geranyl-pyrophosphate and 3-3-dimethylallyl-pyrophosphate

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

In this study, nerol was biosynthesized in the metabolic engineered Escherichia coli from glucose for the first time. Firstly, the truncated neryl diphosphate synthase gene tNDPS1 was expressed that catalyzes isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form neryl diphosphate (NPP), and then the nerol synthase gene GmNES was co-expressed to synthesize the final product nerol from NPP. The engineered strain LZ001 accumulated 0.053 ± 0.015 mg/L of nerol. Secondly, the IDI1, MVD1, ERG8, ERG12, tHMG1 and ERG13 were co-expressed to increase the supply of IPP and DMAPP. Finally, the heterologous ERG10 gene was overexpressed, and the recombinant strain LZ005 produced 1.564 ± 0.102 mg/L of nerol in shaking-flask culture, which represents a 29.51-fold increase over LZ001 strain. This study shows the novel method for the biosynthesis of nerol and provides new metabolic engineering strategy for the production of terpenoids.

Topics: Acyclic Monoterpenes; Escherichia coli; Glucose; Hemiterpenes; Metabolic Engineering; Organophosphorus Compounds; Polyisoprenyl Phosphates; 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

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

Topics: Acyclic Monoterpenes; Cytosol; Diphosphates; Diterpenes; Geranyltranstransferase; Hemiterpenes; Mitochondria; Monoterpenes; Nicotiana; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Picea; Plant Proteins; Plants, Genetically Modified; Plastids; Terpenes; Valerian

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

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

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

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

Topics: Alkyl and Aryl Transferases; Biosynthetic Pathways; Cloning, Molecular; Cyclohexane Monoterpenes; Dimethylallyltranstransferase; Diphosphates; Diterpenes; DNA, Complementary; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Plant; Gene Library; Hemiterpenes; Kinetics; Molecular Sequence Data; Molecular Structure; Monoterpenes; Organophosphorus Compounds; Plant Epidermis; Plant Proteins; Polyisoprenyl Phosphates; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Solanum lycopersicum; Substrate Specificity

Topics: Alkyl and Aryl Transferases; Dimethylallyltranstransferase; Hemiterpenes; Monoterpenes; Organophosphorus Compounds; Plant Epidermis; Plant Proteins; Polyisoprenyl Phosphates; Solanum lycopersicum; Substrate Specificity

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

A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K(m) value for DMAPP was determined to be 650 microM. Due to substrate inhibition, K(m) values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca(2+) enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C--C bonds.

Topics: Aspergillus fumigatus; Carbon; Chromatography, High Pressure Liquid; Cloning, Molecular; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Escherichia coli; Hemiterpenes; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Chemical; Molecular Sequence Data; Organophosphorus Compounds; Substrate Specificity; Tryptophan

Rat geranylgeranyl diphosphate synthase (GGPS) and its deletion mutants from the carboxyl terminus were analysed using Escherichia coli harbouring pACYC-crtIB, which contains crtI and crtB encoding the carotenoid biosynthetic enzymes. Mutants (delta-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (delta-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (delta-4, -8, -12 and -16) were overexpressed in E. coli BL21 (DE3) and purified in a stable form by nickel affinity chromatography except for one mutant (delta-16). The farnesyl-transferring activities of wild-type GGPS, delta-4, -8 and -12 mutants were relatively in a ratio of 1.0, 0.84, 0.26 and 0.0015. Each Km value of the four recombinants were estimated to be 0.71, 2.0 2.8 and 55 microM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 microM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that delta-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and delta-4 mutant. These results suggest that the KMFTEENE residing on the carboxyl-terminal sequence of GGPS stabilizes the active region involved in the substrate binding and catalysis.

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalysis; Diphosphates; Diterpenes; Enzyme Stability; Farnesyltranstransferase; Hemiterpenes; Humans; Mice; Molecular Sequence Data; Organophosphorus Compounds; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Proteins; Substrate Specificity

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His(6)-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K(m) values were 67 microM for DMAPP, and 137 microM for l-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca(2+) enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates l-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

Topics: Alkyl and Aryl Transferases; Aspergillus fumigatus; Cloning, Molecular; Coenzymes; Diphosphates; Diterpenes; DNA, Fungal; Escherichia coli; Gene Expression; Hemiterpenes; Magnetic Resonance Spectroscopy; Mass Spectrometry; Metals; Molecular Sequence Data; Multigene Family; Organophosphorus Compounds; Prenylation; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Tryptophan

Analogs of dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) were prepared and tested as potential substrates of prenyltransferase of the tobacco hornworm, Manduca sexta, and of a sesquiterpene synthase derived from pig liver. Enzyme derived from corpora allata homogenates of both the larval and adult stage of M. sexta coupled each of the DMAPP analogs to produce homologous geranyl and farnesyl diphosphate products in the order (Z)-3-ethyl>(Z)-3-n-propyl>(Z)-3-methyl (DMAPP)>(Z)-3-i-propyl(Z)-3-n-butyl. In competition studies, the ethyl and n-propyl analogs either enhanced or had no effect on DMAPP coupling, whereas the larger analogs were inhibitors. (Z)-7-ethyl and (2Z,6Z)-3,7-diethyl analogs of GPP were as good, if not better substrates of larval prenyltransferase, while the C-3 ethyl analog of GPP, which is precursor to an isomeric form of juvenile hormone (JH) that is not typically found in insects, was poorly coupled by the enzyme. While similarities were seen for whole-cell extracts derived from adult and larval M. sexta, adult prenyltransferase derived from cytosolic and 16,000xg pellet fractions displayed distinct competitive coupling of GPP and its homologs, suggesting differences in substrate specificity as a result of enzyme localization. In contrast to M. sexta, the pig liver enzyme poorly coupled each of the homologous DMAPP derivatives, and the homologous derivatives of GPP were less efficiently coupled than GPP. These results indicate that prenyltransferase in M. sexta possesses high steric latitude at the (Z)-C-3 and C-7 alkyl positions of DMAPP and GPP, respectively, in contrast to other animal prenyltransferases but in keeping with the enzyme's presumptive role in homologous JH metabolism.

Topics: Animals; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Female; Hemiterpenes; Juvenile Hormones; Manduca; Organophosphorus Compounds; Substrate Specificity

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