title: Enterococcus faecalis phosphomevalonate kinase.
abstract: The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.
authors.affiliation: Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907-2063, USA.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Doun
authors.forename: John W
authors.initials: JW
authors.lastname: Burgner
authors.forename: Scott D
authors.initials: SD
authors.lastname: Briggs
authors.forename: Victor W
authors.initials: VW
authors.lastname: Rodwell
mesh_terms: D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D013293:Enterococcus faecalis / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase