Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis
| Autor: | Cynthia V. Stauffacher, John W. Burgner, Autumn Sutherlin, Kevin R. Lehnbeuter, E. Imogen Wilding, Victor W. Rodwell, Damien McDevitt, Marie J. Mazzulla, Pamela Lane, Michael N. Gwynn, Matija Hedl |
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| Rok vydání: | 2002 |
| Předmět: |
7-Dehydrocholesterol reductase
Molecular Sequence Data Hydroxylamine Biology Reductase Microbiology Enterococcus faecalis Substrate Specificity Gene product Hemiterpenes Organophosphorus Compounds Enterococcus hirae Diethyl Pyrocarbonate Amino Acid Sequence Molecular Biology Thiolase Temperature Hydrogen-Ion Concentration biology.organism_classification Enzymes and Proteins Molecular biology Hydroxymethylglutaryl-CoA reductase Kinetics Biochemistry Hydroxymethylglutaryl CoA Reductases Acyl Coenzyme A Mevalonate pathway |
| Zdroj: | Bindley Publications |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.184.8.2116-2122.2002 |
| Popis: | Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE , that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis , Enterococcus faecium , and Enterococcus hirae . The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli . The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, Δ H a and K m values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K i for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme. |
| Databáze: | OpenAIRE |
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