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Published online before print February 6, 2004, 10.1110/ps.03367504
Protein Science (2004), 13:687-693. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Enterococcus faecalis mevalonate kinase

Matija Hedl and Victor W. Rodwell

1 Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063, USA

(RECEIVED August 13, 2003; FINAL REVISION October 16, 2003; ACCEPTED November 10, 2003)



Abstract

Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 µmole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37°C. {Delta}Ha was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. Km values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg2+). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a Ki of 2.7 mM.

Keywords: Enterococcus faecalis; isoprenoid biosynthesis; isopentenyl diphosphate; mevalonate 5-phosphate; mevalonate pathway

Reprint requests to: Victor W. Rodwell, Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN 47907-2063, USA; e-mail: vrodwell{at}purdue.edu; fax: (765) 494-7897.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03367504.


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S. S. Doun, J. W. Burgner II, S. D. Briggs, and V. W. Rodwell
Enterococcus faecalis phosphomevalonate kinase
Protein Sci., May 1, 2005; 14(5): 1134 - 1139.
[Abstract] [Full Text] [PDF]


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