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°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

The Cell Wall-Targeting Antibiotic Stimulon of Enterococcus faecalis

“…IPP is the central precursor of isoprenoids, via the mevalonate pathway [22]. The mevalonate pathway is the sole pathway for IPP biosynthesis in E. faecalis [23], and it is well known that isoprenoids are involved in a wide variety of vital biological processes such as electron transport and peptidoglycan biosynthesis [24]. Of note, four out of the six genes encoding enzymes of the mevalonate pathway were induced by bacitracin treatment (Table S1).…”

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

“…A recombinant form of Streptococcus pneumoniae has been characterized [74] and reported to catalyze a random sequential bi-bi reaction. A recombinant form of Enterococcus faecalis PMK has also been isolated and characterized [75]. The sequence of human PMK has been deduced [76].…”

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

“…Presently, however, it seems that the expression of foreign genes and activities of expressed enzymes were sufficient for the biosynthesis of β-carotene at either temperature, but the supply of IPP was higher at 37 o C due to higher activities of foreign MVA pathway enzymes in the central metabolism of the producing strain. For example, the optimal activities of expressed E. faecalis MVA-dependent enzymes including acetoacetyl-Co A thiolase, HMG-Co A reductase, and phosphomevalonate kinase in E. coli occur at 37 o C [27,28]. The glycolysis flux leading to acetyl CoA is higher at 37 o C [29].…”