farnesyl-pyrophosphate and isopentenyl-pyrophosphate

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.

Topics: Alkyl and Aryl Transferases; Bacteria; Hemiterpenes; Organophosphorus Compounds; Peptidoglycan; Polyisoprenyl Phosphates; Protein Structure, Tertiary; Sesquiterpenes; Terpenes; Transferases

A group of prenyltransferases produce linear lipids by catalyzing consecutive condensation reactions of farnesyl diphosphate (FPP) with specific numbers of isopentenyl diphosphate (IPP), a common building block of isoprenoid compounds. Depending on the stereochemistry of the double bonds formed during IPP condensation, these prenyltransferases are categorized as cis- and trans-types. Undecaprenyl diphosphate synthase (UPPS) that catalyzes chain elongation of FPP by consecutive condensation reactions with eight IPP, to form C₅₅ lipid carrier for bacterial cell wall biosynthesis, serves as a model for understanding cis-prenyltransferases. In this review, the current knowledge in UPPS kinetics, mechanisms, structures, and inhibitors is summarized.

Topics: Alkyl and Aryl Transferases; Bacteria; Bacterial Proteins; Cell Wall; Dimethylallyltranstransferase; Hemiterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Structure, Tertiary; Sesquiterpenes; Structure-Activity Relationship

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

Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants.

Topics: Amino Acid Sequence; Eucommiaceae; Geranyltranstransferase; Hemiterpenes; Kinetics; Models, Molecular; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sequence Homology, Amino Acid; Sesquiterpenes; Substrate Specificity

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

Combinatorial design is an effective strategy to acquire the optimal solution in complex systems. In this study, the combined effects of pathway combination, promoters' strength fine-tuning, copy numbers and integration locus variations caused by δ-integration were explored in Saccharomyces cerevisiae using geranylgeraniol (GGOH) production as an example. Two GGOH biosynthetic pathway branches were constructed. In branch 1, GGOH was converted from isopentenyl pyrophosphate (IPP) and farnesyl diphosphate (FPP). In branch 2, GGOH was derived directly from IPP and dimethylallyl pyrophosphate (DMAPP). Regulated by 10 combinations of 11 diverse promoters, a fusion gene BTS1-ERG20, a heterologous geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius (GGPPSsa) and an endogenous N-terminal truncated gene 3-hydroxyl-3-methylglutaryl-CoA reductase isoenzyme 1 (tHMGR), were incorporated into yeast by δ-integration, leading to a series of GGOH producing strains with yields ranging from 18.45 mg/L to 161.82 mg/L. The yield was further increased to 437.52 mg/L by optimizing the fermentation medium. Consequently, the GGOH yield reached 1315.44 mg/L in a 5-L fermenter under carbon restriction strategy. Our study not only opens large opportunities for downstream diterpenes overproductions, but also demonstrates that pathway optimization based on combinatorial design is a promising strategy to engineer microbes for overproducing natural products with complex structure.

Topics: Bacterial Proteins; Biosynthetic Pathways; Diterpenes; Farnesyltranstransferase; Hemiterpenes; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; Metabolic Engineering; Organophosphorus Compounds; Polyisoprenyl Phosphates; Promoter Regions, Genetic; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sesquiterpenes

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

In vitro synthesis of chemicals and pharmaceuticals using enzymes is of considerable interest as these biocatalysts facilitate a wide variety of reactions under mild conditions with excellent regio-, chemo- and stereoselectivities. A significant challenge in a multi-enzymatic reaction is the need to optimize the various steps involved simultaneously so as to obtain high-yield of a product. In this study, statistical experimental design was used to guide the optimization of a total synthesis of amorpha-4,11-diene (AD) using multienzymes in the mevalonate pathway. A combinatorial approach guided by Taguchi orthogonal array design identified the local optimum enzymatic activity ratio for Erg12:Erg8:Erg19:Idi:IspA to be 100∶100∶1∶25∶5, with a constant concentration of amorpha-4,11-diene synthase (Ads, 100 mg/L). The model also identified an unexpected inhibitory effect of farnesyl pyrophosphate synthase (IspA), where the activity was negatively correlated with AD yield. This was due to the precipitation of farnesyl pyrophosphate (FPP), the product of IspA. Response surface methodology was then used to optimize IspA and Ads activities simultaneously so as to minimize the accumulation of FPP and the result showed that Ads to be a critical factor. By increasing the concentration of Ads, a complete conversion (∼100%) of mevalonic acid (MVA) to AD was achieved. Monovalent ions and pH were effective means of enhancing the specific Ads activity and specific AD yield significantly. The results from this study represent the first in vitro reconstitution of the mevalonate pathway for the production of an isoprenoid and the approaches developed herein may be used to produce other isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) based products.

Topics: Alkyl and Aryl Transferases; Geranyltranstransferase; Hemiterpenes; Mevalonic Acid; Organophosphorus Compounds; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Research Design; Sesquiterpenes

Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8 Å resolution and refined to 2.0 Å resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg(2+) mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; Helicobacter pylori; Hemiterpenes; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Sesquiterpenes

Rubber biosynthesis in plants is a fascinating biochemical system, which evolved at the dawn of the dicotyledoneae and is present in at least four of the dictolydonous superorders. Rubber biosynthesis is catalyzed by a membrane complex in a monolayer membrane envelope, requires two distinct substrates and a divalent cation cofactor, and produces a high-molecular-weight isoprenoid polymer. A solid understanding of this system underpins valuable papers in the literature. However, the published literature is rife with unreliable reports in which the investigators have fallen into traps created by the current incomplete understanding of the biochemistry of rubber synthesis. In this chapter, we attempt to guide both new and more established researchers around these pitfalls.

Topics: Animals; Asteraceae; Enzyme Activation; Enzyme Assays; Enzyme Stability; Hemiterpenes; Hevea; Immunoprecipitation; Kinetics; Latex; Molecular Weight; Organophosphorus Compounds; Photoaffinity Labels; Plant Bark; Plant Proteins; Polyisoprenyl Phosphates; Rubber; Sesquiterpenes; Transferases

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

Topics: Arabidopsis; Carbon-Carbon Double Bond Isomerases; Carboxy-Lyases; Catharanthus; Hemiterpenes; Organophosphorus Compounds; Peroxisomes; Phosphotransferases (Phosphate Group Acceptor); Plant Proteins; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Hevea brasiliensis is one of few higher plants producing the commercial natural rubber used in many significant applications. The biosynthesis of high molecular weight rubber molecules by the higher plants has not been clarified yet. Here, the in vitro rubber biosynthesis was performed by using enzymatically active small rubber particles (SRP) from Hevea. The mechanism of the in vitro rubber synthesis was investigated by the molecular weight distribution (MWD). The highly purified SRP prepared by gel filtration and centrifugation in the presence of Triton((R)) X-100 showed the low isopentenyl diphosphate (IPP) incorporation for the chain extension mechanism of pre-existing rubber. The MWD of in vitro rubber elongated from the pre-existing rubber chains in SRP was analyzed for the first time in the case of H. brasiliensis by incubating without the addition of any initiator. The rubber transferase activity of 70% incorporation of the added IPP (w/w) was obtained when farnesyl diphosphate was present as the allylic diphosphate initiator. The in vitro synthesized rubber showed a typical bimodal MWD of high and low molecular weight fractions in GPC analysis, which was similar to that of the in vivo rubber with peaks at around 10(6) and 10(5) Da or lower. The reaction time independence and dependence of molecular weight of high and low molecular weight fractions, respectively, indicated that the high molecular weight rubber was synthesized from the chain extension of pre-existing rubber molecules whereas the lower one was from the chain elongation of rubber molecules newly synthesized from the added allylic substrates.

Topics: Hemiterpenes; Hevea; Molecular Weight; Organophosphorus Compounds; Plant Proteins; Polyisoprenyl Phosphates; Rubber; Sesquiterpenes

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.

Topics: Culture Media; Escherichia coli; Farnesol; Gene Dosage; Gene Expression; Geranyltranstransferase; Hemiterpenes; Metabolic Networks and Pathways; Mevalonic Acid; Organophosphorus Compounds; Plasmids; Polyisoprenyl Phosphates; Sesquiterpenes

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C(55) product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-(2)H(2)]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985±0.022 measured for UPPS reaction using [1,1-(2)H(2)]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.

Topics: Alkyl and Aryl Transferases; Catalysis; Escherichia coli; Hemiterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C(40) and C(55) products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.

Topics: Alkyl and Aryl Transferases; Chromatography, Thin Layer; Diphosphates; Hemiterpenes; Organophosphates; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases

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

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.

Topics: Alkyl and Aryl Transferases; Diterpenes; Escherichia coli; Gene Deletion; Genes, Bacterial; Geranyltranstransferase; Hemiterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

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

The crystal structure of the geranylgeranyl diphosphate synthase from Sinapis alba (mustard) has been solved in two crystal forms at 1.8 and 2.0 A resolutions. In one of these forms, the dimeric enzyme binds one molecule of the final product geranylgeranyl diphosphate in one subunit. The chainfold of the enzyme corresponds to that of other members of the farnesyl diphosphate synthase family. Whereas the binding modes of the two substrates dimethylallyl diphosphate and isopentenyl diphosphate at the allyl and isopentenyl sites, respectively, have been established with other members of the family, the complex structure presented reveals for the first time the binding mode of a reaction product at the isopentenyl site. The binding geometry of substrates and product in conjunction with the protein environment and the established chemistry of the reaction provide a clear picture of the reaction steps and atom displacements. Moreover, a comparison with a ligated homologous structure outlined an appreciable induced fit: helix alpha8 and its environment undergo a large conformational change when either the substrate dimethylallyl diphosphate or an analogue is bound to the allyl site; only a minor conformational change occurs when the other substrate isopentenyl diphosphate or the product is bound to the isopentenyl site.

Topics: Binding Sites; Catalysis; Crystallography, X-Ray; Diterpenes; Escherichia coli; Farnesyltranstransferase; Hemiterpenes; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Sinapis; Substrate Specificity

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

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Aspartic Acid; Binding Sites; Catalysis; Cell Wall; Conserved Sequence; Crystallization; Crystallography, X-Ray; Escherichia coli; Hemiterpenes; Hydrogen Bonding; Kinetics; Magnesium; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Organophosphorus Compounds; Peptidoglycan; Polyisoprenyl Phosphates; Protein Folding; Sequence Alignment; Sesquiterpenes; Structure-Activity Relationship

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4'-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.

Topics: Bacterial Proteins; Biotechnology; Carotenoids; Cloning, Molecular; Escherichia coli; Escherichia coli Proteins; Farnesyl-Diphosphate Farnesyltransferase; Geranyltranstransferase; Hemiterpenes; Kinetics; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.

Topics: Alkyl and Aryl Transferases; Binding Sites; Catalysis; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Hemiterpenes; Magnesium; Models, Chemical; Octoxynol; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Binding; Protein Structure, Tertiary; Sesquiterpenes; Sulfates

We have identified an omega,E,E-farnesyl diphosphate (omega,E,E-FPP) synthase, encoded by the open reading frame Rv3398c, from Mycobacterium tuberculosis that is unique among reported FPP synthases in that it does not contain the type I (eukaryotic) or the type II (eubacterial) omega,E,E-FPP synthase signature motif. Instead, it has a structural motif similar to that of the type I geranylgeranyl diphosphate synthase found in Archaea. Thus, the enzyme represents a novel class of omega,E,E-FPP synthase. Rv3398c was cloned from the M. tuberculosis H37Rv genome and expressed in Mycobacterium smegmatis using a new mycobacterial expression vector (pVV2) that encodes an in-frame N-terminal affinity tag fusion with the protein of interest. The fusion protein was well expressed and could be purified to near homogeneity, allowing facile kinetic analysis of recombinant Rv3398c. Of the potential allylic substrates tested, including dimethylallyl diphosphate, only geranyl diphosphate served as an acceptor for isopentenyl diphosphate. The enzyme has an absolute requirement for divalent cation and has a K(m) of 43 microM for isopentenyl diphosphate and 9.8 microM for geranyl diphosphate and is reported to be essential for the viability of M. tuberculosis.

Topics: Alkyl and Aryl Transferases; Butanols; Chromatography, Thin Layer; Geranyltranstransferase; Hemiterpenes; Kinetics; Molecular Structure; Mycobacterium tuberculosis; Organophosphorus Compounds; Phylogeny; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes; Stereoisomerism; Substrate Specificity

The genes coding for all the enzymes involved in the conversion of acetyl-CoA to farnesyl diphosphate (FPP) in the zeaxanthin-producing bacterium Paracoccus zeaxanthinifaciens were cloned and characterized. Two genes encoding enzymes catalysing the condensation of two acetyl-CoA molecules to acetoacetyl-CoA were found. The six enzymes involved in the conversion of acetyl-CoA and acetoacetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are grouped in an operon, designated the mevalonate operon. The gene encoding the enzyme catalysing two consecutive condensations, IPP and DMAPP to geranyl diphosphate (GPP) and IPP and GPP to FPP, is not clustered with any other gene encoding an enzyme of the isoprenoid pathway. Genes encoding enzymes involved in the biosynthesis of poly-hydroxyalkanoate and non-carotenoid isoprenoids found in P. zeaxanthinifaciens are also presented.

Topics: Acetyl Coenzyme A; Acyl Coenzyme A; Amino Acid Sequence; Bacterial Proteins; Carbon-Carbon Double Bond Isomerases; DNA, Bacterial; Hemiterpenes; Hydroxymethylglutaryl CoA Reductases; Membrane Proteins; Mevalonic Acid; Molecular Sequence Data; Organophosphorus Compounds; Paracoccus; Polyisoprenyl Phosphates; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sesquiterpenes; Terpenes

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s(-1)) was 100 times larger than the steady-state rate (0.02 s(-1)). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s(-1)) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

Topics: Alkyl and Aryl Transferases; Enzyme Activation; Escherichia coli; Hemiterpenes; Hydrogen-Ion Concentration; Kinetics; Organophosphorus Compounds; Polyethylene Glycols; Polyisoprenyl Phosphates; Sesquiterpenes; Temperature

Human Vgamma2Vdelta2(+) T cells proliferate in vivo during many microbial infections. We have found that Vgamma2Vdelta2(+) T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vgamma2Vdelta2(+) clones and a transfectant responded similarly. Ag docking into a Vgamma2Vdelta2 TCR model reveals a potential binding site in germline regions of the Vgamma2Jgamma1.2 CDR3 and Vdelta2 CDR2 loops. Thus, Vgamma2Vdelta2(+) T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.

Topics: Adult; Antigens; Binding Sites; Cell Line; Clone Cells; Diphosphates; Epitopes, T-Lymphocyte; Hemiterpenes; Humans; Jurkat Cells; Organophosphorus Compounds; Polyisoprenyl Phosphates; Receptors, Antigen, T-Cell, gamma-delta; Sesquiterpenes; T-Lymphocyte Subsets; Transfection

Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPP(i)) was prepared from trimethyl phosphate in four steps. The tris(tetra-n-butylammonium) salt was then used to convert isopentenyl tosylate to (S)-isopentenyl thiodiphosphate (ISPP). (S)-Dimethylallyl (DMASPP), (S)-geranyl (GSPP), (S)-farnesyl (FSPP), and (S)-geranylgeranyl thiodiphosphate (GGSPP) were prepared from the corresponding bromides in a similar manner. ISPP and GSPP were substrates for avian farnesyl diphosphate synthase (FPPase). Incubation of the enzyme with ISPP and GPP gave FSPP, whereas incubation with IPP and GSPP gave FPP. GSPP was a substantially less reactive than GPP in the chain elongation reaction and was an excellent competitive inhibitor, K(I)(GSPP) = 24.8 microM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.

Topics: Animals; Birds; Carbon Radioisotopes; Enzyme Inhibitors; Hemiterpenes; Kinetics; Organophosphorus Compounds; Polyisoprenyl Phosphates; Pyrophosphatases; Sesquiterpenes

Natural rubber was extracted from the fig tree (Ficus carica) cultivated in Korea as part of a survey of rubber producing plants. Fourier transform infrared and (13)C nuclear magnetic resonance analysis of samples prepared by successive extraction with acetone and benzene confirmed that the benzene-soluble residues are natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of fig tree was about 4%, whereas the rubber content in the bark, leaf, and fruit was 0.3%, 0.1%, and 0.1%, respectively. Gel-permeation chromatography revealed that the molecular size of the natural rubber from fig tree is about 190 kD. Similar to rubber tree (Hevea brasiliensis) and guayule (Parthenium argentatum Gray), rubber biosynthesis in fig tree is tightly associated with rubber particles. The rubber transferase in rubber particles exhibited a higher affinity for farnesyl pyrophosphate than for isopentenyl pyrophosphate, with apparent K(m) values of 2.8 and 228 microM, respectively. Examination of latex serum from fig tree by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major proteins of 25 and 48 kD in size, and several proteins with molecular mass below 20 and above 100 kD. Partial N-terminal amino acid sequencing and immunochemical analyses revealed that the 25- and 48-kD proteins were novel and not related to any other suggested rubber transferases. The effect of EDTA and Mg(2+) ion on in vitro rubber biosynthesis in fig tree and rubber tree suggested that divalent metal ion present in the latex serum is an important factor in determining the different rubber biosynthetic activities in fig tree and rubber tree.

Topics: Cations, Divalent; Chromatography, Gel; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Euphorbiaceae; Hemiterpenes; Latex; Magnesium; Magnetic Resonance Spectroscopy; Molecular Weight; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rosales; Rubber; Sesquiterpenes; Spectroscopy, Fourier Transform Infrared

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.

Topics: Alkyl and Aryl Transferases; Buffers; Computer Simulation; Escherichia coli; Hemiterpenes; Kinetics; Octoxynol; Organophosphorus Compounds; Polyisoprenyl Phosphates; Recombinant Fusion Proteins; Sesquiterpenes; Software; Substrate Specificity

Callus cultures of the hornwort Anthoceros punctatus were induced from the apical portions of the gametophytes. Calli can accumulate rosmarinic acid, which is suggested as an intermediate for anthocerotonic acid, a rare phenylpropanoid dimer with a cyclobutane ring, indicating that calli possess the ability to produce secondary metabolites found primarily in intact plants. Biosynthesis of chloroplastidic terpenoids of liverworts showed preferential labeling of the farnesyl diphosphate (FPP)-derived portion in the phytyl side chain of chlorophyll a (1) when calli of A. punctatus are incubated with (2)H- and (13)C-labeled mevalonate. This finding suggests either that cytoplasmic FPP (or isopentenyl diphosphate, IPP) is taken into chloroplasts and condensed with endogenous IPP derived from a nonmevalonate pathway, or that FPP is synthesized within chloroplasts from extraplastidically formed IPP (or mevalonate) and then condensed with endogenous IPP in a different subplastidic fraction.

Topics: Antioxidants; Chlorophyll; Chlorophyll A; Chromatography, Agarose; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cinnamates; Depsides; Hemiterpenes; Isotope Labeling; Magnetic Resonance Spectroscopy; Mevalonic Acid; Optical Rotation; Organophosphorus Compounds; Plants; Polyisoprenyl Phosphates; Rosmarinic Acid; Sesquiterpenes

Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638-14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate. However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50). These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Bacillus subtilis; Catalysis; Dimerization; Dimethylallyltranstransferase; Hemiterpenes; Kinetics; Micrococcus luteus; Molecular Sequence Data; Mutagenesis, Site-Directed; Organophosphorus Compounds; Peptide Fragments; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes

Alendronate (ALN), an aminobisphosphonate compound used for the treatment of osteoporosis and other disorders of bone resorption, has been suggested to act by inhibition of the formation of GGPP. In the present study we used an S(10) homogenate fraction of rat liver to show that ALN causes a dose-dependent inhibition of [(3)H]MVA incorporation into sterols and a concomitant increase in incorporation of radiolabel into IPP and DMAPP. We further show that ALN is a potent inhibitor of cytosolic trans-prenyltransferase (FPP synthase). The inhibition is competitive with respect to allylic pyrophosphate substrates, but not IPP, suggesting that ALN acts as an allylic pyrophosphate analog and binds to the free enzyme. The K(i) is in the 0.5 microM range.

Topics: Alendronate; Alkyl and Aryl Transferases; Animals; Chromatography, High Pressure Liquid; Cytosol; Geranyltranstransferase; Hemiterpenes; Kinetics; Liver; Male; Mevalonic Acid; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes

Pravastatin, a HMG-CoA reductase inhibitor was found to inhibit DNA synthesis of vascular smooth muscle cells (VSMC) in a dose-dependent manner. Flow cytometric analysis demonstrated that pravastatin induced G1 arrest. Mevalonate restored the inhibitory effect of pravastatin on DNA synthesis and on cell cycle progression, suggesting the importance of mevalonate itself and/or its metabolites in VSMC proliferation. The major intermediate metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) and IPP (isopentenyl pyrophosphate) were prepared in the form of liposomes, and the effects of GGPP, FPP and IPP on pravastatin induced inhibition of VSMC proliferation and G1 arrest were examined. Only GGPP restored the pravastatin-induced inhibition of DNA synthesis and G1 arrest. Pravastatin inhibited translocation of Rho small GTPase from cytosol to membrane. By the addition of GGPP, Rho small GTPase are geranylgeranylated and translocated to membranes during G1/S transition. These data suggest that GGPP, rather than FPP or IPP, is an essential metabolite among mevalonic acid metabolites for VSMC proliferation and the G1/S transition.

Topics: Animals; Biological Transport; Cell Division; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; GTP-Binding Proteins; Hemiterpenes; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunoblotting; Liposomes; Membrane Proteins; Muscle, Smooth, Vascular; Organophosphorus Compounds; Polyisoprenyl Phosphates; Pravastatin; Rats; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; S Phase; Sesquiterpenes

We have used the potent squalene synthase inhibitor squalestatin I to investigate the regulation of isoprenoid metabolism in rat liver Fresh-frozen liver pieces from normal rats and rats infused with squalestatin I at 16 micrograms h-1 for 16 h were assayed for farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) by HPLC after dephosphorylation. Levels of FPP and GGPP were 5.4 +/- 1.6 nmol g-1 and 1.6 +/- 0.7 nmol g-1 (n = 13) wet wt., respectively, in control livers and 110 + 41 nmol g-1 and 3.0 +/- 2.2 nmol g-1 (n = 13) in livers from squalestatin I infused rats. In order to determine the relative level of isopentenyl pyrophosphate, liver slices from normal and squalestatin I infused rats were labeled to steady-state with [3H]acetate. Analysis of isoprenoid pyrophosphate intermediates by radio-HPLC after dephosphorylation indicated that squalestatin I brought about a 20-fold increase in the relative level of FPP (confirming direct analysis) and a 5-fold increase in the relative level of IPP. No change in either of these compounds was observed in livers from cholesterol-fed rats. To determine if squalestatin I altered the synthesis of nonsterol products, rats were subjected to long term subcutaneous infusion. After 14 days of infusion of 15 micrograms h-1, the median chain length of hepatic dolichol and dolichyl phosphate increased from C95 to C115 and the levels of these lipids increased approximately 3-fold. In addition, dolichyl phosphate mannose synthase activity in microsomes from squalestatin I treated rats was increased relative to controls when assayed in the absence of dolichyl phosphate. Squalestatin I affected ubiquinone metabolism to a lesser extent: chain lengths shifted from a Q10/Q9 ratio of 0.118 +/- 0.021 in the normal rat to 0.185 +/- 0.016 in the squalestatin I treated animals, and levels rose by approximately 90%. These results suggest that the isoprenoid pyrophosphate intermediates are shared by the cholesterol, dolichol and ubiquinone pathways and further show that the dolichol and ubiquinone pathways are not saturated. Apparently, under normal conditions, the levels of these intermediates are maintained relatively constant by coordinate enzyme regulation, thereby ensuring a constant rate of synthesis of nonsterols.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Hemiterpenes; Liver; Male; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Tricarboxylic Acids

A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39,689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Arabidopsis; Base Sequence; Cloning, Molecular; DNA, Complementary; Ergosterol; Genetic Complementation Test; Geranyltranstransferase; Hemiterpenes; Molecular Sequence Data; Organophosphorus Compounds; Plant Proteins; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Sesquiterpenes; Transferases

Recombinant yeast isopentenyl diphosphate (IPP) isomerase and avian farnesyl diphosphate (FPP) synthase from overproducing strains of Escherichia coli were used to synthesize FPP from IPP and dimethylallyl diphosphate (DMAPP). [2,4,5-13C3]IPP and [2,4,5-13C3]DMAPP were synthesized from ethyl [2-13C]bromoacetate and [1,3-13C2]acetone. Thes compounds were used as substrates for enzymatic synthesis of FPP selectivity labeled at the first or third isoprene residue or at all three.

Topics: Alkyl and Aryl Transferases; Animals; Birds; Carbon Isotopes; Carbon-Carbon Double Bond Isomerases; Escherichia coli; Farnesyltranstransferase; Hemiterpenes; Indicators and Reagents; Isomerases; Isotope Labeling; Magnetic Resonance Spectroscopy; Organophosphorus Compounds; Polyisoprenyl Phosphates; Recombinant Proteins; Saccharomyces cerevisiae; Sesquiterpenes; Transferases

When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.

Topics: Animals; Brain; Chromatography, High Pressure Liquid; Dimethylallyltranstransferase; Hemiterpenes; Liver; Male; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes

Geranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphosphate being 14 and 0.8 microM, respectively. Dimethylallyl diphosphate and geranyl diphosphate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.

Topics: Alkyl and Aryl Transferases; Animals; Brain; Cattle; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Farnesyltranstransferase; Hemiterpenes; Isoelectric Focusing; Kinetics; Magnesium; Manganese; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases

The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.

Topics: Animals; Chromatography, Thin Layer; Farnesyl-Diphosphate Farnesyltransferase; Female; Hemiterpenes; Liver; NADP; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Squalene

The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo.

Topics: Animals; Carbon-Carbon Double Bond Isomerases; Cell-Free System; Chromatography, High Pressure Liquid; Feedback; Hemiterpenes; Isomerases; Mevalonic Acid; Organophosphorus Compounds; Polyisoprenyl Phosphates; Proteins; Rabbits; Sesquiterpenes

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.

Topics: Animals; Brain; Cations, Divalent; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cytosol; Hemiterpenes; Isomerism; Kidney; Kinetics; Liver; Male; Organ Specificity; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Inbred Strains; Sesquiterpenes; Spleen; Testis; Tritium

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.

Topics: Animals; Cations, Divalent; Detergents; Dimethylallyltranstransferase; Dolichols; Hemiterpenes; Intracellular Membranes; Kinetics; Male; Microsomes, Liver; Octoxynol; Organophosphorus Compounds; Polyethylene Glycols; Polyisoprenyl Phosphates; Rats; Rats, Inbred Strains; Sesquiterpenes

The post-translational processing of the yeast a-mating pheromone precursor, Ras proteins, nuclear lamins, and some subunits of trimeric G proteins requires a set of complex modifications at their carboxyl termini. This processing includes three steps: prenylation of a cysteine residue, proteolytic processing, and carboxymethylation. In the yeast Saccharomyces cerevisiae, the product of the DPR1-RAM1 gene participates in this type of processing. Through the use of an in vitro assay with peptide substrates modeled after a presumptive a-mating pheromone precursor, it was discovered that mutations in DPR1-RAM1 cause a defect in the prenylation reaction. It was further shown that DPR1-RAM1 encodes an essential and limiting component of a protein prenyltransferase. These studies also implied a fixed order of the three processing steps shared by prenylated proteins: prenylation, proteolysis, then carboxymethylation. Because the yeast protein prenyltransferase could also prenylate human H-ras p21 precursor, the human DPR1-RAM1 analogue may be a useful target for anticancer chemotherapy.

Topics: Amino Acid Sequence; Cell Compartmentation; Cholesterol; Dimethylallyltranstransferase; DNA Mutational Analysis; Fungal Proteins; Genes, Fungal; Hemiterpenes; Humans; In Vitro Techniques; Mating Factor; Molecular Sequence Data; Oncogene Protein p21(ras); Organophosphorus Compounds; Peptides; Polyisoprenyl Phosphates; Protein Processing, Post-Translational; Restriction Mapping; Saccharomyces cerevisiae; Sesquiterpenes; Transferases

Products of the isoprenoid metabolism were identified upon incubations of extracts from Plasmodium falciparum infected red blood cells with [14C] mevalonate. Uninfected erythrocytes and wild type yeast Saccharomyces cerevisiae extracts were used as controls. In parasitized red blood cells as well as in yeast extracts, mevalonate was converted into the biosynthetic isoprenoid precursors of sterol pathway until farnesyl pyrophosphate. In contrast, no mevalonate conversion was observed in uninfected erythrocyte extracts. The isoprenoid metabolism appeared stage-dependent as shown by the increase of radiolabelled farnesyl pyrophosphate amount at the beginning of the schizogonic phase (30-36 hours).

Topics: Acetates; Animals; Cells, Cultured; Erythrocytes; Hemiterpenes; Humans; Malaria; Mevalonic Acid; Organophosphorus Compounds; Plasmodium falciparum; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sesquiterpenes

Hexaprenyl-diphosphate synthase from Micrococcus luteus B-P 26 has been shown to comprise two essential components, designated as components A and B. Treatment of the synthase with sulphydryl reagents (N-ethylmaleimide, iodoacetamide or p-chloromercuribenzoate) or arginine-specific reagents (2,3-butanedione, 1,2-cyclohexanedione or phenylglyoxal) resulted in a rapid loss of the component B activity. In contrast, component A was resistant to treatment with such reagents, retaining the initial activity almost completely. Farnesyl diphosphate, isopentenyl diphosphate, farnesyl monophosphate and inorganic pyrophosphate protected the synthase against the inactivation by N-ethylmaleimide, farnesyl diphosphate being the most effective. The presence of Mg2+ was essential for the protection by isopentenyl diphosphate and inorganic pyrophosphate. For protection of the synthase activity against the inactivation by 2,3-butanedione, the presence of farnesyl diphosphate, isopentenyl diphosphate and Mg2+ was more effective than that of the individual substrates and Mg2+. Inorganic pyrophosphate provided substantial protection. In the absence of component A, the component B activity was not protected by any substrates or its analogue. These results suggest that the catalytic site of the synthase is formed by cooperative interaction between components A and B, and that cysteine and arginine residues on component B play important roles in the synthase activity.

Topics: Aldehydes; Alkyl and Aryl Transferases; Arginine; Butanones; Chloromercuribenzoates; Cyclohexanes; Cyclohexanones; Diacetyl; Dimethylallyltranstransferase; Diphosphates; Enzyme Activation; Ethylmaleimide; Hemiterpenes; Iodoacetamide; Magnesium; Micrococcus; Organophosphorus Compounds; p-Chloromercuribenzoic Acid; Phenylglyoxal; Polyisoprenyl Phosphates; Sesquiterpenes; Sulfhydryl Reagents; Transferases

Formation of a stable complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26, which represents the catalytically active state of this enzyme, is observed in the presence of a relatively high concentrations of inorganic pyrophosphate or one of the substrates, isopentenyl diphosphate or farnesyl diphosphate. The apparent molecular mass of the complex is estimated to be about 50 kDa by gel filtration with Superose 12.

Topics: Alkyl and Aryl Transferases; Catalysis; Chromatography, Gel; Dimethylallyltranstransferase; Diphosphates; Enzyme Stability; Hemiterpenes; Macromolecular Substances; Micrococcus; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.

Topics: Alkyl and Aryl Transferases; Chromatography, High Pressure Liquid; Chromosome Mapping; Escherichia coli; Geranyltranstransferase; Hemiterpenes; Mutation; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Temperature; Transferases; Ubiquinone

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

Using improved conditions with rat liver microsomes in the presence of 20% glycerol and 2% Triton X-100 at pH 8.5 it was shown that dehydrodolichyl diphosphate and dehydrodolichyl phosphate were synthesized from isopentenyl diphosphate and farnesyl diphosphate. Small amounts of geranylgeranyl diphosphate and geranylgeranyl phosphate were also formed. The carbon chain lengths of the dehydrodolichyl diphosphate and dehydrodolichyl phosphate were identical (C80-C85). A kinetic study showed that dehydrodolichyl diphosphate formed from farnesyl diphosphate and isopentenyl diphosphate was subsequently hydrolyzed to dehydrodolichyl phosphate. As the concentration of isopentenyl diphosphate was increased from 1 to 50 microM, the chain-length distribution of dehydrodolichyl products shifted from C75-C80 to C80-C85. Addition of MgCl2 into the assay mixture decreased product formation, but did not affect the chain-length distribution (C80-C85). The shift of the chain-length distribution to the same as that observed in naturally occurring dolichol derivatives (C90-C95) was observed when Triton X-100 was omitted from the assay mixture, although deletion of the detergent decreased the enzyme activity. These results, which provide insight into optimal conditions for enzymatic synthesis of the dolichol chain, are discussed in the context of the in vivo pathway for dolichol biosynthesis.

Topics: Animals; Dolichol Phosphates; Hemiterpenes; Kinetics; Magnesium; Magnesium Chloride; Male; Microsomes, Liver; Octoxynol; Organophosphorus Compounds; Polyethylene Glycols; Polyisoprenyl Phosphates; Rats; Rats, Inbred Strains; Sesquiterpenes

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

Topics: Animals; Cholesterol; Hemiterpenes; Liver; Mice; Mice, Inbred Strains; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rats; Rats, Inbred Strains; Scintillation Counting; Sesquiterpenes

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

Decaprenyl pyrophosphate synthetase which catalyzes the synthesis of all-trans-decaprenyl pyrophosphate from isopentenyl pyrophosphate and either farnesyl pyrophosphate or geranylgeranyl pyrophosphate has been partially purified from mitochondria of pig liver. This enzyme lacks dimethylallyl-transferring and geranyl-transferring activities.

Topics: Alkyl and Aryl Transferases; Animals; Hemiterpenes; Mitochondria, Liver; Organophosphorus Compounds; Polyisoprenyl Phosphates; Sesquiterpenes; Swine; Transferases

Topics: Acetates; Alkenes; Androstenedione; Animals; Chemical Phenomena; Chemistry; Cholesterol; Dehydroepiandrosterone; Equilin; Estrogens; Farnesol; Female; Hemiterpenes; Horses; Maternal-Fetal Exchange; Mevalonic Acid; Organophosphorus Compounds; Polyisoprenyl Phosphates; Pregnancy; Pregnancy, Animal; Sesquiterpenes; Squalene; Tyrosine

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