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Enterococcus faecalis mevalonate kinase.

Matija Hedl¹, Victor W Rodwell

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

Protein Science : a Publication of the Protein Society

|February 10, 2004

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Researchers investigated the mevalonate pathway in Enterococcus faecalis, identifying and purifying mevalonate kinase. This enzyme is crucial for isoprenoid synthesis in Gram-positive pathogens and a potential drug target.

Area of Science:

Biochemistry
Molecular Biology
Microbiology

Background:

The mevalonate pathway is essential for isoprenoid synthesis in Gram-positive pathogens.
Enzymes in this pathway are potential targets for novel antimicrobial drug development.

Purpose of the Study:

To investigate the mevalonate pathway in Enterococcus faecalis.
To characterize the mevalonate kinase enzyme (mvaK1 gene) from E. faecalis as a potential drug target.

Main Methods:

PCR amplification and cloning of the mvaK1 gene into a pET-28b vector.
Expression and purification of the C-terminally hexahistidine-tagged E. faecalis mevalonate kinase using nickel affinity chromatography.
Enzyme activity assays, sedimentation velocity analysis, and kinetic studies.

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Main Results:

Purified E. faecalis mevalonate kinase demonstrated divalent ion-dependent phosphorylation activity.
The enzyme exists primarily as a monomer (32.2 kD) with optimal activity at pH 10 and 37°C.
Kinetic analysis revealed sequential reaction mechanism with specific K(m) values for mevalonate, ATP, and Mg(2+); unlike mammalian counterparts, it utilized various nucleoside triphosphates.

Conclusions:

E. faecalis mevalonate kinase is a functional enzyme essential for isoprenoid biosynthesis.
Characterization provides a foundation for developing targeted inhibitors against this enzyme in Gram-positive pathogens.