The purification of 3,3-dimethylallyl- and geranyl-transferase and of isopentenyl pyrophosphate isomerase from pig liver

The enzyme catalysing the synthesis of farnesyl pyrophosphate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate, or from geranyl pyrophosphate and isopentenyl pyrophosphate, has been purified 100-fold from homogenates of pig liver. The enzyme has optimum pH 7.9 and requires Mg(2+) as activator in preference to Mn(2+); it is inhibited by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate and phosphate ions in addition to the products of the reaction, inorganic pyrophosphate and farnesyl pyrophosphate. From product-inhibition studies of the geranyltransferase reaction, the order of addition of substrates to and release of products from the enzyme has been deduced: geranyl pyrophosphate combines with the enzyme first, followed by isopentenyl pyrophosphate. Farnesyl pyrophosphate dissociates from the enzyme before inorganic pyrophosphate. The existence of isopentenyl pyrophosphate isomerase in liver is confirmed. Methods for the preparation of the pyrophosphate esters of isopentenol, 3,3-dimethylallyl alcohol, geraniol and farnesol are also described.     

Substrate Binding of avian liver prenyltransferase.

Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

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.     

Partial purification and properties of prenyltransferase from Pisum sativum

Prenyltransferase (EC 2.5.1.1; assayed as farnesyl pyrophosphate synthetase)was purified 106-fold from an homogenate of 3-day-old seedlings of Pisum sativum. Some of the properties of the purified enzyme were determined and these differed in several significant respects from those reported for preparations from other sources, e.g. the apparent MW was 96000 ± 4000 and the preparation could be dissociated into two subunits of MW 45000 ± 3000. The total activity of the extractable enzyme went through a sharp maximum (in the range 1 to 28 days) 3 days after germination. Farnesyl pyrophosphate was formed in cell-free extracts of peas from either isopentenyl pyrophosphate alone, or this together with geranyl pyrophosphate (optimum yields 1.2 and 10% respectively). Use of [1-¹⁴C]- and [4-¹⁴C]-isopentenyl pyrophosphates as the sole substrates and degradation of the products showed that the crude extracts contained a pool of the biogenetic equivalent of 3,3-dimethylallyl pyrophosphate. No analogous pool of geranyl pyrophosphate could be detected.     

Heptaprenyl pyrophosphate synthetase from Bacillus subtilis

Heptaprenyl pyrophosphate synthetase was detected in partially purified extracts of Bacillus subtilis. The enzyme catalyzed the synthesis of all-trans C35 prenyl pyrophosphate from isopentenyl pyrophosphate and farnesyl or geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. The enzyme reaction proceeded with an elimination of 2-pro-R hydrogen of isopentenyl pyrophosphate without accumulation of any prenyl pyrophosphate shorter than C35. The molecular weight of the enzyme was estimated by gel filtration to be 45,000. Michaelis constants for isopentenyl, farnesyl, and geranylgeranyl pyrophosphate were 12.8, 13.3, and 8.3 microM, respectively.     

Lactobacillus plantarum undecaprenyl pyrophosphate synthetase: purification and reaction requirements.

Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ³-[1-¹⁴C]isopentenyl pyrophosphate and Δ³-2R-[2-³H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ exhibiting comparable activities.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Artificial substrates for prenyltransferase

Four out of 16 new allylic pyrophosphates synthesized were found to be artificial substrates for liver prenyltransferase (EC 2.5.1.1). These were the trans-and the cis-3-ethyl-3-methylallyl, the 3,3-diethylallyl and the (mixture of cis and trans) 3-methyl-3-n-propylallyl pyrophosphates. The products synthesized from these substrates and isopentenyl pyrophosphate were the appropriate homo- and bishomo-farnesyl pyrophosphates. Substitution of 3,3-dimethylallyl pyrophosphate at C-2 with a methyl group destroyed its reactivity with the enzyme. Neither the unsubstituted allyl pyrophosphate nor the cis- or trans-3-methylallyl pyrophosphate could be condensed with isopentenyl pyrophosphate. Thus the simplest allylic substrate for prenyltransferase is 3,3-dimethylallyl pyrophosphate.     

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-¹⁴C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn²⁺ which was more effective than Mg²⁺. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C₁₀ hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.Abbreviations DMAPP dimethylallyl pyrophosphate - FPP farnesyl pyrophosphate - GPP geranyl pyrophosphate - IPP isopentenyl pyrophosphate - LPP linalyl pyrophosphate - NPP neryl pyrophosphate     

A new prenyltransferase from Micrococcuslysodeikticus

A new prenyltransferase which catalyzes the synthesis of geranyl pyrophosphate as the only product from dimethylallyl pyrophosphate and isopentenyl pyrophosphate has been separated from other known prenyltransferases from $\text{Micrococcus}\text{lysodeikticus}$. This enzyme fraction is also capable of synthesizing all-$\text{trans}$ geranylgeranyl pyrophosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate though it lacks ability to synthesize farnesyl pyrophosphate.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C5 alcohols (isopentenyl and dimethylallyl) to form C10 and C15 diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.     

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.     

Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis)

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.     

Clomazone does not inhibit the conversion of isopentenyl pyrophosphate to geranyl, farnesyl, or geranylgeranyl pyrophosphate in vitro

Clomazone, an herbicide that reduces the levels of leaf carotenoids and chlorophylls, is thought to act by inhibiting isopentenyl pyrophosphate isomerase or the prenyltransferases responsible for the synthesis of geranylgeranyl pyrophosphate. Cell-free extracts prepared from the oil glands of common sage (Salvia officinalis) are capable of converting isopentenyl pyrophosphate to geranylgeranyl pyrophosphate. Clomazone at 250 micromolar (a level that produced leaf bleaching) had no detectable effect on the activity of the relevant enzymes (isopentenyl pyrophosphate isomerase and the three prenyltransferases, geranyl, farnesyl, and geranylgeranyl pyrophosphate synthases). Thus, inhibition of geranylgeranyl pyrophosphate biosynthesis does not appear to be the mode of action of this herbicide.     

Prenyltransferase from Gossypium hirsutum

A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophosphate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 × 10⁴ to 1.3 × 10⁵. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum.     

Isoprenoid enzyme systems of silkworm. I. Partial purification of isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and geranylgeranyl pyrophosphate synthetase

Isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and geranylgeranyl pyrophosphate synthetase were detected in cell-free extracts of Bombyx mori and were partially purified by hydroxyapatite and Sephadex G-100 chromatography. Two forms of farnesyl pyrophosphate synthetase were chromatographically separated. They were designated as farnesyl pyrophosphate synthetases I and II in the order of their elution from hydroxyapatite. Both enzymes catalyzed the exclusive formation of (E,E)-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. However, they were not interconvertible, unlike the enzyme from pig liver. These two enzymes resembled each other in pH optima and molecular weights but differed in susceptibility to metal ions. Farnesyl pyrophosphate synthetase II was stimulated by Triton X-100 while synthetase I was inhibited by the same reagent.     

Evidence for trans-trans and cis-cis farnesyl pyrophosphate synthesis in Gossipium hirsutum

A protein fraction capable of catalysing the formation of all four geometrical isomers of farnesyl pyrophosphate has been isolated from cotton roots. Using neryl pyrophosphate and isopentenyl pyrophosphate as substrates the product was found to be cis-cis farnesyl pyrophosphate and possibly trans-cis farnesyl pyrophosphate. Geranyl pyrophosphate and isopentenyl pyrophosphate as substrates yielded trans-trans and possible cis-trans farnesyl pyrophosphate. During purification of the active protein fraction, the ratio of utilization of geranyl pyrophosphate and neryl pyrophosphate did not remain constant, indicating that two enzymes may be involved, one specific for cis C₁₀-substrate and the other for trans C₁₀-substrate.