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Enterococcus faecalis phosphomevalonate kinase.

Stephanie S Doun1, John W Burgner, Scott D Briggs

  • 1Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907-2063, USA.

Protein Science : a Publication of the Protein Society
|April 2, 2005
PubMed
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Researchers characterized phosphomevalonate kinase, a key enzyme in isopentenyl diphosphate biosynthesis, from Enterococcus faecalis. This work advances the development of novel antibiotics against drug-resistant bacteria.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • The mevalonate pathway is crucial for isopentenyl diphosphate biosynthesis.
  • Developing new antibiotics against resistant Gram-positive streptococci is a significant health challenge.
  • Previous studies characterized the initial four enzymes of this pathway in Enterococcus faecalis.

Purpose of the Study:

  • To clone, express, and purify phosphomevalonate kinase (E.C. 2.7.4.2) from Enterococcus faecalis.
  • To characterize the biochemical properties of the purified enzyme.
  • To explore potential applications in antibiotic development and drug screening.

Main Methods:

  • Gene cloning using polymerase chain reaction and pET28b(+) vector.
  • Protein expression in Escherichia coli and purification via Ni(++) affinity chromatography.

Related Experiment Videos

  • Enzyme activity assays coupled with NADH oxidation, mass spectrometry, and analytical ultracentrifugation.

    • Main Results:

    • Successfully cloned, expressed, and purified Enterococcus faecalis phosphomevalonate kinase.
    • Determined the enzyme's dimeric structure in solution and optimal activity conditions (pH 8.0, 37°C).
    • Characterized kinetic parameters, including activation energy (5.6 kcal/mol), optimal cation (Mn++), and substrate affinities (Km for ATP and phosphomevalonate).

    Conclusions:

    • Phosphomevalonate kinase from Enterococcus faecalis has been biochemically characterized.
    • The enzyme's properties provide a basis for its use in an immobilized enzyme bioreactor.
    • This research supports the potential of targeting the mevalonate pathway for novel antibiotic development against resistant bacteria.