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°C. The activation energy was ∼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 mol substrate converted per minute per milligram protein.Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. (Paulsen et al. 2003), organisms responsible for infective endocarditis, other nosocomial infections, and food poisoning, has exhausted the armamentarium of antibiotics suitable for control of infections by these Grampositive cocci. As noted by Ziglam and Nathwani (2003), the emergence worldwide of vancomycin-resistant enterococci is a grave concern, and new agents are essential to meet this threat. Inhibiting the biosynthesis of isopentenyl diphosphate (IPP), the building block of all isoprenoids, offers potential for the development of antibiotics that target these bacterial pathogens and also the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. Two distinct pathways form IPP, the mevalonate pathway (Fig. 1 Article published online ahead of print. Article and publication date are at Keywords
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.