Monoterpene Cyclases Research Articles

Oleoresinosis in Grand Fir (Abies grandis) Saplings and Mature Trees (Modulation of this Wound Response by Light and Water Stresses).

The stem content of diterpene resin acids (rosin) increases dramatically following wounding of grand fir (Abies grandis) saplings, but the level of monoterpene olefins (turpentine) in the stem decreases following injury, in spite of a significant increase in monoterpene cyclase (synthase) activity. However, this observation was explained when rapid evaporative losses of the volatile monoterpenes from the wound site was demonstrated by trapping experiments, a finding consistent with a role of turpentine as a solvent for the mobilization and deposition of rosin to seal the injury. Mature forest trees responded to stem wounding by the enhancement of monoterpene cyclization capacity in a manner similar to 2-year-old grand fir saplings raised in the greenhouse. Light and water stresses greatly reduced the constitutive level of monoterpene cyclase activity and abolished the wound-induced response. The diminution in monoterpene biosynthetic capacity was correlated with a dramatic decrease in cyclase protein as demonstrated by immunoblotting. Relief of stress conditions resulted in the restoration of cyclase activity (both constitutive and wound induced) to control levels. The results of these experiments indicate that grand fir saplings are a suitable model for studies of the regulation of defensive oleoresinosis in conifers.

Production and Characterization of Polyclonal Antibodies in Rabbits to 4 S-Limonene Synthase from Spearmint ( Mentha spicata)

Limonene synthase, a monoterpene cyclase from the oil glands of spearmint ( Mentha spicata) leaves that catalyzes the conversion of geranyl pyrophosphate to (−)-4 S-limonene, was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. Immunoblotting analysis revealed that the antibodies were very specific for denatured limonene synthase from all Mentha species tested. However, no immunological cross-reactivity was observed with denatured limonene synthases from Valencia oranges ( Citrus sinensis, Rutacene) or wormseed ( Chenopodium ambrosioides, Chenopodiaceae). Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiacene, Asteracene, and Umbellifereae, or from conifer species, and no cross-reactivity was demonstrated toward several sesquiterpene cyclases of higher plant and fungal origin. Although the antibody preparation was highly selective for denatured limonene cyclase from Mentha, the antibodies did not recognize the native protein in several different types of experiments. Nevertheless, specificity for the target enzyme was unambiguously demonstrated when the antibody preparation was shown to cross-react with the cyclase protein expressed in Escherichia coli that harbored the corresponding limonene synthase cDNA gene from M.spicata.

Biosynthesis of monoterpenes: Inhibition of (+)-pinene and (−)-pinene cyclases by thia and aza analogs of the 4 R- and 4 S-α-terpinyl carbocation

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3 R)-linalyl pyrophosphate and the (4 R)-α-terpinyl cation, to (+)-α-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (−)-pinene cyclase converts the same achiral precursor, via (3 S)-linalyl pyrophosphate and the (4 S)-α-terpinyl cation, to (−)-α-pinene and (−)-β-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and α-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4 R)- and the (4 S)-thia and -aza analogs of the α-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4 R)- and the (4 S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the α-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of ( RS)-α-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discrimintae between enantiomers of α-terpinyl pyrophosphate in this unusual reaction. Either the α-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirment to distinguish the (4 R)- and (4 S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.

Characterization and mechanism of (4 S)-limonene synthase, A monoterpene cyclase from the glandular trichomes of peppermint ( Mentha x piperita)

(4 S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4 S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (−)-(4 S)-limonene (>94% of the total products), plus several other monoterpene oleflns. The general properties of (4 S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg 2+ or Mn 2+, with Mn 2+ preferred. The K m value measured for geranyl pyrophosphate was 1.8 μ m. The activity of (4 S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn 2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.

Antigenic cross-reactivity among monoterpene cyclases from grand fir and induction of these enzymes upon stem wounding

A major wound response in grand fir ( Abies grandis) sapling stems is the rapid increase in monoterpene production at the site of injury. Monoterpene cyclases (synthases) catalyze the formation of monoterpenes from geranyl pyrophosphate, and total cyclase activity increases markedly on wounding. At least six distinct cyclases, producing different monoterpene products, have been isolated from wounded grand fir saplings and characterized. The predominant wound-inducible cyclase produces both α- and β-pinene. This pinene cyclase was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. The antibody preparation was found to crossreact by Western blotting with other grand fir monoterpene cyclases that produce different olefinic products, but not with monoterpene cyclases from related conifer species ( Pinus contorta and P. ponderosa) or from angiosperms ( Mentha piperita and M. spicata). The increase in monoterpene cyclase activity after wounding was closely correlated with the appearance of new cyclase protein as determined by immunoblotting. These results indicate that the wound-dependent increase in monoterpene cyclase activity is a consequence of de novo synthesis of cyclase protein.

Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata).

The p-menthane monoterpenes of the Mentha species are biosynthesized from geranyl pyrophosphate via the monocyclic olefin 4S-limonene. A monoterpene cyclase was isolated from both Mentha x piperita (peppermint) and Mentha spicata (spearmint) that catalyzes the cyclization of geranyl pyrophosphate to 4S-limonene. This enzyme, 4S-limonene synthase, was purified to apparent homogeneity by dye ligand, anion exchange, and hydrophobic interaction chromatography. Since the monoterpenes of Mentha are synthesized and secreted in modified epidermal hairs called glandular trichomes, an extract of isolated glandular trichome cells was used as the source of this enzyme. A combination of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that purified 4S-limonene synthase had a native molecular weight of 56,000 and was monomeric. The principal product of the enzyme was enantiomerically pure (-)-4S-limonene, and a catalytic constant of 0.3/s was determined. The basic properties of 4S-limonene synthase from both M. x piperita and M. spicata are identical and, in general, are similar to those of other monoterpene, sesquiterpene, and diterpene cyclases isolated from microorganisms and higher plants.

Wound-inducible pinene cyclase from grand fir: Purification, characterization, and renaturation after SDS-PAGE

The major wound-inducible monoterpene synthase (cyclase) of grand fir ( Abies grandis) stems transforms geranyl pyrophosphate to both (−)-α-pinene (40%) and (−)-β-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn 2+ ( K m = 30 μM) for activity, exhibited a K m value of 6 μ m for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 °C, and was inhibited by pyrophosphate ( I 50 = 0.17 mM), orthophosphate ( I 50 = 51 μM), and α-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate ( I 50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate ( I 50 = 1.9 mM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.

Characterization of the constitutive and wound-inducible monoterpene cyclases of grand fir ( Abies grandis)

Monoterpene cyclase activity is greatly increased in grand fir ( Abies grandis) sapling stems in response to wounding and the composition of the cyclic olefin mixture generated differs from that produced constitutively as determined by radio gas-liquid chromatography. Cell-free extracts from wounded stems and from non-wounded controls were systematically compared for monoterpene cyclase activities following partial purification and separation of these enzymes by anion-exchange chromatography (Mono Q FPLC) and native PAGE. The increase in monoterpene cyclase activity following wounding represents both the apparent enhancement of constitutive cyclase activities and the appearance of novel cyclization enzymes that are absent in nonwounded controls. A pinene cyclase was shown to be the major wound-inducible enzyme directly responsible for oleoresin monoterpene formation and was tentatively identified as a 62-kDa protein by SDSPAGE.

Isotopically sensitive branching as a tool for evaluating multiple product formation by monoterpene cyclases

The deuterated substrates [4- 2H 2,1- 3H]geranyl pyrophosphate and [10- 2H 3,1- 3H]geranyl pyrophosphate were employed to examine isotopically sensitive branching in the biosynthesis of monoterpene olefin isomers. By this method, (−)-α-pinene and (−)-β-pinene were shown to be synthesized via a common intermediate by a single cyclization enzyme from grand fir ( Abies grandis), as were (−)-(α-phellandrene and (−)-β-phellandrene by a single cyclase from lodgepole pine ( Pinus contorta). Kinetic isotope effects were determined for the various deprotonations leading to the pinenes and phellandrenes.

Purification and characterization of the monoterpene cyclase γ-terpinene synthase from Thymus vulgaris

The monoterpene cyclase, γ-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h · mg protein (turnover number of ~0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of M r ~ 55,000. The protein was very hydrophobic, and possessed a p I value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 m m Mg 2+; 5 m m Mn 2+ could support catalysis, albeit at a much lower rate. The K m value for the substrate, geranyl pyrophosphate, was 2.6 μ m. Cyclase activity was inhibited by cysteineand histidine-directed reagents. Purified γ-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of α-thujene and to lesser quantities of myrcene, α-terpinene, limonene, linalool, terpinen-4-ol, and α-terpineol, all of which appear to be coproducts of the reaction sequence leading to γ-terpinene. In general properties, the γ-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.

Defense Mechanisms of Conifers

Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue.

Defense Mechanisms of Conifers

Levels of monoterpene cyclase activity were determined in extracts from wounded and unwounded saplings of 10 conifer species to assess whether oleoresin biosynthesis is induced by stem wounding. Species of Abies and Picea, with low to moderate levels of constitutive monoterpene cyclase activity, exhibited a five- to 15-fold increase in cyclase activity 7 days after wounding relative to unwounded controls. In contrast, species of genera such as Pinus, with high levels of constitutive cyclase activity, did not significantly respond to wounding by alteration in the level of cyclase activity. The highest fold increase in monoterpene cyclase activity was consistently observed in Abies grandis, and the time-course of induction of activity following stem wounding in this species demonstrated a threefold increase at 2 days relative to unwounded controls, rising to a maximum increase in the response at 9 days (greater than 10-fold) followed by an apparent decline. The wound response was localized, and both bark (phloem) and wood (xylem) tissues displayed increased cyclase activity at the wound site. The magnitude of the increase in cyclase activity was dependent on the severity of the wound.

Immobilization of cyclase enzymes for the production of monoterpenes and sesquiterpenes

The first steps in the biosynthesis of most monoterpenes and sesquiterpenes are catalysed by a novel class of enzymes termed cyclases. Several techniques were tested for the immobilization of a partially purified monoterpene cyclase from Foeniculum vulgare [geranyl pyrophosphate: (-)-endo-fenchol cyclase] and a partially purified sesquiterpene cylase from Pogostemon cablin [farnesyl pyrophosphate:(-)-patchoulol cyclase]. Although both enzymes could be immobilized by entrapment and adsorption methods, the most efficient immobilization, in terms of activity recovery, was achieved by covalent attachment involving coupling of the protein amino groups to N-hydroxysuccinimide-activated 6-aminohexanoic acid Sepharose 4B. The immobilized forms of these cyclases were markedly more stable in storage than the free forms.

Purification and characterization of the sesquiterpene cyclase patchoulol synthase from Pogostemon cablin

The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a p I value of about 5.0, as determined by isoelectric and chromatofocusing. SDSPAGE showed the enzyme to be composed of two apparently identical subunits of M r ~ 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg 2+ ( K m ~ 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The K m value for the substrate, farnesyl pyrophosphate, was 6.8 μ m. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (α- and β-patchoulene, α-bulnesene, and α-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of ( E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-ol, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage ( Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K i ′ values of 3.2 and 4.7 μ m, respectively. These values are close to the corresponding K m values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2( trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.

Monoterpene cyclases: Physicochemical features required for pyrophosphate binding determined from inhibition by structural analogs

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C 10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate: (+)-α-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage ( Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.

インドールアルカロイド及びイリドラクトンの生合成におけるイリダン骨格形成機構

Four biosynthetic intermediates for non-tryptophan moieties of indole alkaloids, 10-oxogeraniol (27a), 10-hydroxygeranial (27b), 10-oxogeranial (28) and iridodial (3), were isolated from the incubation system of 10-hydroxygeraniol (16) and crude enzyme extracts from Rauwolfia serpentina cell suspension cultures. This finding enabled us to establish the mechanism of iridane skeleton formation from 16 in the biosynthesis of indole alkaloids. Furthermore, a novel type of monoterpene cyclase catalyzing the iridodial (3) formation from 28 in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) was partially purified and characterized. Systematic research has not yet been performed on the biosynthesis of iridolactones. However, feeding experiments of several 3H-labelled putative precursors to Nepeta cataria and Patrinia gibbosa suggested that some of iridolactones in these plants are biosynthesized via somewhat different route from those for the usual iridoids.

Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate.

To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10-2H3,1-3H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.

Direct demonstration of the isomerization component of the monoterpene cyclase reaction using a cyclopropylcarbinyl pyrophosphate substrate analog.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene cyclases has precluded direct observation of the topologically required isomerization step leading from geranyl pyrophosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl pyrophosphate, which ultimately cyclizes to the various monoterpene skeletons. By using a partially purified monoterpene cyclase preparation and 2,3-cyclopropylgeranyl pyrophosphate, a substrate analog designed to uncouple the reaction sequence, the production of the corresponding tertiary homoallylic pyrophosphate isomer was demonstrated. This provides direct evidence for the usually cryptic isomerase component of the overall catalytic cycle. A number of other related products generated by reaction of cyclase with the analog were also identified, the structures and proportions of which were consistent with the intermediacy in catalysis of a cyclopropylcarbinyl cation X pyrophosphate anion pair. Kinetic parameters for the analog were compared with those of the natural substrate geranyl pyrophosphate. The results presented confirm mechanistic similarities in the enzymatic ionization and subsequent transformation of allylic pyrophosphate and cyclopropylcarbinyl pyrophosphate intermediates of isoprenoid metabolism.

Monoterpene cyclases. Stereoelectronic requirements for substrate binding and ionization.

Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.