Phosphomevalonate Kinase Research Articles

Pig liver phosphomevalonate kinase: Kinetic mechanism

Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate to diphosphomevalonate by ATP, one of the initial steps in the biosynthesis of steroids and isoprenoids. In previous studies, the enzyme from pig liver was purified and characterized, and preliminary work showed that the enzyme follows hyperbolic kinetics and a sequential mechanism. The present work is a more detailed analysis of its kinetic mechanism, using initial velocity and isotope exchange at equilibrium measurements. The results are compatible with a Bi Bi sequential ordered mechanism with phosphomevalonate as the first substrate and ADP the last product. The K m values estimated are 43 ± 7 μM for Mg-ATP and 12 ± 3 μM for phosphomevalonate, with a V max of 51 ± 2 μmol min −1 mg of protein −1.

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.

CHARACTERIZATION & FUNCTIONAL INVESTIGATION OF HUMAN PHOSPHOMEVALONATE KINASE

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. cDNA encoding human PMK has been used to construct expression plasmids encoding his-tagged enzyme. Expression of N-his-tagged PMK is much more efficient than C-his-tagged PMK, thus N-tagged enzyme has been selected for detailed study. Kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggest an N-terminal basic residue rich sequence as an unusual “Walker A”ATP site motif. The functions of four basic (K17, R18, K19, K22) and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate Km values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast R18Q exhibits a Vmax decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10,000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer. DK53766

Phosphomevalonate Kinase: Functional Investigation of the Recombinant Human Enzyme

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. To expedite functional and structural study of this enzyme, an expression plasmid encoding His-tagged human PMK has been constructed and recombinant enzyme isolated in an active, stable form. PMK catalyzes a reversible reaction; kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. Animal and invertebrate PMKs are not orthologous to plant, fungal, or bacterial PMKs, limiting the information available from sequence alignment analysis. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggests an N-terminal basic residue rich sequence as a possible "Walker A" ATP binding motif. The functions of four basic (K17, R18, K19, K22) residues and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate K(m) values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast, R18Q exhibits a V(max) decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer.

335. Global Transcriptional Profiles of Murine C2C12 Myoblasts Treated with IGF-1

These studies were intended to characterize the mechanisms of action of insulin-like growth factor (IGF-1) in myoblasts. Toward that end, we exposed cultures of murine C2C12 myoblasts to IGF-1 and examined the global gene expression profiles of the cells using a high-density oligonucleotide array platform (Affymetrix, mouse 430A) containing about 22,600 probe sets. We identified approximately 94, 180 and 221 genes that were up-regulated at least two fold relative to control cells (zero time point) at 1, 2 and 4 hours respectively. In addition, we identified approximately 48, 111 and 107 genes that were down-regulated more than two fold at the same times following IGF-1 treatment. Of special interest is the fact that the up-regulated genes include several genes that belong to the cholesterol biosynthetic pathway, such as hydroxysteroid (17 beta) dehydrogenase 7, lanosterol synthase, CYP 51, isopentenyl-diphosphate delta isomerase, 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 1, phosphomevalonate kinase and farnesyl diphosphate farnesyl transferase 1, all of which are induced by 3-5 fold, suggesting the involvement of a common trans-acting factor(s). Two candidates for such a common trans-acting factor(s) are suggested by the slight but reproducible (ca. 1.5 fold) degree of over-expression following IGF-1 treatment of Sterol Regulatory Element Binding Protein (SREBP2) and SREBP Cleavage Associated Protein (SCAP) mRNAs. Even a slight change in the expression of these trans-acting factors themselves might elicit a more marked change in the expression of their target genes. We are further characterizing the effect IGF-1 on the steroid and other selected pathways in C2C12 cells. These studies were intended to characterize the mechanisms of action of insulin-like growth factor (IGF-1) in myoblasts. Toward that end, we exposed cultures of murine C2C12 myoblasts to IGF-1 and examined the global gene expression profiles of the cells using a high-density oligonucleotide array platform (Affymetrix, mouse 430A) containing about 22,600 probe sets. We identified approximately 94, 180 and 221 genes that were up-regulated at least two fold relative to control cells (zero time point) at 1, 2 and 4 hours respectively. In addition, we identified approximately 48, 111 and 107 genes that were down-regulated more than two fold at the same times following IGF-1 treatment. Of special interest is the fact that the up-regulated genes include several genes that belong to the cholesterol biosynthetic pathway, such as hydroxysteroid (17 beta) dehydrogenase 7, lanosterol synthase, CYP 51, isopentenyl-diphosphate delta isomerase, 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 1, phosphomevalonate kinase and farnesyl diphosphate farnesyl transferase 1, all of which are induced by 3-5 fold, suggesting the involvement of a common trans-acting factor(s). Two candidates for such a common trans-acting factor(s) are suggested by the slight but reproducible (ca. 1.5 fold) degree of over-expression following IGF-1 treatment of Sterol Regulatory Element Binding Protein (SREBP2) and SREBP Cleavage Associated Protein (SCAP) mRNAs. Even a slight change in the expression of these trans-acting factors themselves might elicit a more marked change in the expression of their target genes. We are further characterizing the effect IGF-1 on the steroid and other selected pathways in C2C12 cells.

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

For Abstract see ChemInform Abstract in Full Text.

Enterococcus faecalis phosphomevalonate kinase

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 degrees C. The activation energy was approximately 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 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.

Molecular Functions of Conserved Aspects of the GHMP Kinase Family

The sequences and three-dimensional structures of the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family were compared to identify highly conserved surface residues. The functions of these solvent-accessible residues were assessed by determining the effects of their substitution, via mutagenesis, on the initial-rate parameters of a representative member of the GHMP kinase family, phosphomevalonate kinase from Streptococcus pneumoniae. What emerges from this study is a profile of the conserved surface-linked functions of the family. Certain substitutions produce highly selective effects on the steady-state affinity of a particular substrate, while one residue, Asp150, appears to be a pure k(cat) effector. Substitutions elsewhere affect multiple initial-rate parameters with varying, and sometimes compensatory, patterns. An alpha-helix that repositions during catalysis was substituted along its length to assess how its different segments contribute to catalysis-the substrate-proximal edge of the helix affects ATP recognition and k(cat), while the distal edge affects recognition of both substrates without affecting turnover. GHMP kinase mutations at the conserved surface residues corresponding to Ser291 and Ala293 in phosphomevalonate kinase are linked to mevalonic acid deficiency, which can lead to early fatality, and galactokinase deficiency, which causes cataracts. Our results suggest that the molecular basis for this particular galactokinase deficiency is an increase in the K(m) for galactose.

Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases.

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.

Phosphomevalonate kinase is a cytosolic protein in humans

In the past decade, a predominant peroxisomal localization has been reported for several enzymes functioning in the presqualene segment of the cholesterol/isoprenoid biosynthesis pathway. More recently, however, conflicting results have been reported raising doubts about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study, we have determined the subcellular localization of human phosphomevalonate kinase using a variety of biochemical and microscopic techniques, including conventional subcellular fractionation studies, digitonin permeabilization studies, immunofluorescence, and immunoelectron microscopy. We found an exclusive cytosolic localization of both endogenously expressed human phosphomevalonate kinase (in human fibroblasts, human liver, and HEK293 cells) and overexpressed human phosphomevalonate kinase (in human fibroblasts, HEK293 cells, and CV1 cells). No indication of a peroxisomal localization was obtained. Our results do not support a central role of peroxisomes in isoprenoid biosynthesis.

Sites for Interaction between Gal80p and Gal1p in Kluyveromyces lactis: Structural Model of Galactokinase based on Homology to the GHMP Protein Family

The induction of transcription of the galactose genes in yeast involves the galactose-dependent binding of ScGal3p (in Saccharomyces cerevisiae) or KlGal1p (in Kluyveromyces lactis) to Gal80p. This binding abrogates Gal80's inhibitory effect on the activation domain of Gal4p, which can then activate transcription. Here, we describe the isolation and characterization of new interaction mutants of K. lactis GAL1 and GAL80 using a two-hybrid screen. We present the first structural model for Gal1p to be based on the published crystal structures of other proteins belonging to the GHMP (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) kinase family and our own X-ray diffraction data of Gal1p crystals at 3 Å resolution. The locations of the various mutations in the modelled Gal1p structure identify domains involved in the interaction with Gal80p and provide a structural explanation for the phenotype of constitutive GAL1 mutations.

Cloning, purification, crystallization and preliminary crystallographic analysis of galactokinase fromPyrococcus furiosus

Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222(1), with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 2.9 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.

Cholesterol biosynthesis is not defective in peroxisome biogenesis defective fibroblasts

To evaluate the presumed peroxisomal involvement in cholesterol/isoprenoid biosynthesis, we determined the protein levels and activities of five different enzymes of the presqualene segment of the cholesterol/isoprenoid biosynthetic pathway in primary skin fibroblasts of selected patients with a peroxisomal biogenesis disorder (PBD). These five enzymes all have been reported to be partly or exclusively peroxisomal and include HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and isopentenyl pyrophosphate isomerase. To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, have differential effects on the activity of the cholesterol biosynthetic enzymes and on de novo cholesterol biosynthesis, we chose fibroblasts of patients with defined defects in one of four different PEX genes leading to Zellweger syndrome ( PEX1, PEX5, PEX16 or PEX19). We found that all enzymes measured are at least as active in the peroxisome-deficient cells cultured in cholesterol-depleted medium as in identically cultured control cells. This indicates that if these presumed peroxisomal proteins are mislocalized to the cytosol they do not loose their activity, nor get degraded unlike most other authentic peroxisomal proteins. We also measured de novo cholesterol synthesis from radio-labeled acetate in all cell lines and found similar or even elevated rates for the PBD cells when compared to controls. Our results imply that functional peroxisomes are not a prerequisite for the functioning of enzymes involved in cholesterol/isoprenoid biosynthesis and as such raise doubts about the true involvement of peroxisomes therein.

Kinaselike Structures in Sexual Specification Proteins

The specification of sex involves either the presence or absence of a particular chromosome (the Y chromosome in mammals) along with dosage compensation, or the number of X chromosomes (for example, in the model organisms nematode and fruit fly). In C. elegans XOL-1 protein governs gender--high levels of XOL-1 produce males and low levels produce hermaphrodites. The presence of two X chromosomes in the hermaphrodites results in the expression of xol-1 repressors. Beyond the genetic evidence linking xol-1 to sexual differentiation, little is know regarding the function of the XOL-1 protein. Luz et al. crystallized XOL-1 to gain insight into its function. Surprisingly, despite no primary sequence similarity to GHMP kinases, the protein folded into a structure very similar to that of GHMP kinases, which catalyze small molecular metabolism. Indeed, the family is named for the first members identified, galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase. Despite the overall structural similarity, differences in specific residues predict that XOL-1 will not bind adenosine triphosphate (ATP), and in vitro, no ATP binding or hydrolysis by XOL-1 was detected. The structural differences suggested that XOL-1 may be regulated by a small molecule ligand. In yeast, GHMP-like kinases have been identified that act as transcriptional regulators or even as bifunctional proteins with transcription regulatory activity and kinase activity. Thus, the GHMP-like structure appears to be a dual-function motif, with roles outside of metabolism, involved in development and sexual specification. J. G. Luz, C.A. Hassig, C. Pickle, A. Godzik, B. J. Meyer, I. A. Wilson, XOL-1, primary determinant of sexual fate in C. elegans , is a GHMP kinase family member and a structural prototype for a class of developmental regulators. Genes Dev. 17 , 977-990 (2003). [Abstract] [Full Text]

The Kinetic Mechanism of Phosphomevalonate Kinase

Phosphomevalonate kinase catalyzes an essential step in the so-called mevalonate pathway, which appears to be the sole pathway for the biosynthesis of sterols and other isoprenoids in mammals and archea. Despite the well documented importance of this pathway in the cause and prevention of human disease and that it is the biosynthetic root of an enormous diverse class of metabolites, the mechanism of phosphomevalonate kinase from any organism is not yet well characterized. The first structure of a phosphomevalonate kinase from Streptococcus pneumoniae was solved recently. The enzyme exhibits an atypical P-loop that is a conserved defining feature of the GHMP kinase superfamily. In this study, the kinetic mechanism of the S. pneumoniae enzyme is characterized in the forward and reverse directions using a combination of classical initial-rate methods including alternate substrate inhibition using ADPbetaS. The inhibition patterns strongly support that in either direction the substrates bind randomly to the enzyme prior to chemistry, a random sequential bi-bi mechanism. The kinetic constants are as follows: k(cat(forward)) = 3.4 s(-1), K(i(ATP)) = 137 microm, K(m(ATP)) = 74 microm, K(i(pmev)) = 7.7 microm, K(m(pmev)) = 4.2 microm; k(cat(reverse)) = 3.9 s(-1), K(i(ADP)) = 410 microm, K(m(ADP)) = 350 microm, K(i(ppmev)) = 14 microm, K(m(ppmev)) = 12 microm, where pmev and ppmev represent phosphomevalonate and diphosphomevalonate, respectively.

Peroxisome deficiency does not result in deficiency of enzymes involved in cholesterol biosynthesis.

To unravel conflicting literature data on the dependence of cholesterol biosynthesis on functional peroxisomes, we have determined activities and levels of selected cholesterol biosynthetic enzymes in livers of a PEX5 knock-out mouse, a well-characterized animal model for human Zellweger syndrome. All enzymes measured, including putative peroxisomal enzymes, were normally active in the peroxisome-deficient mice indicating that mislocalization of these enzymes to the cytosol does not lead to decreased activity or degradation. Since these findings were in apparent contrast to previously reported data in human livers, we re-examined cholesterol biosynthetic enzymes in livers of Zellweger patients and control subjects. We found that the previously reported deficient enzymes mevalonate kinase and phosphomevalonate kinase are inactivated rapidly in a time-and temperature-dependent manner. This inactivation is not due to degradation as a result of mislocalization since normal levels of protein can be detected. Therefore, the deficiencies of these enzymes in livers of Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data explain the conflicting findings in literature and demonstrate that great care should be taken in the interpretation of data obtained in postmortem material (1). To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, might influence the activity of selected cholesterol biosynthetic enzymes, we also have determined activities and levels of these enzymes in human fibroblasts of patients belonging to various different complementation groups. We included fibroblasts of patients with mutations in genes encoding PEX1, PEX5, PEX7, PEX16, and PEX19, resulting in selective deficiencies in import of PTS1 or PTS2 containing proteins or in a more general defect in peroxisome biogenesis resulting in the complete absence of peroxisomes or peroxisomal ghosts. We measured normal protein levels and activities of enzymes involved in cholesterol biosynthesis in all cell lines. We conclude that peroxisomes are not a prerequisite for cholesterol biosynthesis in humans.

Hyperinsulinemia and hyperglycemia downregulate lipoprotein lipase activity

To evaluate the presumed peroxisomal involvement in cholesterol/isoprenoid biosynthesis, we determined the protein levels and activities of five different enzymes of the presqualene segment of the cholesterol/isoprenoid biosynthetic pathway in primary skin fibroblasts of selected patients with a peroxisomal biogenesis disorder (PBD). These five enzymes all have been reported to be partly or exclusively peroxisomal and include HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and isopentenyl pyrophosphate isomerase. To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, have differential effects on the activity of the cholesterol biosynthetic enzymes and on de novo cholesterol biosynthesis, we chose fibroblasts of patients with defined defects in one of four different PEX genes leading to Zellweger syndrome (PEX1, PEX5, PEX16 or PEX19). We found that all enzymes measured are at least as active in the peroxisome-deficient cells cultured in cholesterol-depleted medium as in identically cultured control cells. This indicates that if these presumed peroxisomal proteins are mislocalized to the cytosol they do not loose their activity, nor get degraded unlike most other authentic peroxisomal proteins. We also measured de novo cholesterol synthesis from radio-labeled acetate in all cell lines and found similar or even elevated rates for the PBD cells when compared to controls. Our results imply that functional peroxisomes are not a prerequisite for the functioning of enzymes involved in cholesterol/isoprenoid biosynthesis and as such raise doubts about the true involvement of peroxisomes therein.

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.

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.