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PubMed1982

Modulation of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity by reversible phosphorylation.

We have previously reported that the enzymic activity of rat liver-3-hydroxy-3-methyl-glutaryl-CoA reductase (NADPH) (HMG-CoA reductase) is modulated in vitro by a phosphorylation-dephosphorylation reaction sequence. The in vitro phosphorylation of HMG-CoA reductase was further studied by utilizing purified HMG-CoA reductase and reductase kinase. Analysis of 32P-labeled HMG-CoA reductase revealed 1 mol of phosphate per subunit. Purified [32P]HMG-CoA reductase could be dephosphorylated with phosphoprotein phosphatase. To demonstrate the in vivo phosphorylation, rats were injected with 32P and hepatic HMG-CoA reductase was isolated by immunoprecipitation and also by purification of the enzyme to homogeneity. Analysis of [32P]HMG-CoA reductase by sodium dodecyl sulfate gel electrophoresis revealed a single peak of radioactivity comigrating with HMG-CoA reductase. Administration of glucagon enhances the in vivo phosphorylation of both HMG-CoA reductase and reductase kinase. In response to glucagon, HMG-CoA reductase activity is decreased whereas reductase kinase activity is increased. These results support our concept that the enzymic activity of HMG-CoA reductase is modulated by a bicyclic cascade system involving phosphorylation-dephosphorylation. The enzymic activity of HMG-CoA reductase has also been shown to be modulated by cholesterol and mevalonolactone by both short-term and long-term mechanisms. The effects of cholesterol and mevalonolactone are twofold. Rapid inhibition of HMG-CoA reductase activity is due to increased phosphorylation of the enzyme; the long-term effect of HMG-CoA reductase is achieved by reduction in enzyme concentration by modulation of enzyme synthesis and/or degradation. Regulation of HMG-CoA reductase by mevalonolactone is of major importance in cellular metabolism because mevalonate serves as precursor for four separate metabolic pathways, including the formation of cholesterol, ubiquinone, dolichols, and isopentenyl tRNA.

AMP-Activated Protein Kinases↗
PubMed1975

Relationship between sterol synthesis and DNA synthesis in phytohemagglutinin-stimulated mouse lymphocytes.

Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.

Animals↗
PubMed1986

Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a 13C nuclear magnetic resonance study.

13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.

Acetates↗
PubMed2005

Pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: candidate mechanisms for anti-lipid deposition in blood vessels.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are considered first-line therapeutic agents for the prevention of coronary heart disease and atherosclerotic disorders related to hypercholesterolemia. Statins inhibit lipid deposition in the aortic endothelium. Although it has been accepted that the statins are potent inhibitors of cholesterol biosynthesis in the liver and that they lower circulating cholesterol levels, several cholesterol-independent (pleiotropic) effects have been reported. The cholesterol-independent effects of statins involve normalization of the nitric oxide (NO)-NO synthase system, anti-inflammatory effects through the inhibition of cytokine/chemokine production, inhibition of vascular smooth muscle cell proliferation and migration, and inhibition of platelet thrombus formation/reduction of the thrombotic response. Some pleiotropic effects of statins may depend on the inhibition of the biosynthesis of farnesyl- and geranylgeranyl-nonsterol compounds from mevalonate in the cells. The Rho/Rho kinase pathway and the phospatidylinositol-3 kinase/Akt pathway mediate the pleiotropic effects of statins. As variations occur in absorption, metabolism, and excretion mechanisms due to the characteristics of specific statins including their hydrophilicity and lipophilicity, there are differences in the transfer mechanisms of statins into tissues. However, the pleiotropic effects occur regardless of statin hydrophilicity and lipophilicity. This review summarizes the pleiotropic effects of statins on lipid deposition in blood vessels.

Animals↗
PubMed1987

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver.

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.

Animals↗
PubMed1987

Cell cycle-specific requirement for mevalonate, but not for cholesterol, for DNA synthesis in glial primary cultures.

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria.

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.

Amino Acid Sequence↗
PubMed1995

Developmental regulation of sterol biosynthesis in Zea mays.

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods from Zea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-(14)C]mevalonic acid, [2-(14)C]acetate, and [2-(3)H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylgutaryl CoA reductase is not "the" rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24 alpha-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an active de novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3 beta-ol. Examination of the mass spectrum and 1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.

Acetates↗
PubMed1991

Inhibition of growth factor signaling pathways by lovastatin.

Human fibroblasts treated with the antihypercholesterolaemic drug, lovastatin, displayed a diminished signaling response to epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Supplementing the culture medium with mevalonic acid restored the signaling response. Not all growth factor signaling pathways were impaired, however, as platelet-derived growth factor (PDGF-BB) and basic fibroblast growth factor (bFGF) responses were refractory to lovastatin treatment. These results suggest the involvement of product(s) of mevalonate metabolism (e.g., prenylated proteins such as p21ras or G proteins) in the signal transduction of EGF, insulin and IGF-I. The inhibition of cell growth by lovastatin may be caused by the inability of the cell to enter the S phase of the cell cycle due to obstruction of the signaling of progression factors.

Cells, Cultured↗
PubMed2004

Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.

Arabidopsis↗
PubMed2002

Farnesol and geranylgeraniol: prevention and reversion of lovastatin-induced effects in NIH3T3 cells.

Mevalonic acid-derived intermediates in the cholesterol biosynthetic pathway have been recognized as being critical to the isoprenylation of a variety of growth-regulating proteins, including those of the RAS superfamily. Treatment of cells with lovastatin, a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, depletes cells of mevalonic acid and thus blocks the isoprenylation of proteins in the RAS superfamily. In NIH3T3 cells pretreated with lovastatin, subsequent addition of farnesol (FOH), but not geranylgeraniol (GGOH), reverses lovastatin's block of RAS isoprenylation. Neither FOH nor GGOH prevents lovastatin-induced inhibition of RAS isoprenylation when added to cells concurrently with lovastatin. In intact cells, 167 microM FOH and 125 microM GGOH decrease incorporation of [14C]acetate into cholesterol by approximately 50 and 75%, respectively. Results suggest that the radio-label from either [3H]FOH or [3H]GGOH is incorporated into cholesterol. Co-treatment of cells with lovastatin or mevalonic acid did not significantly alter [3H]FOH or [3H]GGOH incorporation into cholesterol. Lovastatin induces cell rounding; GGOH, but not FOH, both prevents and reverses lovastatin-induced cell rounding. These results provide additional evidence for the existence of a novel "isoprenoid shunt" that differentially utilizes FOH and GGOH as metabolic precursors for isoprenoids that have been depleted by lovastatin treatment.

3T3 Cells↗
PubMed1987

Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides.

The biosynthesis of dolichol and dolichol-linked saccharide intermediates in glycoprotein synthesis was studied in an embryonic Drosophila cell line (Kc) that lacks the squalene-cholesterol branch of the polyisoprenoid biosynthetic pathway. Kc cells were labeled with [5-3H]mevalonic acid and the radioactive lipids formed were analyzed. Although the major labeled product was coenzyme Q, dolichol and a variety of dolichol derivatives could be readily detected. On the basis of their chromatographic and chemical properties, these derivatives were identified as dolichyl phosphate, glucosylphosphoryldolichol, mannosylphosphoryldolichol, and oligosaccharylpyrophosphoryldolichol. Both short term (4-h) and steady state (4-day) labeling experiments with mevalonate, rather than sugars as previously used, were performed to assess the level of these intermediates. The results of these studies, using a precursor common to all the intermediates, reveal that the early intermediates, N-acetylglucosaminylpyrophosphoryldolichol and N,N'-diacetylchitobiosylpyrophosphoryldolichol, are present at very low levels (less than 5%) relative to the other intermediates on the pathway to oligosaccharylpyrophosphoryldolichol. The total amount of dolichol intermediates remained essentially constant during the chase phase of pulse-chase experiments, indicating the absence of a major catabolic pathway for the polyisoprenoid backbone. As expected, however, the sugar moiety, studied with mannosylphosphoryldolichol, underwent rapid turnover. These results are discussed in the context of our current understanding of the pathway whereby dolichol derivatives participate in glycoprotein synthesis.

Acetylglucosamine↗
PubMed1996

Regulation of fatty acid biosynthesis by intermediates of the cholesterol biosynthetic pathway.

The biosynthesis of cholesterol and fatty acid (FA) proceeds by independent pathways. Information is lacking on potential interaction that could provide feedback regulation between these pathways. In an attempt to search for a new approach to produce a dual effect on regulation of these two processes, we have identified mevalonate-5-diphosphate (MevPP) decarboxylase, an enzyme of the cholesterol biosynthesis pathway, the inhibition of which leads not only to the suppression of cholesterol but also FA biosynthesis. Mechanistic studies with Hep G2 cells using specific inhibitors for MevPP decarboxylase and related enzymes reveal that the inhibitory effect on FA biosynthesis is mainly due to the accumulation of MevPP, resulting from MevPP decarboxylase inhibition. The present study proposes a new mechanism through which interpathway regulation could take place between the cholesterol and FA cascades.

Anticholesteremic Agents↗
PubMed1996

Monoterpenes as regulators of malignant cell proliferation.

Limonene and related monoterpenes display compelling anticarcinogenic activity. The mechanism(s) that underline this activity is/are as yet unknown. One attractive possibility is that the monoterpenes interact with the RAS signal transduction pathway. The monoterpenes have been shown to impair incorporation of mevalonic acid-derived isoprene compounds, that is farnesyl pyrophosphate, into RAS and RAS-related proteins. As farnesylation is critical for RAS's membrane localization and function, the isoprenylation pathways have received attention as potential targets of anti-RAS pharmacological maneuvers. We have expanded on prior studies and demonstrate that one of limonene's metabolic derivatives, perillyl alcohol, decreases the levels of antigenic RAS in the human-derived myeloid THP-1 and lymphoid RPMI-8402 leukemia cell lines. Both limonene and perillyl alcohol decrease levels of 35[S] methionine labeled RAS proteins in cells that have been pulsed with radiolabeled methionine for four hours. In contrast, lovastatin, which inhibits hydroxymethylglutaryl coenzyme A reductase and thus depletes cells of farnesyl pyrophosphate, does not diminish levels of total antigenic RAS but rather results in a shift in the RAS protein; levels of farnesylated RAS decrease whereas levels of unmodified/unfarnesylated RAS increase. As limonene and perillyl alcohol do not induce such a shift we conclude that these monoterpenes decrease farnesylated RAS protein levels by a mechanism that is clearly distinct from that of either depleting cells of farnesyl pyrophosphate or inhibiting the enzyme farnesyl protein transferase that catalyzes the posttranslational farnesylation of RAS. These findings are discussed with respect to implications for the monoterpenes to alter RAS protein synthesis and degradation. The results of these studies will likely impact the inclusion of the monoterpenes in clinical anticancer trials.

Animals↗
PubMed2003

Frontalin: De novo biosynthesis of an aggregation pheromone component by Dendroctonus spp. bark beetles (Coleoptera: Scolytidae).

The pheromone component, frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane) is thought to be formed in Dendroctonus spp. bark beetles through the cyclization of oxygenated 6-methyl-6-hepten-2-one (6-MHO). Unlike many of the isoprenoid pheromone components of bark beetles, there is no obvious immediate host conifer precursor for 6-MHO or frontalin. To elucidate the biosynthetic pathway of frontalin, juvenile hormone-treated male Dendroctonus jeffreyi were injected separately with [1-(14)C]acetate, [2-(14)C]mevalonolactone, [1-(14)C]isopentenol, [1-(14)C]:[1-(3)H]isopentenol, and [4,5-(3)H]leucine. Subsequently volatiles were collected on Porapak Q from these males and abdominal tissues were extracted. Radio-HPLC analyses of extracts from males injected with each radiolabeled substrate showed that radioactivity from the injected precursors eluted in a peak with a retention time that matches that of unlabeled frontalin. In all cases, HPLC fractions containing radiolabel that eluted at the same time as a frontalin standard were analyzed by GC-FID and GC-MS to confirm the presence of frontalin. In a separate study, male D. jeffreyi were injected with [1-(13)C]acetate and an abdominal tissue extract from these insects was analyzed by tandem gas chromatography-isotope ratio monitoring-mass spectrometry (GC-IRM-MS), which unequivocally showed incorporation of (13)C into frontalin. Because mevalonate is the key intermediate in the isoprenoid pathway, its incorporation (as mevalonolactone) into frontalin provides compelling evidence that the biosynthesis of frontalin involves that pathway in some form. In the experiment with [1-(14)C]:[1-(3)H]isopentenol, there was no significant difference in the mean percentage incorporation of either radioisotope into frontalin. This supports the role of the classical isoprenoid pathway, as tritium would be lost if only a hybrid pathway were involved. Confirming that de novo synthesis may be general to all Dendroctonus spp., (14)C-acetate was also incorporated into frontalin by females of D. rufipennis and D. simplex. A radiolabeled precursor/pathway inhibitor study showed that the fatty acid synthase inhibitor, 2-octynoic acid, increased (although not significantly) the mass of frontalin produced and significantly increased the percentage incorporation of radioactivity from [1-(14)C]acetate into frontalin. This suggests that as fatty acid biosynthesis is blocked, an increased amount of acetate is funneled into frontalin production via the isoprenoid pathway.

Acetates↗
PubMed1990

Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver.

Mevalonate kinase [ATP:(R)-mevalonate 5-phosphotransferase, EC 2.7.1.36] may be a regulatory site in the cholesterol biosynthetic pathway, and a mutation in the gene coding for this enzyme is thought to cause the genetic disease mevalonic aciduria. To characterize this enzyme, a rat liver cDNA library was screened with a monospecific antibody, and a 1.7-kilobase cDNA clone coding for mevalonate kinase was isolated. The complete DNA sequence was determined, and the longest open reading frame coded for a protein containing 395 amino acids with a deduced molecular weight of 41,990. Identification of the cDNA clone was confirmed by expression of enzyme activity in yeast and by protein sequence data obtained from sequencing purified rat mevalonate kinase. The deduced amino acid sequence of mevalonate kinase contained a motif for the ATP-binding site found in protein kinases, and it also showed sequence homology to the yeast RAR1 protein. The size of mevalonate kinase mRNA in rat liver was approximately 2 kilobases. Treatment with diets containing cholesterol-lowering agents caused an increase in both mevalonate kinase activity and mRNA levels, whereas diets containing 5% cholesterol lowered the levels of both enzyme activity and mRNA. These data indicate that long-term regulation of enzyme activity in rat liver is controlled by changes in the levels of mevalonate kinase mRNA.

Amino Acid Sequence↗
PubMed2006

3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase is involved in biosynthesis of isovaleryl-CoA in the myxobacterium Myxococcus xanthus during fruiting body formation.

Isovaleryl-coenzyme A (IV-CoA) is the starting unit for some secondary metabolites and iso-odd fatty acids in several bacteria. According to textbook biochemistry, IV-CoA is derived from leucine degradation, but recently an alternative pathway that branches from the well-known mevalonate-dependent isoprenoid biosynthesis has been described for myxobacteria. A double mutant was constructed in Myxococcus xanthus by deletion of genes involved in leucine degradation and disruption of mvaS encoding the 3-hydroxy-3-methylglutaryl-coenzyme A synthase. A dramatic decrease of IV-CoA-derived iso-odd fatty acids was observed for the mutant, confirming mvaS to be involved in the alternative pathway. Additional quantitative real-time reverse transcription-PCR experiments indicated that mvaS is transcriptionally regulated by isovalerate. Furthermore, feeding studies employing an intermediate specific for the alternative pathway revealed that this pathway is induced during fruiting body formation, which presumably increases the amount of IV-CoA available when leucine is limited.

Acyl Coenzyme A↗
PubMed1999

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis.

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.

Amino Acid Sequence↗