Biochemical and genetic aspects of mevalonate kinase and its deficiency

Mevalonate kinase (MK) is an essential enzyme in the mevalonate pathway which produces numerous cellular isoprenoids. The enzyme has been characterized both at the biochemical and the molecular level in a variety of organisms. Despite the fact that mevalonate kinase is not the rate-limiting enzyme in isoprenoid biosynthesis, its activity is subject to feedback regulation by the branch-point intermediates geranyldiphosphate, farnesyldiphosphate and geranylgeranyldiphosphate. Recently, the importance of mevalonate kinase was demonstrated by the identification of its deficiency as the biochemical and molecular cause of the inherited human disorders mevalonic aciduria and hyperimmunoglobulinemia D and periodic fever syndrome. The pathophysiology of these disorders is not yet understood, but eventually will give insight into the in vivo role of mevalonate kinase and isoprenoid biosynthesis with respect to the acute phase response and fever. The subcellular localization of mevalonate kinase is still a matter of debate. The enzyme could be localized predominantly in the cytosol, or in peroxisomes, or it is associated differentially with peroxisomes. Here we review the biochemical and molecular properties of MK, and discuss its biological significance, the regulation of its enzyme activity and finally its subcellular localization.     

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.     

Effect of a disulfide bond on mevalonate kinase

Mevalonate kinase is one of ATP-dependent enzymes in the mevalonate pathway and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. In animal cells, it plays a key role in regulating biosynthesis of cholesterol, while in microorganisms and plants, it is involved in the biosynthesis of isoprenoid derivatives that are one of the largest groups of natural products. Crystal structure and sequence alignment show that a unique disulfide bond exists in mevalonate kinase of thermostable species Methanococcus jannaschii, but not in rat mevalonate kinase. In the present study, we investigated the effect of the disulfide bond in M. jannaschii mevalonate kinase and an engineered disulfide bond in rat mevalonate kinase mutant A141C on the properties of enzymes through characterization of their wild-type and variant enzymes. Our result suggests that the Cys107-Cys281 disulfide bond is important for maintaining the conformation and the thermal activity of M. jannaschii mevalonate kinase. Other interactions could also have contributions. The thiol-titration and fluorescence experiment further indicate that rat mevalonate kinase A141C variant enzyme has a new disulfide bond, which makes the variant protein enhance its thermal activity and resist to urea denaturation.     

Expression of the yeast mevalonate kinase gene in trangenic tobacco

The coding region of the yeast mevalonate kinase gene (ERG12), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, has been inserted in tobacco (Nicotiana tabacum cv. Paraguay Bell) using an Agrobacterium tumefaciens binary vector system. Integration and expression of the ERG12 chimaeric gene was demonstrated in several independent transformants in which specific mevalonate kinase (MK) activity in young plantlets was increased by about 60% on average. The expression of this MK gene was accompanied by phenotypical modifications, such as acceleration of regenerating processes, lateral bud growth, and peculiar flowering behaviour. A higher chlorophyll content all along the plant development, paralleled by an unusual starch accumulation in the leaves of young plantlets and, later, in roots of full-grown plants, was also detected. Overexpression of the MK gene led also to a stronger inhibition of cytokinin-induced plant growth by methyl jasmonate in transgenic plants. All these events may be interpreted as a possible modification of the hormonal balance in transgenic tobaccos.     

Identification of a mutation cluster in mevalonate kinase deficiency, including a new mutation in a patient of Mennonite ancestry.

Mevalonate kinase (MKase) deficiency (MKD) is a rare autosomal recessive disorder in the pathway of cholesterol and nonsterol isoprenoid biosynthesis. Thus far, two disease-causing missense alleles have been identified, N301T and A334T. We report four additional mutations associated with MKD: L264F, T243I, L265P, and I268T, the last found in a patient of Mennonite ancestry. Electrophoretic analysis of bacterially expressed wild-type and mutant MKase indicated that I268T and T243I mutants produced normal or somewhat reduced amounts of MKase protein; conversely, L264F and L265P mutations resulted in considerably decreased, or absent, MKase protein. Immunoblot analysis of MKase from all patients suggested that the MKase polypeptide was grossly intact and produced in amounts comparable to control levels. Three mutations resulted in significantly diminished MKase enzyme activity (<2%), whereas the I268T allele yielded approximately 20% residual enzyme activity. Our results should allow more-accurate identification of carriers and indicate a mutation "cluster" within amino acids 240-270 of the mature MKase polypeptide.     

Purification and regulation of mevalonate kinase from rat liver

Mevalonate kinase may play a key role in regulating cholesterol biosynthesis because its activity may be regulated via feedback inhibition by intermediates in the cholesterol biosynthetic pathway. To study the regulation of mevalonate kinase, the enzyme was purified to homogeneity from rat liver, and monospecific antibody against mevalonate kinase was prepared. The purified mevalonate kinase had a dimeric structure composed of identical subunits, and the Mr of the enzyme determined by gel chromatography was 86,000. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit Mr was 39,900. The pI for mevalonate kinate was 6.2. The levels of mevalonate kinase protein and enzyme activity were determined in the livers of rats treated with either cholesterol-lowering agents (cholestyramine, pravastatin, and lovastatin) or with dietary modifications. Diets containing cholestyramine alone or cholestyramine and either pravastatin or lovastatin increased mevalonate kinase activity 3-6-fold. Mevalonate kinase activity decreased approximately 50% in rats treated with diets containing either 5% cholesterol or 5% cholesterol and 0.5% cholic acid. Fasting did not significantly change mevalonate kinase activity. The amount of mevalonate kinase protein in the liver was quantitated using immunoblots, and the changes in the levels of kinase activity induced by either drug treatment or by cholesterol feeding were correlated with similar changes in the levels of mevalonate kinase protein. Therefore, under these experimental conditions, mevalonate kinase activity in the liver was regulated principally by changes in the rates of enzyme synthesis and degradation.     

Inhibition of rat liver mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase by phenyl and phenolic compounds.

1. Mevalonate pyrophosphate decarboxylase of rat liver is inhibited by various phenyl and phenolic acids. 2. Some of the phenyl and phenolic acids also inhibited mevalonate phosphate kinase. 3. Compounds with the phenyl-vinyl structure were more effective. 4. Kinetic studies showed that some of the phenolic acids compete with the substrates, mevalonate 5-phosphate and mevalonate 5-pyrophosphate, whereas others inhibit umcompetitively. 5. Dihydroxyphenyl and trihydroxyphenyl compounds and p-chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. 6. Of the three mevalonate-metabolizing enzymes, mevalonate pyrophosphate decarboxylase has the lowest specific activity and is probably the rate-determining step in this part of the pathway.     

The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis latex

1. Evidence has been produced for the formation of 5-phosphomevalonate from potassium dl-mevalonate by the latex of Hevea brasiliensis and by reconstituted freeze-dried serum obtained from this latex. 2. The enzyme, mevalonate kinase, catalysing the formation of 5-phosphomevalonate from potassium dl-mevalonate and ATP has been partially purified. 3. 5-Phosphomevalonate formed by the purified mevalonate kinase from potassium [2-(14)C]mevalonate has been shown to be incorporated by latex into rubber to about 2.4 times the extent of dl-mevalonate. 4. The enzyme can utilize inosine triphosphate as effectively as adenosine triphosphate as a phosphate donor and is also slightly active with uridine triphosphate. 5. The enzyme was fairly stable to a range of pH values and temperatures, the activity being optimum at pH7.5 and 60-70 degrees . The energy of activation was 10.7kcal./mole. The K(m) values were 0.13mm for potassium dl-mevalonate and 2.0mm for ATP at 30 degrees . 6. The enzyme required the presence of Mn(2+) (1mm) for maximum activity; this could be replaced by Mg(2+) (4mm), which was less effective, and by Ca(2+), which was far less effective. 6. Although the enzyme did not require cysteine or reduced glutathione for activation in aerobic conditions, it was inhibited by reagents known to react with thiol groups.     

The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.     

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.     

Phosphorylase kinase deficiency

Phosphorylase kinase deficiency in I strain mice and in humans both show X-chromosomal inheritance. Neither deficient adult humans nor deficient mice show any sign of disease. Thus the two conditions resemble each other. However, there are differences. The enzyme is only partially deficient in human patients in liver, muscle, and blood cells; in mice the deficiency is complete and seems to be confined to the muscles.This paper was presented at a symposium entitled Genetic Control of Mammalian Metabolism held at The Jackson Laboratory, Bar Harbor, Maine, June 30–July 2, 1969. The symposium was supported in part by an allocation from NIH General Research Support Grant FR 05545 from the Division of Research Resources to The Jackson Laboratory.Supported by grant AM 13359 of the National Institutes of Health.     

Partial purification and properties of mevalonate kinase from neonatal chick liver

Mevalonate kinase from neonatal chick liver has been partially purified by ammonium sulphate precipitation and Sephadex G100 and DEAE-cellulose fractionation. The kinetic characteristics agreed with the sequential mechanism suggested for the enzyme and provided apparent Km values of 0.01 mM for mevalonic acid and 0.25 mM for ATP. Partially purified mevalonate kinase from neonatal chick liver showed an absolute specificity for ATP. Mn2+ was a better activator than Mg2+ at low concentrations (0.1-1.0 mM). Higher Mn2+ concentrations produced a clear inhibition of mevalonate kinase. Likewise, addition of EDTA, with or without metal ions, clearly inhibited the enzymatic reaction.     

Cloning,expression, and purification of His-tagged rat mevalonate kinase

Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells. Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity. The purified rat mevalonate kinase had a dimeric structure composed of identical subunits. Based on SDS-PAGE, the subunit was 42 kDa. The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer. The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively. The V(max) was determined to be 38.7 micromol/min/mg. The overexpression of rat mevalonate kinase in E. coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.