title: Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis.
abstract: 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, DeltaH(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.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
authors.forename: Matija
authors.initials: M
authors.lastname: Hedl
authors.forename: Autumn
authors.initials: A
authors.lastname: Sutherlin
authors.forename: E Imogen
authors.initials: EI
authors.lastname: Wilding
authors.forename: Marie
authors.initials: M
authors.lastname: Mazzulla
authors.forename: Damien
authors.initials: D
authors.lastname: McDevitt
authors.forename: Pamela
authors.initials: P
authors.lastname: Lane
authors.forename: John W
authors.initials: JW
authors.lastname: Burgner
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Lehnbeuter
authors.forename: Cynthia V
authors.initials: CV
authors.lastname: Stauffacher
authors.forename: Michael N
authors.initials: MN
authors.lastname: Gwynn
authors.forename: Victor W
authors.initials: VW
authors.lastname: Rodwell
mesh_terms: D000214:Acyl Coenzyme A / Q000502:physiology*; D000595:Amino Acid Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000214:Acyl Coenzyme A; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C010667:acetoacetyl CoA; D019811:Hydroxylamine; C004809:isopentenyl pyrophosphate; D006903:Hydroxymethylglutaryl CoA Reductases; D004047:Diethyl Pyrocarbonate