Enterococcus faecalis mevalonate kinase

Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Properties and partial purification of mevalonate kinase from Agave americana

Mevalonate kinase activity was demonstrated in acetone powder extracts from Agave americana leaves, flowers and scape. ATP was the most effective phosphate donor. The enzyme had an optimum pH of 7.9 in Tris-HCl buffer. Dialysis decreased the ability to phosphorylate mevalonic acid (MVA). Partially purified mevalonate kinase reached maximum activity in the presence of 2 mM Mn²⁺ or 6–8 mM Mg²⁺. Higher concentrations of Mn²⁺ were inhibitory, whereas higher concentrations of Mg²⁺ produced only a small decrease in the activity. The amount of mevalonate-5-phosphate (MVAP) formed depended on protein concentration and incubation time. During short incubations, the MVAP formed increased as protein concentration rose, whereas during prolonged incubations (1–6 hr), there was a decrease in the MVAP formed when a certain amount of protein was exceeded. It is suggested that MVAP formed was hydrolysed by a phosphatase present in the extracts. This interfering activity was eliminated when mevalonate kinase is partially purified. The apparent K_m values of the enzyme from leaves were 0.05 mM for MVA and 0. 14 mM for ATP. Similar K_m values are obtained with partially purified mevalonate kinase. The enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 filtration and DEAE-Sephadex A-50 fractionation.     

An improved purification procedure, an alternative assay and activation of mevalonate kinase by ATP

An improved procedure for the purification of pig liver mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) is described. A high-voltage electrophoresis assay was developed for mevalonate kinase. The procedure separates mevalonate from phosphomevalonate and also from diphosphomevalonate so that it can be used to measure the subsequent enzyme, phosphomevalonate kinase (EC 2.7.4.2). The assay has allowed the reassessment of the metal ion and nucleotide specificity of the pig liver enzyme. Some of the previously reported properties reflected those of the enzymes in the coupling assay rather than mevalonate kinase itself. A series of compounds were tested as activators or inhibitors of mevalonate kinase. It was found that ATP4-, arsenate and, to a smaller extent, inorganic phosphate activated the enzyme. At fixed MgATP2- (1 mM) concentrations the activation of mevalonate kinase by free ATP4- at pH 8.0 was observed at concentrations at up to 10-fold that of MgATP2- before causing any inhibition. The presence of free ATP4- resulted in a biphasic Lineweaver-Burke plot with apparent Km values for MgATP2- being 0.14 mM and 60 microM, respectively. Fluorescence measurements were consistent with the notion that the binding of excess ATP4- to the enzyme caused a conformational change.     

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).     

Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase,an enzyme of isopentenyl diphosphate biosynthesis

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.     

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.     

Pig liver phosphomevalone kinase. 1. Purification and properties

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.     

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.     

Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase

An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).     

Crystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonate

Streptococcus pneumoniae, a ubiquitous gram-positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 A and in complex with DPM.Mg(2+) in the active-site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary-complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK-binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency.     

STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from Catharanthus roseus suspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis-Menten kinetics with respect to ATP and their specific substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n = 3) is always less than 2.5% and in between determinations (n = 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay.     

Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthesis of isopentenyl diphosphate

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the "missing" phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.     

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.     

Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.     

Multienzyme mevalonate pathway bioreactor

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.     

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis . The gene was overexpressed in Escherichia coli , which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci . At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.     

Mevalonate kinase is localized in rat liver peroxisomes.

Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.