Neryl Pyrophosphate Research Articles

Conversion of [1- 3H 2,G- 14C]geranyl pyrophosphate to cyclic monoterpenes without loss of tritium

Soluble enzyme preparations from Salvia officinalis convert the acyclic precursor [1- 3H 2,G- 14C]geranyl pyrophosphate to cyclic monoterpenes of the pinane (α-pinene,β-pinene), isocamphane (camphene), p-menthane (limonene,1,8-cineole), and bornane (bornyl pyrophosphate, determined as borneol) type without loss of tritium, and without significant conversion to other free acyclic intermediates. Similarly, [1- 3H 2,G- 14C]geraniol is converted in intact S. officinalis leaves to the cyclic monoterpene olefins and 1,8-cineole, as well as to isothujone and camphor, without loss of tritium from C(1). These results clearly eliminate transcis isomerization of geranyl pyrophosphate to neryl pyrophosphate via aldehyde intermediates prior to cyclization, and they support a scheme whereby the trans precursor is cyclized directly by way of a bound linaloyl intermediate.

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 × 10 4 to 1.3 × 10 5. 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.

Biosynthesis of monoterpenes: Preliminary characterization of l-endo-fenchol synthetase from fennel ( Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product

A soluble enzyme preparation from the leaves of fennel ( Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys. 200, 524–533) . To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn 2+ (1 m m) for catalytic activity. Co 2+ can partially substitute for Mn 2+, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.

Biosynthesis of monoterpenes: Conversion of the acyclic precursors geranyl pyrophosphate and neryl pyrophosphate to the rearranged monoterpenes fenchol and fenchone by a soluble enzyme preparation from fennel ( Foeniculum vulgare)

(+)-(1 S)-Fenchone, an irregular bicyclic monoterpene ketone thought to be derived via rearrangement of a bicyclic precursor, is one of the major terpenoids of the volatile oil of fennel ( Foeniculum vulgare M.). A soluble enzyme preparation obtained from homogenates of young fennel leaves converted the acyclic precursors geranyl pyrophosphate and neryl pyrophosphate to fenchol in the presence of Mn 2+, and in the presence of pyridine nucleotide, dehydrogenated the fenchol formed to fenchone. Particulate enzyme fractions were inactive in the synthesis of fenchane-type monoterpenes. The identities of the biosynthetic products as fenchol and fenchone were confirmed by radiochromatographic analysis and by the synthesis of crystalline derivatives. Differential crystallization of the fenchyl hydrogen phthalates and the fenchyl p-nitrobenzoates demonstrated that the initial cyclic product was endo-[ 3H]fenchol and not the exo-epimer, while resolution of [ 3H]fenchone oxime via the diastereomeric l-menthyloxymethyl ethers confirmed the stereochemistry of the labeled cyclic products as (−)-(1 S)- endo-fenchol and (+)-(1 S)-fenchone. Biosynthetic l-endo-[ 3H]fenchol derived from the [1- 3H]-labeled acyclic precursors was chemically converted to (+)-(1 R)-α-fenchocamphorone, and exchange of the α-protons of this derivative established that 3H was located specifically on the bridge carbon (C-7) of the original fenchane nucleus. Thus, the bicyclic fenchane skeleton was shown to be derived by rearrangement of a pinane-type intermediate. These results provide strong evidence that fenchone is derived by the cyclization of geranyl pyrophosphate or neryl pyrophosphate to endo-fenchol followed by dehydrogenation of this bicyclic alcohol, and they constitute the first demonstration of the biosynthesis of a rearranged monoterpene in a cell-free system.

Preferential participation of linaloyl pyrophosphate rather than neryl pyrophosphate in biosynthesis of cyclic monoterpenoids in higher plants

Comparisons of the incorporation of [1-3H2]nerol, [1-3H2]geraniol, and [1-3H2]linalool and their pyrophosphates into some cyclic monoterpenoids have been made in both intact plants and cell-free systems; the preferential incorporation of linalool and its pyrophosphate into the cyclic monoterpenoids suggests that they are better precursors than nerol or its pyrophosphate for the biological formation of the cyclic monoterpenoids.

Biosynthesis of monoterpenes: Hydrolysis of bornyl pyrophosphate, an essential step in camphor biosynthesis, and hydrolysis of geranyl pyrophosphate, the acyclic precursor of camphor, by enzymes from sage ( Salvia officinalis)

Soluble enzyme preparations from sage ( Salvia officinalis) leaves catalyze the hydrolysis of (+)-bornyl pyrophosphate to (+)-borneol, which is an essential step in the biosynthesis of the cyclic monoterpene (+)-camphor [(1 R,4 R)-bornan-2-one] in this tissue. Chromatography of the preparation on Sephadex G-150 allowed the separation of two regions of bornyl pyrophosphate hydrolase activity. One region was further separated into a pyrophosphate hydrolase and a monophosphate hydrolase by chromatography on hydroxylapatite, but the other contained pyrophosphate and monophosphate hydrolase activities which were inseparable by this or any other chromatographic technique tested. Each phosphatase and pyrophosphatase activity was characterized with respect to molecular weight, pH optimum, response to inhibitors, K m for bornyl phosphate or bornyl pyrophosphate, and substrate specificity, and each activity was distinctly different with regard to these properties. One pyrophosphatase activity was specific for pyrophosphate esters of sterically hindered monoterpenols such as bornyl pyrophosphate. The other preferred pyrophosphate esters of primary allylic alcohols such as geranyl pyrophosphate and neryl pyrophosphate, which are precursors of cyclic monoterpenes, and it hydrolyzed geranyl pyrophosphate at faster rates than neryl pyrophosphate. The monophosphate hydrolase activities were similar in substrate specificity, showing a preference for phosphate esters of primary allylic alcohols. The terpenyl pyrophosphate hydrolase exhibiting specificity for bornyl pyrophosphate may be involved in camphor biosynthesis in vivo, while the terpenyl pyrophosphate hydrolase more specific for geranyl pyrophosphate was shown to be a source of potential interference in studies on monoterpene cyclization processes.

Biosynthesis of monoterpenes: Preliminary characterization of bornyl pyrophosphate synthetase from sage ( Salvia officinalis) and demonstration that geranyl pyrophosphate is the preferred substrate for cyclization

Previous studies with soluble enzyme preparations from sage ( Salvia officinalis) demonstrated that the monoterpene ketone (+)-camphor was synthesized by the cyclization of neryl pyrophosphate to (+)-bornyl pyrophosphate followed by hydrolysis of this unusual intermediate to (+)-borneol and then oxidation of the alcohol to camphor (R. Croteau, and F. Karp, 1977, Arch. Biochem. Biophys. 184, 77–86) . Preliminary investigation of the (+)-bornyl pyrophosphate synthetase in crude preparations indicated that both neryl pyrophosphate and geranyl pyrophosphate could be cyclized to (+)-bornyl pyrophosphate, but the presence of high levels of phosphatases in the extract prevented an accurate assessment of substrate specificity. The competing phosphatases were removed by combination of gel filtration on Sephadex G-150, chromatography on hydroxylapatite, and chromatography on O-(diethylaminoethyl)-cellulose. In these fractionation steps, activities for the cyclization of neryl pyrophosphate and geranyl pyrophosphate to bornyl pyrophosphate were coincident, and on the removal of competing phosphatases, the synthetase was shown to prefer geranyl pyrophosphate as substrate ( V K m for geranyl pyrophosphate was 20-fold that of neryl pyrophosphate). No interconversion of geranyl and neryl pyrophosphates was detected. The partially purified bornyl pyrophosphate synthetase had an apparent molecular weight of 95,000, and required Mg 2+ for catalytic activity ( K m for Mg 2+ ~ 3.5 m m). Mn 2+ and other divalent cations were ineffective in promoting the formation of bornyl pyrophosphate. The enzyme exhibited a pH optimum at 6.2 and was strongly inhibited by both p-hydroxymercuribenzoate and diisopropylfluorophosphate. Bornyl pyrophosphate synthetase is the first monoterpene synthetase to be isolated free from competing phosphatases, and the first to show a strong preference for geranyl pyrophosphate as substrate. A mechanism for the cyclization of geranyl pyrophosphate to bornyl pyrophosphate is proposed.

γ-Terpinene synthetase: A key enzyme in the biosynthesis of aromatic monoterpenes

Previous studies with thyme ( Thymus vulgaris L.) leaf slices indicated that γ-terpinene (1,4- p-menthadiene) is the precursor of the aromatic monoterpenes p-cymene (4-isopropyl toluene) and thymol (5-methyl-2-isopropyl phenol) (Poulose, A. and Croteau, R. (1978) Arch. Biochem. Biophys. 187, 307–314). A 105,000 g supernatant obtained from an extract of young thyme leaves catalyzed the cyclization of both [1- 3H]neryl pyrophosphate and [1- 3H]geranyl pyrophosphate to γ-[3- 3H]terpinene. No evidence for the interconversion of the acyclic precursors was obtained, and isotopic dilution experiments suggested that γ-terpinene was synthesized directly from these acyclic precursors without the involvement of any free intermediates. Competing phosphatase activity in the soluble preparation was removed by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150. In these fractionation steps, γ-terpinene synthetase activity co-purified with small amounts of α-thujene (1-isopropyl-4-methylbicyclo[3.1.0]-hex-3-ene) and α-terpineol ( p-menth-1-en-8-ol) synthetase activities, and these three activities could not be resolved by subsequent hydroxylapatite chromatography, anion exchange chromatography on QAE-Sephadex, or affinity chromatography on neroic acid-substituted agarose. All the enzymatic products were identified by radio chromatography and by the synthesis of derivatives followed by radio chromatography or crystallization to constant specific activity. γ-Terpinene synthetase has an apparent molecular weight of 96,000, shows a pH optimum at about 6.8, and requires Mg 2+ for catalytic activity. Mn 2+ can partially substitute for Mg 2+, but other divalent cations are ineffective. Estimated values of V and K m are 3.5 nmol/h/mg and 9 μ m, respectively, for neryl pyrophosphate, and 3.0 nmol/h/mg and 14 μ m, respectively, for geranyl pyrophosphate. Enzymic activity is inhibited by sulfhydryl-directed reagents and inorganic pyrophosphate, but not by γ-terpinene, p-cymene, or thymol. Based on the specific location of tritium in the product, a mechanism is proposed which involves the cyclization of the acyclic precursor, loss of a proton from C5 to form the Δ 4 double bond, and a 1,2-hydride shift from C4 to C8 to give γ-terpinene. A similar mechanism, but with loss of the proton from C6 and the formation of a cyclopropane ring, would yield α-thujene.

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol

A soluble enzyme preparation obtained from young sage ( Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH 4) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.

Biosynthesis of monoterpene hydrocarbons from [1- 3H]neryl pyrophosphate and [1- 3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum

A soluble enzyme preparation from the flavedo of Citrus limonum transforms [1- 3H 1]neryl pyrophosphate or [1- 3H 1]geranyl pyrophosphate into β-pinene, sabinene, α-pinene, and limonene. The enzyme has been partially purified and stabilized by precipitation with polyethyleneglycol. The enzymic cyclization requires the presence of Mn 2+, which cannot be replaced with Mg 2+. The addition of reagents containing sulfhydryl groups is essential for optimal activity. Allylic C 10 monophosphates do not act as substrates, but they inhibit hydrocarbon formation. Inorganic pyrophosphate has a similar inhibitory effect. No interconversion of neryl and geranyl pyrophosphate has been observed. Possible pathways for the enzymic cyclization reactions are proposed.

Biosynthesis of monoterpenes: Partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage ( Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl 2 (1 m m). ZnCl 2 was nearly as effective as MnCl 2, and MgCl 2 could substitute for MnCl 2 only at tenfold higher concentrations. The apparent K m and V of the enzyme were 10 −5 m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10 −4 m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10 −5 m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.

Biosynthesis of monoterpenes: Enzymatic conversion of neryl pyrophosphate to 1,8-cineole, α-terpineol, and cyclic monoterpene hydrocarbons by a cell-free preparation from sage ( Salvia officinalis)

The volatile oil of sage ( Salvia officinalis) leaf contains primarily cyclic monoterpenes, and a cell-free enzyme system prepared from immature sage leaves catalyzed the conversion of [1- 3H]neryl pyrophosphate to cyclic monoterpenes, including 1,8-cineole, α-terpineol, and limonene. The identity of these products was confirmed by the preparation of derivatives, chemical degradation studies, and radiochromatographic analyses. Enzymatic activity capable of 1,8-cineole and hydrocarbon formation was located primarily in the soluble fraction of the leaf homogenate, whereas activity responsible for α-terpineol formation, and phosphatase activity, was distributed among the soluble fraction and the 3000g particles. The formation of all cyclic products was stimulated in the presence of MnCl 2 (1–2 m m) and MgCl 2 (5–10 m m) and by inclusion of phosphatase inhibitors, such as NaF, in the incubation medium. Enzymatic activity increased linearly with protein concentration and time and was maximum at about pH 6.5. As α-terpineol could give rise to limonene by dehydration and yield 1,8-cineole by intramolecular attack of the hydroxyl at the double bond, the possible intermediary role of α-terpineol was examined. Inclusion of 1 m m (±)-α-terpineol in the incubation medium suppressed 1,8-cineole and hydrocarbon formation from neryl pyrophosphate by approximately 35%, suggesting that this alcohol could be an intermediate. However, the inhibition appeared to be nonspecific as several other monoterpene alcohols were similarly effective. In order to provide a more direct examination of the pathway, (±)-[3- 3H]α-terpineol was prepared and tested as a substrate with the enzyme system. (±)-[3- 3H]α-Terpineol was not converted to 1,8-cineole or to cyclic hydrocarbons, indicating that this alcohol was not an intermediate in the biosynthesis of the structurally related compounds. Furthermore, labeled α-terpineol was not incorporated into other monoterpenes in sage leaf slices, whereas the acyclic alcohols, [1- 3H]nerol and [1- 3H]geraniol, were readily incorporated into the characteristic cyclic monoterpenes of sage leaf. Similar lines of evidence excluded 1,8-cineole, limonene, and terpinolene as intermediates in the formation of cyclic products from neryl pyrophosphate. Thus, all of these results suggest that 1,8-cineole, α-terpineol, and limonene are derived independently from neryl pyrophosphate, rather than as free intermediates of a common reaction sequence. Additionally, this is the first report on the biosynthesis of 1,8-cineole in a cell-free system.

Enzymatic synthesis of camphor from neryl pyrophosphate by a soluble preparation from sage ( Salvia Officinalis)

Camphor, a bicyclic monoterpene ketone, is one of the major components of the volatile oil of sage ( Salvia officinalis ). A soluble enzyme preparation obtained from homogenates of young sage leaves converts the acyclic precursor neryl pyrophosphate to borneol in the presence of Mg ++ , and in the presence of NAD, dehydrogenates the borneol formed to camphor. The biosynthetic products were identified via the synthesis of derivatives and radiochromatographic analysis. The results presented strongly suggest that camphor is derived by the cyclization of neryl pyrophosphate to horneol followed by dehydrogenation of this bicyclic alcohol. This is the first report on the enzymes involved in the biosynthesis of camphor.

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 C 10-substrate and the other for trans C 10-substrate.

Enzymatic cyclization of neryl pyrophosphate to α-terpineol by cell-free extracts from peppermint

A cell-free system prepared from peppermint ( Mentha piperita L.) shoot tips catalyzed the cyclization of neryl pyrophosphate to α-terpineol. Cyclization could be demonstrated in the absence of added cofactors, but addition of NaF inhibited competing phosphatase/pyrophosphatase activity, resulting in much higher levels of α-terpineol formation. Under certain conditions cyclization was stimulated by Mg ++. Similar enzyme preparations were obtained from spearmint ( Mentha spicata L.) leaves and carrot ( Daucus carota L.) storage organ. The cyclization of neryl pyrophosphate to α-terpineol appears to be a key reaction in the biosynthesis of cyclohexanoid monoterpenes.

Stereospecificity of isopentenylpyrophosphate isomerase and prenyl transferase from Pinus and Citrus

Isopentenylpyrophosphate isomerases in cell-free extracts from Pinus radiata and from Citrus sinensis eliminate the C-4 pro-S proton from mevalonic acid. Prenyl transferase from Pinus forms geranyl pyrophosphate, neryl pyrophosphate, 2,6 trans, trans farnesyl pyrophosphate and 2 cis, 6 trans farnesyl pyrophosphate from mevalonic acid. The 4-pro-S proton of mevalonic acid is eliminated in the formation of both trans and of cis double-bonds. The pattern is similar for the Citrus enzyme. Isopentenyl pyrophosphate isomerase from Pinus is completely inhibited by 15 m m iodoacetamide, which does not affect prenyl transferase. The pattern is the same for the Citrus isomerase, but less clear for the transferase. Previous work has ruled out direct trans-cis isomerization of C 10 or C 15 prenylpyrophosphates. Alternative mechanisms are proposed for the stereospecific elimination of the 4-pro-S proton of mevalonic acid in the biosynthesis of both cis and trans prenyl pyrophosphates.

Synthesis of isomeric farnesols by soluble enzymes from Pinus radiata seedlings

Water soluble enzymes obtained from Pinus radiata seedlings form two sesquiterpene alcohols from 2- 14C mevalonic acid. They have been identified as 2,6- trans,trans-farnesol and 2- cis,6- trans-farnesol. The pyrophosphate of the former prenol could be isolated, but there was no evidence of the presence of phosphorylated derivatives of the cis isomer. The same pair of sesquiterpene alcohols and trans-farnesyl pyrophosphate are formed from isopentenyl pyrophosphate plus geranyl pyrophosphate (2- trans). Neryl pyrophosphate (2- cis) is completely inactive as a precursor of farnesols. Isomerization of trans- to cis-farnesyl pyrophosphate did not occur in this system.

Terpene biosynthesis from geranyl and neryl pyrophosphates by enzymes from orange flavedo

A cell free extract from orange flavedo forms phosphorylated derivatives of isopentenol, dimethylallyl alcohol and farnesol, as well as small amounts of hydrocarbons from 2- 14C mevalonic acid. Isopentenyl pyrophosphate 4- 14C is transformed into phosphorylated derivatives of dimethylallyl alcohol, nerol, geraniol, linalool and α-terpineol. The eventual formation of linaloyl pyrophosphate is discussed. Limonene is formed from 2- 14C neryl or geranyl pyrophosphate to the extent of 1–3%. α-Pinene is formed only from the cis isomer 2- 14C neryl pyrophosphate. There are differences between this system and a similar one from P. radiata seedlings. No interconversion of neryl and geranyl pyrophosphate was observed.

Biosynthesis of Artemisia Ketone

MEVALONIC acid (MVA) is generally believed to be converted in higher plants into isopentenyl and 3,3-dimethylallyl pyrophosphates which in turn condense to form geranyl and neryl pyrophosphates, the presumed precursors of acyclic and cyclic monoterpenes respectively. Some of the individual steps have been demonstrated1–3; radioactive MVA has been incorporated without scrambling of tracer into several monoterpenes4–6; and the various classes of monoterpenes are thought to be biosynthesized from geranyl and neryl pyrophosphates by routes outlined by Ruzicka et al.7.

Biosynthesis of Monoterpenes in Rose Flowers

ALTHOUGH mevalonic acid (MVA) is generally assumed to be an obligatory precursor of terpenes in higher plants, incorporation of 2-14C-MVA into the principal monoterpenes of numerous species is almost invariably less than 1 per cent (ref. 1). Monoterpene biosynthesis is nevertheless assumed to occur via conversion of MVA into isopentenyl pyrophosphate (IPP) and 3,3-dimethyl-allylpyrophosphate (DMAPP) which condense to form, directly or in sequence, geranyl or neryl pyrophosphates (GPP and NPP), the presumed precursors of straight chain and cyclic monoterpenes respectively. More than 80 per cent of the total label from 2-14C-MVA, however, has been found to pass into that part of several thujane derivatives2, camphor3, pulegone4 and artemisia ketone4, and of certain sesquiterpenes5 also, which was derived from IPP. In only one example6 has a monoterpene, other than a methyl cyclopentanoid monoterpene, biosynthesized from 2-14C-MVA by a higher plant been shown to contain radioactivity comparably distributed between the moieties derived from IPP and DMAPP. All these experiments involved feeding leaf and stem tissue for 1–5 days, and less than 0.1 per cent of the applied tracer was incorporated during this period.