Mevalonate 5-diphosphate Decarboxylase Research Articles

Cycloheximide as a tool to investigate protein import in peroxisomes: A case study of the subcellular localization of isoprenoid biosynthetic enzymes

Cytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes.

Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis

The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.1 revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.2 This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.3 Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.

Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta

In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.

Simulation of structural and functional properties of mevalonate diphosphate decarboxylase (MVD)

Mevalonate 5-diphosphate decarboxylase (MVD) is an important enzyme in the mevalonate pathway catalyzing the ATP-dependent decarboxylation of mevalonate 5-diphosphate (MDP) to yield isopentynyl diphosphate (IPP) which is an ubiquitous precursor for isoprenoids and sterols. Although there are studies to show the involvement of certain amino acid residues in MVD activity, the structure and the function of the active site is yet to be investigated. Therefore the objectives of this study were to elucidate the active site of Saccharomyces cerevisiae MVD (scMVD) using a molecular docking and simulation-based approach. The Cartesian coordinates of scMVD retrieved from the PDB database were used in the docking procedure. 3D atomic coordinates of MDP, ATP and an inhibitor trifluoromevalonate (TFMDP) were generated using Gaussian 98. ATP, MDP and TFMDP were docked into the potential active site identified by sequence analyses using Hex 4.2. The complexes obtained from docking procedure were subjected to 1.5 ns simulation by GROMACS 3.2. Investigation of complexes revealed that Ala15, Lys18, Ser121 & Ser155; Lys22, Ser153 & Ser155 and Tyr19, Ser121, Ser153, Gly154 & Thr209 of MVD are within hydrogen bond forming distances of MDP, ATP and TFMDP, respectively indicating their possible involvement in active site formation through H-bond formation. The presence of a water molecule between the carboxyl group of Asp302, a previously characterized active site residue and C3 region of MDP at a distance of 3 A suggests that deprotonation of the hydroxyl of the C3 takes place via a water molecule. Conjunction with reported crucial catalytic activity of Ser121 of MVD and our finding of the presence of this residue in hydrogen bond forming distance to MDP suggests that this hydrogen bond helps in proper orienting of MDP for phosphorylation /decarboxylation. We further suggest that the reported greater RMS deviation of Pro(79)- Leu mutated MVD with respect to native MVD of temperature sensitive mutant phenotype of S. cerevisiae is due to partial unfolding of MVD as a result of mutation. Finally, this study provides a tantalizing glimpse about hitherto unknown structural and functional properties of the active site of MVD.

Mutation and inhibition studies of mevalonate 5-diphosphate decarboxylase

Mevalonate 5-diphosphate decarboxylase plays an important role in regulating cholesterol biosynthesis, which was studied through incubation with various synthetic substrate analogs and characterization of mutated enzymes. The results are potentially useful for further developing inhibitors that block the mevalonate pathway which is a drug target for treating cardiovascular disease and cancer.

GHMP Kinases - Structures, Mechanisms and Potential for Therapeutically Relevant Inhibition

The GHMP kinases are a structurally related family of small molecule kinases named after four of its members - galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase. The group also includes the enzymes N-acetylgalactosamine kinase, arabinose kinase, mevalonate 5-diphosphate decarboxylase, archeal shikimate kinase and 4-(cytidine 5-diphospho)-2-c-methyl-D-erythritol kinase. In addition the group includes two members not known to be catalytically active, the Caenorhabditis elegans sex-fate determining protein XOL-1 and the Saccharomyces cerevisiae transcriptional activator Gal3p. Two catalytic mechanisms have been proposed for GHMP kinases. The structure of mevalonate kinase suggests that an aspartate residue acts as an active site base, removing a proton from the substrate to facilitate attack on the γ- phosphate of MgATP. In contrast, in homoserine kinase there is no potential catalytic base and it is proposed that catalysis is driven by transition state stabilisation. Potential chemotherapeutic interventions against GHMP kinases fall into three main categories: inhibition of galactokinase to assist suffers of galactosemia, inhibition of mevalonate kinase or mevalonate 5-diphosphate decarboxylase to reduce flux through the cholesterol biosynthesis pathway and inhibition of bacterial GHMP kinases for novel anti-microbial therapies. These are in the early stages of development, but the accumulation of structural and mechanistic data will assist future progress. Keywords: Galactokinase, homoserine kinase, mevalonate kinase, cholesterol metabolism, carbohydrate metabolism, galactosemia, mevalonic aciduria, hyperimmunoglobulinemia D syndrome (HIDS), anti-microbial chemotherapy, enzyme mechanism

Inhibition of mevalonate 5-diphosphate decarboxylase by fluorinated substrate analogs

Mevalonate 5-diphosphate decarboxylase (MDD) is a peroxisomal enzyme in the cholesterol biosynthetic pathway, which plays an important role in regulating cholesterol biosynthesis. In the present study, rat MDD was cloned and purified to apparent homogeneity. Two fluorinated MDD substrate analogs, P′-geranyl 2-fluoromevalonate 5-diphosphate (4) and 2-fluoromevalonate 5-diphosphate (6), were synthesized, and both were found to be irreversible inhibitors of rat MDD. These two inhibitors were characterized, and mechanisms of the inactivation process were proposed. Kinetic studies indicate both analogs only bind into mevalonate binding-site of MDD. Compound 4 shows competitive inhibition on mevalonate kinase (MVK), and its IC 50 value was determined to be comparable with that of geranyl diphosphate. Further kinetic studies indicate compound 4 only bind into ATP binding-site of MVK. These studies provide an example for a single inhibitor to carry out sequential blocking of two enzymes in cholesterol biosynthesis, which may provide useful information for drug discovery for the purpose of treating cardiovascular disease and cancer or for pest control.

Bifunctional Inhibitors of Mevalonate Kinase and Mevalonate 5-Diphosphate Decarboxylase

[structure: see text] A bifunctional inhibitor of mevalonate kinase and mevalonate 5-diphosphate decarboxylase was synthesized. Both enzymes are in the cholesterol biosynthetic pathway and play an important role in regulating cholesterol biosynthesis. The molecule may become a useful lead compound for further development for treating cardiovascular disease and cancer. This study provides a novel example of a single inhibitor blocking two sequential steps simultaneously in the cholesterol biosynthetic pathway.

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae).

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

Structure of the Methanococcus jannaschii Mevalonate Kinase, a Member of the GHMP Kinase Superfamily

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

Substrate binding order in mevalonate 5-diphosphate decarboxylase from chicken liver

The substrate binding order of chicken liver mevalonate 5-diphosphate decarboxylase was investigated by using competitive inhibitors of the substrates. Mevalonate and mevalonate 5-phosphate showed mixed inhibition when ATP was the varied substrate. Both analogues of ATP, adenosine 5′- O-(3-thiotriphosphate) and β-τ methylene adenosine 5′-triphosphate showed uncompetitive inhibition against mevalonate 5-diphosphate. These results are in accordance with an ordered sequential mechanism with mevalonate 5-diphosphate as the first substrate to bind to the enzyme.

Inactivation of chicken liver mevalonate 5-diphosphate decarboxylase by sulfhydryl-directed reagents: evidence of a functional dithiol.

Chicken liver mevalonate 5-diphosphate decarboxylase (ATP:(R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5'-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 +/- 0.1).10(-5) microM-2.s-1, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pKapp values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 +/- 0.1 and 7.6 +/- 0.3, respectively. From the data presented, it is concluded that the enzyme possesses a functional dithiol that is important for substrate binding.

Physiological aspects and mechanism of action of mevalonate 5-diphosphate decarboxylase

1. 1. This work reviews the present knowledge of the physiological role and mechanism of action of mevalonate 5-diphosphate decarboxylase, the third enzyme involved in the biosynthesis of cholesterol from mevalonic acid. 2. 2. Published evidence indicates that this and other enzymes of the cholesterol biosynthetic pathway present coordinate fluctuations in activity in rat liver. A possible regulatory role for the brain decarboxylases from chicken and rat has been proposed. 3. 3. From kinetic and stereochemical studies with the chicken liver enzyme it has been proposed that the reaction is initiated by the abstraction of a proton from the 3-hydroxyl group of mevalonate 5-diphosphate by a basic group in the enzyme, followed by the nucleophilic attack of the C-3 oxygen on P γ of the λ isomer of the β, γ bidentate MgATP 2- in a S N2(P) reaction that goes with inversion of configuration at P.

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg 2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg 2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP 2- is the true substrate. The relative efficiencies of Mg 2+, Mn 2+, Cd 2+, and Zn 2+ as activating metal ions were evaluated in terms of V/ K m for the corresponding (metal-ATP) 2- complexes, and the relative ratios were: Mn 2+ 100, Cd 2+ 37, Mg 2+ 14, Zn 2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg 2+, and were in the order Zn 2+ > Cd 2+ > Mn 2+.