Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate biosynthesis.
Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.
Sutherlin
Autumn
A
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Hedl
Matija
M
Sanchez-Neri
Barbara
B
Burgner
John W
JW
Stauffacher
Cynthia V
CV
Rodwell
Victor W
VW
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013293:Enterococcus faecalis / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D006904:Hydroxymethylglutaryl-CoA Synthase; D007700:Kinetics; D015636:Magnesium Chloride; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature
D000942:Antigens, Bacterial; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D015636:Magnesium Chloride; C004809:isopentenyl pyrophosphate; D000105:Acetyl Coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase; D003066:Coenzyme A Ligases; D008798:Mevalonic Acid