geranyl-pyrophosphate and farnesyl-pyrophosphate

Statins have demonstrated substantial benefits in supporting cardiovascular health. Older individuals are more likely to experience the well-known muscle-related side effects of statins compared with younger individuals. Elderly females may be especially vulnerable to statin-related muscle disorder. This review will collate and discuss statin-related muscular effects, examine their molecular and genetic basis, and how these apply specifically to elderly women. Developing strategies to reduce the incidence of statin-induced myopathy in older adult women could contribute to a significant reduction in the overall incidence of statin-induced muscle disorder in this vulnerable group of patients. Reducing statin-related muscle disorder would likely improve overall patient compliance, thereby leading to an increase in improved short- and long-term outcomes associated with appropriate use of statins.

Topics: Aged; Aging; Cell Death; Comorbidity; Drug Interactions; Female; Genetic Predisposition to Disease; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Incidence; Muscular Diseases; Myositis; Polyisoprenyl Phosphates; Quality of Life; Rhabdomyolysis; Sesquiterpenes; Sex Factors; Ubiquinone

The isoprenoid biosynthetic pathway is the source of a wide array of products. The pathway has been highly conserved throughout evolution, and isoprenoids are some of the most ancient biomolecules ever identified, playing key roles in many life forms. In this review we focus on C-10 mono-, C-15 sesqui-, and C-20 diterpenes. Evidence for interconversion between the pathway intermediates farnesyl pyrophosphate and geranylgeranyl pyrophosphate and their respective metabolites is examined. The diverse functions of these molecules are discussed in detail, including their ability to regulate expression of the beta-HMG-CoA reductase and Ras-related proteins. Additional topics include the mechanisms underlying the apoptotic effects of select isoprenoids, antiulcer activities, and the disposition and degradation of isoprenoids in the environment. Finally, the significance of pharmacological manipulation of the isoprenoid pathway and clinical correlations are discussed.

Topics: Animals; Humans; Hydroxymethylglutaryl CoA Reductases; Molecular Structure; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Topics: Animals; Dimethylallyltranstransferase; Humans; Polyisoprenyl Phosphates; Protein Binding; Protein Prenylation; Sesquiterpenes

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.

Topics: Diphosphates; Humans; Polyisoprenyl Phosphates; Terpenes

Topics: Biocatalysis; Crystallography, X-Ray; Density Functional Theory; Methyltransferases; Models, Molecular; Molecular Structure; Polyisoprenyl Phosphates; Sesquiterpenes; Streptomyces; Substrate Specificity

Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a S

Topics: Amino Acid Motifs; Bacterial Proteins; Binding Sites; Biocatalysis; Bridged Bicyclo Compounds; Cloning, Molecular; Cyclization; Escherichia coli; Gene Expression; Genetic Vectors; Methylation; Methyltransferases; Molecular Docking Simulation; Mutagenesis, Site-Directed; Octanes; Polyisoprenyl Phosphates; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Serratia; Sesquiterpenes; Substrate Specificity

Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C

Topics: Binding Sites; Crystallography, X-Ray; Diphosphates; Diterpenes; Geranyltranstransferase; Hemiterpenes; Humans; Leishmania major; Leishmaniasis, Cutaneous; Models, Molecular; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Conformation; Sesquiterpenes; Substrate Specificity

Viperin is a radical SAM enzyme that possesses antiviral properties against a broad range of enveloped viruses. Here, we describe the activity of human viperin with two molecules of the mevalonate pathway, geranyl pyrophosphate, and farnesyl pyrophosphate, involved in cholesterol biosynthesis. We postulate that the radical modification of these two molecules by viperin might lead to defects in cholesterol synthesis, thereby affecting the composition of lipid rafts and subsequent enveloped virus budding.

Topics: Biocatalysis; Cholesterol; Humans; Models, Molecular; Molecular Docking Simulation; Oxidoreductases Acting on CH-CH Group Donors; Polyisoprenyl Phosphates; Proteins; Sesquiterpenes; Substrate Specificity; Virus Release

Isoprenoids (IsoP) are an important class of molecules involved in many different cellular processes including cholesterol synthesis. We have developed a sensitive and specific LC-MS/MS method for the quantitation of three key IsoPs in bio-matrices, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). LC-MS/MS analysis was performed using a Nexera UPLC System connected to a LCMS-8060 (Shimadzu Scientific Instruments, Columbia, MD) with a dual ion source. The electrospray ionization source was operated in the negative MRM mode. The chromatographic separation and detection of analytes was achieved on a reversed phase ACCQ-TAG Ultra C18 (1.7 µm, 100 mm × 2.1 mm I.D.) column. The mobile phase consisted of (1) a 10 mM ammonium carbonate with 0.1% ammonium hydroxide in water, and (2) a 0.1% ammonium hydroxide in acetonitrile/methanol (75/25). The flow rate was set to 0.25 mL/min in a gradient condition. The limit of quantification was 0.04 ng/mL for all analytes with a correlation coefficient (r2) of 0.998 or better and a total run time of 12 min. The inter- and intra-day accuracy (85⁻115%) precision (<15%), and recovery (40⁻90%) values met the acceptance criteria. The validated method was successfully applied to quantitate basal concentrations of GPP, FPP and GGPP in human plasma and in cultured cancer cell lines. Our LC-MS/MS method may be used for IsoP quantification in different bio-fluids and to further investigate the role of these compounds in various physiological processes.

Topics: Calibration; Cell Line, Tumor; Chromatography, Liquid; Humans; Pancreatic Neoplasms; Polyisoprenyl Phosphates; Reproducibility of Results; Sensitivity and Specificity; Sesquiterpenes; Tandem Mass Spectrometry

Topics: Diphosphates; Diterpenes; Escherichia coli; Escherichia coli Proteins; Genetic Enhancement; Geranyltranstransferase; Hydrocarbons; Monoterpenes; Mutation; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes

Topics: Acyclic Monoterpenes; Camptotheca; Diphosphates; Diterpenes; DNA, Complementary; Escherichia coli; Geranyltranstransferase; Hemiterpenes; Monoterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Polymerase Chain Reaction; Sesquiterpenes; Terpenes

Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Diphosphates; Diterpenes; Gene Expression Regulation, Fungal; Geranyltranstransferase; Hemiterpenes; Kinetics; Metabolic Engineering; Molecular Docking Simulation; Organophosphorus Compounds; Point Mutation; Polyisoprenyl Phosphates; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Saccharomyces cerevisiae; Sequence Alignment; Sequence Homology, Amino Acid; Sesquiterpenes; Substrate Specificity; Terpenes; Thermodynamics

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from Zea mays exhibit isotopically sensitive branching in the formation of mono- and sesquiterpene volatiles. The impact of the kinetic isotope effects and the stabilization of the reactive intermediates by hyperconjugation along with the shift of products from alkenes to alcohols are discussed.

Topics: Alkyl and Aryl Transferases; Deuterium; Diphosphates; Diterpenes; Kinetics; Plant Proteins; Polyisoprenyl Phosphates; Sesquiterpenes; Zea mays

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from maize (Zea mays) catalyze the conversion of farnesyl diphosphate (FDP) and geranyl diphosphate (GDP) into a complex mixture of sesquiterpenes and monoterpenes, respectively. Various isotopic and geometric isomers of natural substrates like (2Z)-[2-(2)H]- and [2,4,4,9,9,9-(2)H6]-(GDP) and (2Z,6E)-[2-(2)H]- and [2,4,4,13,13,13-(2)H6]-(FDP) were synthesized analogous to presumptive reaction intermediates. On incubation with labeled (2Z) substrates, TPS4 and TPS5 showed much lower kinetic isotope effects than the labeled (2E) substrates. Interestingly, the products arising from the deuterated (2Z)-precursors revealed a distinct preference for cyclic products and exhibited an enhanced turnover on comparison with natural (2E)-substrates. This increase in the efficiency due to (2Z) configuration emphasizes the rate limiting effect of the initial (2E) → (2Z) isomerization step in the reaction cascade of the multiproduct terpene synthases. Apart from turnover advantages, these results suggest that substrate geometry can be used as a tool to optimize the biosynthetic reaction cascade towards valuable cyclic terpenoids.

Topics: Alkyl and Aryl Transferases; Cyclization; Diphosphates; Diterpenes; Monoterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Stereoisomerism; Substrate Specificity; Zea mays

The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

Topics: Amino Acid Sequence; Cloning, Molecular; Diatoms; DNA, Complementary; Escherichia coli; Gas Chromatography-Mass Spectrometry; Geranyltranstransferase; Isomerism; Life Cycle Stages; Molecular Sequence Data; Phylogeny; Polyisoprenyl Phosphates; Recombinant Proteins; Sequence Alignment; Sesquiterpenes; Terpenes

Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

Topics: Arabidopsis; Arabidopsis Proteins; Archaea; Cytosol; Gene Expression Regulation, Plant; Gene Knockout Techniques; Genes, Plant; Hemiterpenes; Kinetics; Metabolic Networks and Pathways; Mevalonic Acid; Nicotiana; Organophosphorus Compounds; Phosphotransferases (Alcohol Group Acceptor); Plants, Genetically Modified; Plastids; Polyisoprenyl Phosphates; Sequence Homology, Amino Acid; Sesquiterpenes; Terpenes

Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects.. We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg(2+), the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP).. AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.

Topics: Adenosine Triphosphate; Amino Acid Motifs; Amino Acid Sequence; Animals; Catalysis; Cations; Corpora Allata; Culicidae; Diphosphates; Diterpenes; Female; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Juvenile Hormones; Magnesium; Mevalonic Acid; Molecular Sequence Data; Nucleotides; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Polyisoprenyl Phosphates; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid; Sesquiterpenes; Terpenes; Thorax

Both norepinephrine (NE) and connective tissue growth factor (CTGF) contribute to vascular fibrosis during hypertension. Recent studies indicate that farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiac remodeling in hypertension. However, the role of FPPS in NE-induced fibrotic responses and related molecular mechanisms is unknown. Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were stimulated with NE. The fibrotic responses were assessed by measuring CTGF, hydroxyproline (hyp), and α-1 procollagen I levels using Western blot, a hydroxyproline test kit, and real-time quantitative PCR assays, respectively. Ras activity was determined by a pull-down assay using a Ras activation assay kit and detected by Western blot. NE dose-dependently increased fibrosis in SHR-VSMCs, and this increase was significantly reduced by ibandronate, an inhibitor of FPPS. The addition of farnesol, but not geranylgeraniol, partially reversed the inhibitory effects of ibandronate. Furthermore, the anti-fibrotic effects of ibandronate could be mimicked by FTI-276 but not by GGTI-286. A pull-down assay showed that ibandronate reduced the NE-induced Ras activation. Moreover, ibandronate inhibited the NE-induced activation of p38, JNK, and ERK1/2. Only SB203580 (specific inhibitor of p38) diminished the NE-induced CTGF production. These results demonstrated that inhibiting FPPS prevents NE-induced fibrotic responses in SHR-VSMCs and that the Ras kinase and p38 pathways were the underlying mechanisms involved in this process.

Topics: Amyloid beta-Protein Precursor; Animals; Cell Proliferation; Cells, Cultured; Dimethylallyltranstransferase; Diphosphonates; Enzyme Activation; Enzyme Inhibitors; Fibrosis; Genes, ras; Hydroxyproline; Hypertension; Ibandronic Acid; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Norepinephrine; Peptide Fragments; Polyisoprenyl Phosphates; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sesquiterpenes

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily are involved in the biosynthesis of secondary metabolites and show broad substrate specificity towards their aromatic substrates with a high regioselectivity for the prenylation reactions. Most members of this superfamily accepted as prenyl donor exclusively dimethylallyl diphosphate (DMAPP). One enzyme, AnaPT from Neosartorya fischeri, was reported recently to use both DMAPP and geranyl diphosphate (GPP) as prenyl donors. In this study, we demonstrate the acceptance of DMAPP, GPP and farnesyl diphosphate (FPP) by a new member of this superfamily, BAE61387 from Aspergillus oryzae DSM1147, for C-prenylations of hydroxynaphthalenes.

Topics: Aspergillus oryzae; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Hemiterpenes; Naphthols; Organophosphorus Compounds; Polyisoprenyl Phosphates; Prenylation; Sesquiterpenes

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co(2+) or Mn(2+) as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg(2+). In reaction with Co(2+), PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg(2+), PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co(2+), Mn(2+), or Mg(2+) in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Animals; Cobalt; Coleoptera; Diphosphates; Diterpenes; Insect Proteins; Manganese; Molecular Sequence Data; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

Topics: Acetates; Alkyl and Aryl Transferases; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Cyclohexane Monoterpenes; Cyclohexenes; Cyclopentanes; Gas Chromatography-Mass Spectrometry; Gossypium; Intramolecular Lyases; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Phytoalexins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Sesquiterpenes, Guaiane; Terpenes; Volatile Organic Compounds

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.

Topics: Animals; Apicoplasts; Atorvastatin; Diphosphates; Diterpenes; Farnesyltranstransferase; Female; Gene Knockdown Techniques; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Mice, Inbred BALB C; Polyisoprenyl Phosphates; Protozoan Proteins; Pyrroles; Sesquiterpenes; Toxoplasma; Toxoplasmosis

Cotton (Gossypium hirsutum L.) plants damaged by insects emit a blend of volatiles, including monoterpenes and sesquiterpenes, which can directly repel herbivores and/or indirectly protect the plant by attracting natural enemies of the herbivores. To understand the molecular basis of terpene biosynthesis and regulation in cotton, two terpene synthase genes, GhTPS1 and GhTPS2, were heterologously expressed and characterized. Recombinant GhTPS1 accepted farnesyl pyrophosphate as substrate and produced (E)-β-caryophyllene and α-humulene. GhTPS2 was characterized as a monoterpene synthase which formed α-pinene and β-pinene using geranyl pyrophosphate as substrate. Quantitative real-time PCR analysis revealed that GhTPS1 and GhTPS2 gene expression was elevated after methyl jasmonate (MeJA) treatment in cotton leaves. Moreover, feeding of the green plant bug Apolygus lucorum, a major cotton pest in northern China, resulted in increased GhTPS2 expression in young leaves, suggesting that GhTPS2 might be involved in plant defense in cotton.

Topics: Acetates; Adaptation, Physiological; Alkyl and Aryl Transferases; Animals; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Carbon-Oxygen Lyases; China; Cyclopentanes; Gene Expression; Genes, Plant; Gossypium; Herbivory; Insecta; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Plant Diseases; Plant Leaves; Plant Proteins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.

Topics: Actinidia; Acyclic Monoterpenes; Alkyl and Aryl Transferases; Arabidopsis; Base Sequence; Diphosphates; Diterpenes; Farnesol; Flowers; Gene Expression Regulation, Plant; Kinetics; Molecular Sequence Data; Monoterpenes; Nicotiana; Oils, Volatile; Phylogeny; Plant Leaves; Plant Proteins; Polyisoprenyl Phosphates; Recombinant Proteins; Sequence Analysis, DNA; Sesquiterpenes; Substrate Specificity

Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of the C(5) terpenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, to form geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes-monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to detect the short-chain prenyl diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C(10) enzyme products (geranyl diphosphate and neryl diphosphate) and three C(15) products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could determine their IDS activity in a reproducible fashion.

Topics: Alkyl and Aryl Transferases; Arabidopsis; Chromatography, Liquid; Diphosphates; Diterpenes; Enzyme Activation; Enzyme Assays; Nicotiana; Phosphorylation; Picea; Plant Extracts; Plant Proteins; Polyisoprenyl Phosphates; Populus; Sensitivity and Specificity; Sesquiterpenes; Tandem Mass Spectrometry; Terpenes

Short-chain prenyltransferases are responsible for biosynthesis of the C(10)-C(20) precursors of a variety of isoprenoids. We previously isolated two different short-chain prenyltransferases from the green peach aphid, Myzus persicae (MpIPPS1 and MpIPPS2). In this study, the activity of the two aphid prenyltransferases was analyzed in vitro. Kinetic analysis using recombinant enzymes showed that both prenyltransferases could efficiently catalyze the formation of C(10) geranyl diphosphate (GPP) and C(15) farnesyl diphosphate (FPP) from the C(5) substrates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and MpIPPS2 had higher catalytic activity than MpIPPS1. Product analysis by gas chromatography-mass spectrometry demonstrated that FPP was generated as the major product, but GPP could be detected at low enzyme concentrations. Molecular docking revealed that MpIPPS2 had higher binding affinity with the substrates DMAPP, IPP, and GPP than MpIPPS1, which supported the experimentally determined kinetic parameters. Molecular docking also identified an amino acid residue (K266) critical to the catalytic activity of both MpIPPS1 and MpIPPS2. This prediction was subsequently confirmed by site-directed mutagenesis, in which a point mutation (K266I) abolished the activity of both MpIPPS1 and MpIPPS2. Our data illustrate that both aphid short-chain prenyltransferases are active forms, which is in contrast to the previously reported results.

Topics: Animals; Aphids; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Kinetics; Molecular Docking Simulation; Polyisoprenyl Phosphates; Protein Binding; Recombinant Proteins; Sesquiterpenes

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the "missing" GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.

Topics: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Cell Wall; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Farnesol; Flame Ionization; Gas Chromatography-Mass Spectrometry; Geranyltranstransferase; Hemiterpenes; Isomerism; Kinetics; Molecular Sequence Data; Mycobacterium tuberculosis; Organophosphorus Compounds; Polyisoprenyl Phosphates; Recombinant Proteins; Sequence Alignment; Sesquiterpenes; Substrate Specificity

Norway spruce (Picea abies) defends itself against herbivores and pathogens by formation of traumatic resin ducts filled with terpenoid-based oleoresin. An important group of enzymes in terpenoid biosynthesis are the short-chain isoprenyl diphosphate synthases which produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of monoterpenes, sesquiterpenes, and diterpene resin acids, respectively. After treatment with methyl jasmonate (MJ) we investigated the expression of all isoprenyl diphosphate synthase genes characterized to date from Norway spruce and correlated this with formation of traumatic resin ducts and terpene accumulation. Formation of traumatic resin ducts correlated with higher amounts of monoterpenes, sesquiterpenes and diterpene resin acids and an upregulation of isoprenyl diphosphate synthase genes producing geranyl diphosphate or geranylgeranyl diphosphate. Among defense hormones, jasmonate and jasmonate-isoleucine conjugate accumulated to higher levels in trees with extensive traumatic resin duct formation, whereas salicylate did not. Jasmonate and ethylene are likely to both be involved in formation of traumatic resin ducts based on elevated transcripts of genes encoding lipoxygenase and 1-aminocyclopropane-1-carboxylic acid oxidase associated with resin duct formation. Other genes involved in defense signalling in other systems, mitogen-activated protein kinase3 and nonexpressor of pathogenesis-related gene1, were also associated with traumatic resin duct formation. These responses were detected not only at the site of MJ treatment, but also systemically up to 60 cm above the site of treatment on the trunk.

Topics: Acetates; Alkyl and Aryl Transferases; Cyclopentanes; Diphosphates; Diterpenes; Oxylipins; Picea; Plant Growth Regulators; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

Topics: Amino Acid Sequence; Cloning, Molecular; Farnesyltranstransferase; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Picea; Plant Extracts; Plant Proteins; Plants, Genetically Modified; Polyisoprenyl Phosphates; RNA, Plant; Sequence Alignment; Sequence Analysis, DNA; Sesquiterpenes; Terpenes

Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Chromatography, High Pressure Liquid; Diphosphates; Diphosphonates; Diterpenes; DNA Ligases; Hemiterpenes; Mevalonic Acid; Organophosphorus Compounds; Osteoclasts; Polyisoprenyl Phosphates; Polyphosphates; RNA Ligase (ATP); Sesquiterpenes; Substrate Specificity

In addition to providing lipid chains for protein prenylation, short-chain isoprenyl diphosphate synthases (scIPPSs) play a pivotal role in the biosynthesis of numerous mevalonate pathway end-products, including insect juvenile hormone and terpenoid pheromones. For this reason, they are being considered as targets for pesticide development. Recently, we characterized an aphid scIPPS displaying dual geranyl diphosphate (GPP; C(10))/farnesyl diphosphate (FPP; C(15)) synthase activity in vitro. To identify the mechanism(s) responsible for this dual activity, we assessed the product selectivity of aphid scIPPSs bearing mutations at Gln107 and/or Leu110, the fourth and first residue upstream from the "first aspartate-rich motif" (FARM), respectively. All but one resulted in significant changes in product chain-length selectivity, effectively increasing the production of either GPP (Q107E, L110W) or FPP (Q107F, Q107F-L110A); the other mutation (L110A) abolished activity. Although some of these effects could be attributed to changes in steric hindrance within the catalytic cavity, molecular dynamics simulations identified other contributing factors, including residue-ligand Van der Waals interactions and the formation of hydrogen bonds or salt bridges between Gln107 and other residues across the catalytic cavity, which constitutes a novel product chain-length determination mechanism for scIPPSs. Thus the aphid enzyme apparently evolved to maintain the capacity to produce both GPP and FPP through a balance between these mechanisms.

Topics: Animals; Aphids; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Geranyltranstransferase; Models, Molecular; Mutation; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity

Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.

Topics: Cells, Cultured; Diphosphates; Diterpenes; Humans; K562 Cells; Limit of Detection; Polyisoprenyl Phosphates; Sesquiterpenes

alpha-Zingiberene synthase (ZIS), a sesquiterpene synthase gene that was isolated from lemon basil (Ocimum basilicum L.), encodes an enzyme that catalyzes the formation of alpha-zingiberene, and other sesquiterpenes, from farnesyl diphosphate. Transgenic tomato fruits overexpressing ZIS under the control of the fruit ripening-specific tomato polygalacturonase promoter (PG) accumulated high levels of alpha-zingiberene (224-1000 ng g(-1) fresh weight) and other sesquiterpenes, such as alpha-bergamotene, 7-epi-sesquithujene, beta-bisabolene and beta-curcumene, whereas no sesquiterpenes were detected in non-transformed control fruits. The ZIS-transgenic fruits also produced monoterpenes, such as alpha-thujene, alpha-pinene, beta-phellandrene and gamma-terpinene (1-22 ng g(-1) fresh weight), which were either not detected or were found only in minute concentrations in control fruits. Recombinant ZIS overexpressed in Escherichia coli catalyzed the formation of these monoterpenes from geranyl diphosphate. As the ZIS protein apparently lacks a transit peptide, and is localized in the cytosol, the production of monoterpenes in the transgenic tomatoes suggests that a pool of geranyl diphosphate is available in the cytosol. The phenotype of the ZIS-transgenic tomatoes was the same as that for wild-type tomatoes, with regard to plant vigor and shape, but transgenic plants exhibited a small decrease in lycopene content. This study thus showed that the synthesis of both mono- and sesquiterpenes can be enhanced by the ectopic expression of a single transgene in tomato fruit, and it further demonstrated the interconnection between the pools of terpenoid precursors in the plastids and the cytosol.

Topics: Alkyl and Aryl Transferases; Carotenoids; Cloning, Molecular; Diphosphates; Diterpenes; Escherichia coli; Fruit; Gas Chromatography-Mass Spectrometry; Genetic Vectors; Monocyclic Sesquiterpenes; Monoterpenes; Ocimum basilicum; Oils, Volatile; Phytosterols; Plants, Genetically Modified; Polyisoprenyl Phosphates; RNA, Plant; Sesquiterpenes; Solanum lycopersicum; Transformation, Genetic

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Heveabrasiliensis and Parthenium argentatum. Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz-DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz-DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.

Topics: Asteraceae; Benzophenones; Ficus; Hemiterpenes; Hevea; Latex; Molecular Structure; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rubber; Sesquiterpenes; Substrate Specificity; Transferases

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells. We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Melanoma; Polyisoprenyl Phosphates; RNA, Messenger; Sesquiterpenes; Simvastatin

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product- or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPS- and UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.

Topics: Alkyl and Aryl Transferases; Binding Sites; Crystallography, X-Ray; Dimerization; Diphosphates; Diphosphonates; Diterpenes; Farnesyltranstransferase; Hydrophobic and Hydrophilic Interactions; Isoenzymes; Ligands; Models, Chemical; Models, Molecular; Molecular Structure; Polyisoprenyl Phosphates; Protein Structure, Secondary; Saccharomyces cerevisiae; Sesquiterpenes; Stereoisomerism; Substrate Specificity; Transferases

Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyl-diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.

Topics: Alternative Splicing; Amino Acid Motifs; Amino Acid Sequence; Animals; Baculoviridae; Bone Density Conservation Agents; Catalysis; Cloning, Molecular; Diphosphates; Diphosphonates; Diterpenes; Dolichols; Drug Design; Enzyme Inhibitors; Escherichia coli; Farnesyltranstransferase; Gene Expression; Gene Expression Regulation, Enzymologic; Geranyltranstransferase; Isoenzymes; Molecular Sequence Data; Polyisoprenyl Phosphates; Protein Prenylation; Protozoan Proteins; Quantitative Trait Loci; Recombinant Proteins; Sesquiterpenes; Sterols; Toxoplasma; Toxoplasmosis; Ubiquinone

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cholesterol; Extracellular Signal-Regulated MAP Kinases; Hydroxycholesterols; JNK Mitogen-Activated Protein Kinases; Lipid Metabolism; Lipoproteins; Mice; NIH 3T3 Cells; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Polyisoprenyl Phosphates; Protein Kinase Inhibitors; rhoA GTP-Binding Protein; Sesquiterpenes; Transfection

Coenzyme Q(10) (CoQ(10)), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells' survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ(10) production, as indicated by the amount of CoQ(9) formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, omega,E,E-farnesyl diphosphate (FPP), and omega,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V (max) (pmol/min), K (M) (microM), k (cat) (1/min), and k (cat) /K (M) of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.

Topics: Agrobacterium tumefaciens; Alkyl and Aryl Transferases; Chromatography, Thin Layer; Cloning, Molecular; Coenzymes; Diphosphates; Diterpenes; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hemiterpenes; Kinetics; Molecular Sequence Data; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rhodobacter sphaeroides; Sequence Analysis, DNA; Sesquiterpenes; Substrate Specificity; Ubiquinone

UPPS (undecaprenyl pyrophosphate synthase) catalyses consecutive condensation reactions of FPP (farnesyl pyrophosphate) with eight isopentenyl pyrophosphates to generate C55 UPP, which serves as a lipid carrier for bacterial peptidoglycan biosynthesis. We reported the co-crystal structure of Escherichia coli UPPS in complex with FPP. Its phosphate head-group is bound to positively charged arginine residues and the hydrocarbon moiety interacts with hydrophobic amino acids including L85, L88 and F89, located on the alpha3 helix of UPPS. We now show that the monophosphate analogue of FPP binds UPPS with an eight times lower affinity (K(d)=4.4 microM) compared with the pyrophosphate analogue, a result of a larger dissociation rate constant (k(off)=192 s(-1)). Farnesol (1 mM) lacking the pyrophosphate does not inhibit the UPPS reaction. GGPP (geranylgeranyl pyrophosphate) containing a larger C20 hydrocarbon tail is an equally good substrate (K(m)=0.3 microM and kcat=2.1 s(-1)) compared with FPP. The shorter C10 GPP (geranyl pyrophosphate) displays a 90-fold larger K(m) value (36.0+/-0.1 microM) but similar kcat value (1.7+/-0.1 s(-1)) compared with FPP. Replacement of L85, L88 or F89 with Ala increases FPP and GGPP K(m) values by the same amount, indicating that these amino acids are important for substrate binding, but do not determine substrate specificity. With GGPP as a substrate, UPPS still catalyses eight isopentenyl pyrophosphate condensation reactions to synthesize C60 product. Computer modelling suggests that the upper portion of the active-site tunnel, where cis double bonds of the product reside, may be critical for determining the final product chain length.

Topics: Alkyl and Aryl Transferases; Binding Sites; Escherichia coli Proteins; Farnesol; Hemiterpenes; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Molecular Weight; Mutagenesis, Site-Directed; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Conformation; Recombinant Fusion Proteins; Sesquiterpenes; Substrate Specificity

Statins were recently shown to possess anti-inflammatory activities, which might be responsible for their favourable effects in cardiovascular or CNS disorders independently from the inhibition of hydroxy-methyl-glutaryl CoA reductase. Here we investigated the effects of the statins lovastatin and mevastatin on prostanoid production in primary cultures of rat cortical microglia and astrocytes. We found that both statins significantly reduce prostaglandin E2 (PGE2) release from microglia, either under basal conditions or after stimulation by interleukin-1beta. Lovastatin also tends to reduce, although not in a significant manner, basal and interleukin-1beta-stimulated PGE2 release from astrocytes. Precursors and intermediates in cholesterol biosynthesis--mevalonic acid and geranyl and farnesyl pyrophosphate--also reduce PGE2 production, and potentiate the inhibitory effects of statins, suggesting that the latter might not depend on the inhibition of hydroxy-methyl-glutaryl CoA reductase.

Topics: Animals; Anti-Inflammatory Agents; Astrocytes; Autoimmune Diseases of the Nervous System; Cells, Cultured; Coculture Techniques; Cytokines; Dinoprostone; Down-Regulation; Drug Synergism; Encephalitis; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-1; Lovastatin; Mevalonic Acid; Microglia; Polyisoprenyl Phosphates; Prostaglandins; Rats; Sesquiterpenes

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi. The protein (T. cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T. cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. Heterologous expression of TcFPPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth. The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents.

Topics: Alkyl and Aryl Transferases; Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Birds; Blotting, Northern; Blotting, Southern; Calcium Channel Blockers; Cations; Cells, Cultured; Cloning, Molecular; Crystallography, X-Ray; Diphosphonates; Dose-Response Relationship, Drug; Escherichia coli; Etidronic Acid; Geranyltranstransferase; Hydrogen-Ion Concentration; Models, Chemical; Models, Molecular; Molecular Sequence Data; Polyisoprenyl Phosphates; Protein Binding; Recombinant Proteins; Risedronic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sesquiterpenes; Trypanosoma cruzi

New phosphorylated microbial metabolites referred to as phosphoantigens activate immune responses in humans. Although these molecules have leading applications in medical research, no direct method allows their rapid and unambiguous structural identification. Here, we interfaced online HPAEC (high performance anion-exchange chromatography) with ESI-ITMS (electrospray ionization ion trap mass spectrometry) to identify such pyrophosphorylated molecules. A self-regenerating anion suppressor located upstream of electrospray ionization enabled the simultaneous detection of pyrophosphoester by conductimetry, UV and MS. By HPAEC-ITMS and HPAEC-ITMS2, a single run permitted characterization of reference phosphoantigens and of related structures. Although all compounds were resolved by HPAEC, MS enabled their detection and identification by [M-H]- and fragment ions. Isobaric phosphoantigen analogues were also separated by HPAEC and distinguished by MS2. The relevance of this device was demonstrated for phosphoantigens analysis in human urine and plasma. Furthermore, identification of natural phosphoantigens by automatically generated 2D mass spectra from nano-ESI-ITMS is presented. This last technique permits the simultaneous performance of molecular screening of natural phosphoantigen extracts and their identification.

Topics: Antigens; Chromatography, Ion Exchange; Epoxy Compounds; Humans; Mycobacteriaceae; Organophosphorus Compounds; Phosphorylation; Polyisoprenyl Phosphates; Sesquiterpenes; Spectrometry, Mass, Electrospray Ionization

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.

Topics: Animals; Blotting, Western; Cell Culture Techniques; Decapoda; Genes, ras; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Hydrolysis; Mammals; Polyisoprenyl Phosphates; Sesquiterpenes; Transfection

The biochemical mechanism(s) by which Nm23 proteins/nucleoside diphosphate kinases suppress tumor metastasis, inhibit cell motility, and affect cellular differentiation are not known. Here we report that Nm23 proteins can phosphorylate geranyl and farnesyl pyrophosphates to give triphosphates. Wild type Nm23-H1 had higher geranyl and farnesyl pyrophosphate kinase activities than did mutants of Nm23-H1 that do not inhibit cell motility. The phosphorylation of farnesyl pyrophosphate appears to occur in vivo as cells with an elevated level of Nm23-H1 contained more farnesyl triphosphate than did control cells. To our knowledge, this is the first report that farnesyl triphosphate exists in cells. The phosphorylation of farnesyl pyrophosphate by Nm23 proteins could alter isoprenoid metabolism, and cells with an elevated level of Nm23 proteins were found to contain more farnesylated 46- and 24-kDa proteins than did control cells. The phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins provides a novel mechanism by which these proteins might exert their biological effects.

Topics: Animals; Escherichia coli; Kinetics; Liver; Monomeric GTP-Binding Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phosphorylation; Polyisoprenyl Phosphates; Protein Prenylation; Rats; Sesquiterpenes; Time Factors; Transcription Factors

To assess the potential for feedback inhibition by isoprene intermediates in the cholesterol and nonsterol isoprene biosynthetic pathway, we expressed human cDNAs encoding mevalonate kinase (MKase), phosphomevalonate kinase (PMKase), and mevalonate diphosphate decarboxylase (MDDase) as fusion proteins in Escherichia coli DH5alpha, and purified these proteins by affinity chromatography. Several phosphorylated and non-phosphorylated isoprenes were analyzed as inhibitors of the enzymes using a standard spectrophotometric assay. Of the three proteins, only MKase was inhibited through competitive interaction at the ATP-binding site. The intermediates studied (and their relative inhibitory capacity) were: geranylgeranyl-diphosphate (GGPP, C20) > farnesyl-diphosphate (FPP, C15) > geranyl-diphosphate (GPP, C10) > isopentenyl-diphosphate (IPP, C5) > or = 3,3-dimethylallyl-diphosphate (DMAPP, C5) > farnesol (C15) > dolichol-phosphate (DP, C(80-100)). Mevalonate-diphosphate, geraniol, and dolichol were not inhibitors. Our data further define the spectrum of physiologic inhibitors of MKase, and provide the first evidence for feedback inhibition of MKase by a nonsterol isoprene produced by the branched pathway, dolichol-phosphate. These results provide additional evidence that MKase may occupy a central regulatory role in the control of cholesterol and nonsterol isoprene biosynthesis.

Topics: Carboxy-Lyases; Cholesterol; Dolichol Phosphates; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Escherichia coli; Gene Expression; Humans; Kinetics; Mevalonic Acid; Phosphotransferases (Alcohol Group Acceptor); Phosphotransferases (Phosphate Group Acceptor); Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes; Terpenes

The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48,500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.

Topics: Amino Acid Sequence; Base Sequence; Ergosterol; Genes, Fungal; Molecular Sequence Data; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Polyisoprenyl Phosphates; Promoter Regions, Genetic; Restriction Mapping; Saccharomyces cerevisiae; Sesquiterpenes

Cell-free homogenates from sage (Salvia officinalis) leaves convert dimethylallyl pyrophosphate and isopentenyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [1-14C]dimethylallyl pyrophosphate and [1-3H]isopentenyl pyrophosphate was verified as [14C,1-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradative studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the C10 precursor for the production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases.

Topics: Cell-Free System; Chromatography; Chromatography, Thin Layer; Dimethylallyltranstransferase; Hemiterpenes; Hydrogen-Ion Concentration; Organophosphorus Compounds; Plants; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes; Transferases

A prenyltransferase purified from the commercial rubber tree, Hevea brasiliensis, that elongates existing cis-polyisoprene rubber molecules also catalyzes the formation of all trans-farnesyl pyrophosphate (t,t-FPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In assays of the latter activity trans-geranyl pyrophosphate is the only other product identified. In contrast to this limited addition of IPP to DMAPP, we measured 7000 additions of isoprene per rubber molecule in a previous titration of active allylic ends of rubber molecules by purified prenyltransferase (Light, D. R., and Dennis, M. S. (1989) J. Biol. Chem. 264, 18589-18597). In order to confirm that purified prenyltransferase extensively elongates rubber molecules, doubly labeled [1-14C]isopentenyl [U-32P]pyrophosphate ([14C,32P]IPP) was synthesized. Using this reagent we show that both prenyltransferase purified from H. brasiliensis and prenyltransferase purified from avian liver (FPP synthase) add greater than 15 isoprene units to existing rubber molecules, consistent with the previous titration data. For confirmation that the prenyltransferase purified from H. brasiliensis adds isoprene units to rubber to make cis-polyisoprene, chirally tritiated [14C]IPP ([14C,2S-3H]IPP) was synthesized. Retention of the tritium label in FPP synthesized from [14C,2S-3H]IPP and DMAPP, geranyl pyrophosphate, or neryl pyrophosphate by prenyltransferase from H. brasiliensis or avian liver confirms trans addition to these substrates. In contrast, when [14C,2S-3H]IPP is incubated with serum-free rubber particles and prenyltransferase purified from H. brasiliensis, avian liver, or yeast, no tritium is incorporated into the rubber particles indicating cis addition. Thus, rubber particles have the ability to alter the stereoselective removal of the 2R-prochiral proton in favor of the removal of the 2S-prochiral proton. This apparent inversion of carbon 2 of IPP during the proton abstraction step by rubber particles represents a novel example of a switch in enzyme stereospecificity. In addition to being enzymatically similar to other prenyltransferases, rubber transferase also appears to be related immunologically to FPP synthases, since polyclonal antibodies to the H. brasiliensis prenyltransferase cross-react with the purified yeast prenyltransferase. In order to investigate potential primers of greater molecular weight than that of FPP, cis-undecaprenyl pyrophosphate (C55PP) was syn

Topics: Animals; Chickens; Dimethylallyltranstransferase; Hemiterpenes; Liver; Molecular Weight; Organophosphorus Compounds; Phosphates; Plants; Polyisoprenyl Phosphates; Rubber; Sesquiterpenes; Stereoisomerism; Substrate Specificity; Transferases; Trees

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.

Topics: Amino Acid Sequence; Chromatography; Chromatography, High Pressure Liquid; Dimethylallyltranstransferase; Dithiothreitol; Hemiterpenes; Kinetics; Latex; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Organophosphorus Compounds; Plants; Polyisoprenyl Phosphates; Rubber; Sesquiterpenes; Substrate Specificity; Transferases; Trees

Topics: Chemical Phenomena; Chemistry; Models, Molecular; Molecular Conformation; Polyisoprenyl Phosphates; Sesquiterpenes; Structure-Activity Relationship; Terpenes

Upon rehydration of lyophilized Escherichia coli cells with phosphate buffer containing [14C]isopentenyl pyrophosphate (IPP), 14C was incorporated into the cells. Radioactivity was found in ubiquinone-8, an unidentified precursor of ubiquinone-8, demethylmenaquinone-8 and phosphate esters of all-trans-octaprenol and cis, trans-polyprenols. On rehydration of the cells with the buffer containing geranyl pyrophosphate or farnesyl pyrophosphate in combination with [14C]IPP, higher radioactivity was incorporated into the above products and some radioactivity was found in free prenols. Fractionation of the 14C-labeled cells by sucrose-density gradient centrifugation before and after recultivation indicated that the size of 14C-labeled cells had changed during the recultivation. This shows that radioactivity of [14C]IPP was incorporated into live cells but not into dead cells. The metabolism of the radioactive products in the recultivated cells was examined. It was found that the unidentified precursor was converted to ubiquinone-8, but demethylmenaquinone-8 was not converted to menaquinone-8. "Lipid intermediates" in peptidoglycan synthesis increased in the logarithmic growth phase and decreased in the stationary phase. In the stationary phase, however, an increase in cis,trans-polyprenyl monophosphates was observed. These observations suggest the operation of the lipid cycle of peptidoglycan synthesis.

Topics: Carbon Radioisotopes; Cells, Cultured; Centrifugation, Density Gradient; Chromatography, Thin Layer; Escherichia coli; Freeze Drying; Hemiterpenes; Isomerism; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes

Topics: Diphosphates; Hemiterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes

Topics: Cell-Free System; Chemical Phenomena; Chemistry; Molecular Conformation; Plants; Polyisoprenyl Phosphates; Sesquiterpenes; Streptomyces; Terpenes

Topics: Adenosine Triphosphate; Animals; Cytosol; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Liver; Microsomes, Liver; NADP; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Polyisoprenyl Phosphates; Rats; Sesquiterpenes

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.

Topics: Alkenes; Bacillus subtilis; Bacitracin; Detergents; Dimethylallyltranstransferase; Farnesol; Hemiterpenes; Magnesium; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Sulfhydryl Reagents; Transferases