activity that accompanies phosphorylation of a unique serine, namely, charge-charge interaction with the catalytic histidine. 3) Generate mutant proteins to facilitate visualization of the three- dimensional structures of the """"""""third domain"""""""" of the P. mevalonii enzyme. 4. Overproduce homogeneous hamster HMG-CoA reductase to determine conditions for crystallization and ultimate structure determination. 5) Identify regions of bacterial and hamster HMG-CoA reductase which participate in substrate recognition and binding. 6) Characterize an archaebacterial HMG-CoA reductase. Health Relatedness. The rate-limiting enzyme of human cholesterogenesis, HMG-CoA reductase represents an established target for chemotherapy of hypercholesterolemias. Benefits from the proposed research include improved understanding of the mechanisms of catalysis and of phosphorylation-mediated control of HMG-CoA reductase activity. Practical benefits include enhanced capability for rational design of drugs which inhibit HMG-CoA reductase activity. The compounds in wide use to lower cholesterol levels in hypercholesterolemic individuals are widely held to be active site-directed inhibitors. However, recent work from the P.I. 's laboratory has located the regulatory serine at the active site, and has shown that phosphorylation blocks the function of the catalytic histidine. Existing and future drugs thus might act by interfering with dephosphorylation of the regulatory serine. Research Design and Methodologies. The research will employ HMG-CoA reductases from all three phylogenetic kingdoms: eukaryotes, eubacteria, and the archae. Inter-kingdom chimeric enzymes will also be generated and characterized. Selection of amino acids to be mutated will guided by the now available 3 A resolution crystal structure of P. mevalonii HMG-CoA reductase. Mutant enzymes will be overexpressed, purified, and their enzymic and biophysical properties characterized. Methods to be employed encompass molecular biological, biochemical, and biophysical techniques: site-directed mutagenesis and PCR; overexpression; enzyme purification; enzymic and physical characterization of mutant enzymes; reconstitution of active enzymes by co-expression of mutually complementary inactive mutant enzymes; construction of chimeric enzymes; crystallographic determination of three-dimensional structures.
|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|
Showing the most recent 10 out of 27 publications