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Journal of Bacteriology, August 2002, p. 4065-4070, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4065-4070.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis{dagger}

AUG_only

Departments of Biochemistry,1 Biological Sciences, Purdue University, West Lafayette, Indiana 479072

Received 28 February 2002/ Accepted 29 April 2002

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 Ni2+-agarose to apparent homogeneity and a specific activity of 10 µmol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s20,w, 5.3). Optimal activity occurred in 2.0 mM MgCl2 at 37oC. The {Delta}Ha was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pKa 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 Km for the hydrolysis of acetyl-CoA was 10 µM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.

* Corresponding author. Mailing address: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153. Phone: (765) 494-1608. Fax: (765) 494-7897. E-mail: vrodwell{at}purdue.edu.

{dagger} Journal paper 16732 from the Purdue Agricultural Experiment Station.

Journal of Bacteriology, August 2002, p. 4065-4070, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4065-4070.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Voynova, N. E., Rios, S. E., Miziorko, H. M. (2004). Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway. J. Bacteriol. 186: 61-67 [Abstract] [Full Text]  


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