Biomedical subjects

R Croteau

Publications and source records attributed to R Croteau.

At least 91 records · Page 5Linked to original sources

PubMed1994

Monoterpene biosynthesis: isotope effects associated with bicyclic olefin formation catalyzed by pinene synthases from sage (Salvia officinalis).

The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pinene and (+)-beta-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. [1-3H,4-2H2]- and [10-2H2]-geranyl pyrophosphates were prepared and used in conjunction with 1-3H- and 1-3H,10-2H3-labeled geranyl precursors to examine the isotope effects attending the C4- and C10-deprotonation steps in the enzymatic synthesis of the pinenes. The observation of isotopically sensitive branching within each set of stereochemically related bicyclic olefins confirmed that each product set was synthesized by the respective pinene synthase by partitioning of common carbocationic intermediates along different reaction channels at the active site. The changes in product distribution resulting from deuterium substitution at C4 and C10 of the substrate were used to determine kinetic isotope effects (KIEs) for the terminating deprotonations; these observed KIEs represent the lower limits of the intrinsic isotope effects. The intramolecular isotope effects for the methyl-methylene elimination in beta-pinene formation by cyclases II and III were also evaluated with [10-2H2]geranyl pyrophosphate as substrate and by MS analysis of the olefin products. The intramolecular KIEs (kH/kD = 3.0 and 3.5) were significantly higher than the observed KIEs determined from product ratios (kH/kD = 1.7 and 2.6) since the former involves considerably less masking of the intrinsic isotope effects.

Alkenes↗

PubMed1994

Stereochemistry of the proton elimination in the formation of (+)- and (-)-alpha-pinene by monoterpene cyclases from sage (Salvia officinalis).

The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pinene and (+)-beta-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. (1R)-4-2H1- and (1S)-4-2H1-labeled geranyl pyrophosphates were prepared and used to examine the stereochemistry of the C3-proton elimination from the pinyl cation intermediates in the formation of the alpha-pinene enantiomers. Mass spectrometric analysis of the biosynthetic products derived from the chirally deuterated substrates revealed that cyclase I and cyclase III removed the C4-proR-hydrogen of the substrate (C3 proton trans to the dimethyl bridge of the pinyl nucleus) with a stereoselectivity exceeding 94% in the formation of (+)-alpha-pinene. Similarly, cyclase II removed the C4-proS-hydrogen of the substrate (C3-trans proton of the corresponding pinyl cation) with a stereoselectivity exceeding 78% in the formation of (-)-alpha-pinene. The stereoselectivity of these C3-axial hydrogen eliminations is rationalized on the basis of a stereochemical model for the electrophilic isomerization-cyclization reaction sequence catalyzed by the pinene cyclases. The changes in the overall rates of olefin biosynthesis by these enzymes and in the product ratios resulting from deuterium substitution also permitted confirmation of isotopically sensitive branching in pinene biosynthesis and allowed the observation of primary kinetic isotope effects in isolation.

Alkenes↗

PubMed1994

Diterpenoid resin acid biosynthesis in conifers: characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis.

Abietic acid is a major component of the rosin fraction of oleoresin synthesized by grand fir (Abies grandis), lodgepole pine (Pinus contorta), and many other conifer species as a defensive secretion against insect and pathogen attack. The diterpenoid resin acid is derived from geranylgeranyl pyrophosphate via abietadiene, with subsequent oxidation of the C18-methyl of this olefin to abietadienol, abietadienal, and abietic acid. The pathway was confirmed by administering [1,2-14C]acetic acid to grand fir stems which incorporated the radiolabel into abietadiene, the corresponding alcohol and aldehyde, as well as abietic acid. Three different enzymatic activities, catalyzing the sequential oxidation of the olefin to abietic acid, were demonstrated in cell-free stem extracts of both grand fir and lodgepole pine. The first two oxidation steps were catalyzed by the microsomal fraction and required both oxygen and a reduced pyridine nucleotide (NADPH preferred). Both activities were strongly inhibited by CO (blue light reversible) and were differentially sensitive to several substituted N-heterocyclic inhibitors, suggesting that these two enzymes are distinct, microsomal cytochrome P450-dependent monooxygenases. A third enzymatic activity, catalyzing the oxidation of abietadienal to abietic acid, was located in the soluble protein fraction. This oxidation reaction employed NAD+ as cofactor, but did not require oxygen and was not inhibited by CO, indicating that this last step of abietic acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase.

Abietanes↗

PubMed1993

Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway.

Procedures for the isolation and analysis of all 11 intermediates of the mevalonic acid pathway from acetyl-CoA through geranylgeranyl pyrophosphate were developed. Both acid-labile and base-labile metabolites are simultaneously extracted with good recoveries into 7 M urea at neutral pH and low temperature to minimize hydrolytic and degradative enzyme losses, and the extract is partially purified by adsorption and desorption in high yield from an anion-exchange membrane. With the use of internal standards, the pathway intermediates are subsequently separated and quantified by reversed-phase ion-pair HPLC with on-line radiodetection. Permeable secretory cells specialized for monoterpene biosynthesis were isolated from peppermint (Mentha x piperita) leaves and employed as a model system to test the analytical protocols by examining the incorporation of [14C]pyruvate, [14C]mevalonate, and [3H]isopentenyl pyrophosphate as precursors. This simple new method should be readily adaptable to a wide range of cell and tissue types that can be administered basic metabolic precursors, and should allow measurements of both flux and steady-state levels of the intermediates of the mevalonic acid pathway.

Acetyl Coenzyme A↗

PubMed1993

4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase.

The committed step in the biosynthesis of monoterpenes in mint (Mentha) species is the cyclization of geranyl pyrophosphate to the olefin (-)-4S-limonene catalyzed by limonene synthase (cyclase). Internal amino acid sequences of the purified enzyme from spearmint oil glands were utilized to design three distinct oligonucleotide probes. These probes were subsequently employed to screen a spearmint leaf cDNA library, and four clones were isolated. Three of these cDNA isolates were full-length and were functionally expressed in Escherichia coli, yielding a peptide that is immunologically recognized by polyclonal antibodies raised against the purified limonene synthase from spearmint and that is catalytically active in generating from geranyl pyrophosphate a product distribution identical to that of the native enzyme (principally limonene with small amounts of the coproducts alpha- and beta-pinene and myrcene). The longest open reading frame is 1800 nucleotides and the deduced amino acid sequence contains a putative plastidial transit peptide of approximately 90 amino acids and a mature protein of about 510 residues corresponding to the native enzyme. Several nucleotide differences in the 5'-untranslated region of all three full-length clones suggest the presence of several limonene synthase genes and/or alleles in the allotetraploid spearmint genome. Sequence comparisons with a sesquiterpene cyclase, epi-aristolochene synthase from tobacco, and a diterpene cyclase, casbene synthase from castor bean, demonstrated a significant degree of similarity between these three terpenoid cyclase types, the first three examples of this large family of catalysts to be described from higher plants.

Amino Acid Sequence↗

PubMed1993

Production and characterization of polyclonal antibodies in rabbits to 4S-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 (-)-4S-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, Rutaceae) or wormseed (Chenopodium ambrosioides, Chenopodiaceae). Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiaceae, Asteraceae, 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.

Animals↗

PubMed1993

Hydride shifts in the biosynthesis of the p-menthane monoterpenes alpha-terpinene, gamma-terpinene, and beta-phellandrene.

The biosynthesis of several monoterpenes from the acyclic precursor geranyl pyrophosphate requires the migration of positive charge from the isopropyl side chain into the cyclohexenoid ring of the universal alpha-terpinyl cation intermediate of the reaction. The hydride shifts responsible for charge migration in the formation of three p-menthane olefin isomers were examined in a range of plant species: alpha-terpinene in American wormseed, gamma-terpinene in thyme, and (-)-beta-phellandrene in lodgepole pine. The experimental approach utilized soluble cell-free enzyme systems and specifically labeled geranyl substrates, with the determination of the labeling patterns in the resulting cyclic products. The results were consistent with stereoelectronic features of the cyclization and support the general model for monoterpene formation.

Cell-Free System↗

PubMed1993

Biosynthesis of monoterpenes: regio- and stereochemistry of (+)-3-carene biosynthesis.

Incubation of [1-3H1]geraniol with stem disks of Douglas fir (Pseudotsuga menziesii) and incubation of [1-3H1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine (Pinus contorta) resulted in the production of (+)-3-[3H] carene. Subsequent conversion of the product to car-3-en-5-one and to 4-isocaranone followed by base-catalyzed exchange of the alpha-hydrogens established that the 3H located at C1 in the geranyl substrate resided at C5 of (+)-3-carene. Incubation of the (+)-3-carene synthase preparation with (S)-[5-3H1, 4-14C]geranyl pyrophosphate resulted in the production of (+)-3-carene without loss of tritium, indicating that the 5-proR hydrogen is eliminated during cyclopropyl ring closure. Analysis of the conformational requirements for this 1,3 elimination involving the 5-proR hydrogen suggested that cyclopropyl ring formation occurs via a (4S)-alpha-terpinyl cation derived from the anti-endo cyclization of a (3S)-linalyl pyrophosphate intermediate. Kinetic analyses of the conversion of (1Z,3R)-[1-3H1]linalyl pyrophosphate, (1Z, 3S)-[1-3H1]linalyl pyrophosphate and [1-3H1]geranyl pyrophosphate by (+)-3-carene synthase revealed that the velocity of the reaction with the (3S)-linalyl enantiomer was 25-fold greater than the velocity with the (3R)-enantiomer and twice that of the natural substrate, geranyl pyrophosphate, thereby confirming this stereochemical prediction and also indicating that the cyclization of the linalyl intermediate is faster than the coupled isomerization and cyclization of the geranyl substrate. From these results, a model that details the regio- and stereochemistry of the enzymatic conversion of geranyl pyrophosphate to (+)-3-carene is proposed.

Bicyclic Monoterpenes↗

PubMed1993

Irreversible inactivation of monoterpene cyclases by a mechanism-based inhibitor.

Monoterpene synthases (cyclases) catalyze the divalent metal ion-dependent transformation of geranyl pyrophosphate to representative of the various monocyclic and bicyclic skeletal types by an electrophilic reaction mechanism involving coupled isomerization and cyclization steps. An analogue of the geranyl substrate, in which the terminal gem-dimethyl groups were joined to form a cyclopropyl function (6-cyclopropylidene-3E-methyl-hex-2-en-l-yl pyrophosphate) was shown to be a potent inhibitor of (-)-4S-limonene synthase from Mentha spicata and of several other monoterpene cyclases from diverse plant species. Inhibition was concentration and time dependent (pseudo-first-order kinetics), as well as absolutely contingent on the presence of the divalent metal ion cofactor. A double reciprocal plot of kinactivation versus inhibitor concentration gave an apparent Ki of approximately 0.3 microM and a maximum rate of inactivation of about 0.3 min-1 with limonene synthase. As expected for an active-site-directed process, the natural substrate, geranyl pyrophosphate, afforded protection against inactivation by the cyclopropylidene analogue. Selectivity of the inhibition was demonstrated with [1-3H]6-cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate by specific labeling of limonene synthase in crude enzyme extracts as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radio-fluorography, and immunoblotting. The radioactive cyclase-inactivator complex was formed with 1:1 stoichiometry and was stable to extended dialysis and boiling in 2% sodium dodecyl sulfate, suggesting irreversible covalent modification of the enzyme involving a chemical reaction between cyclase and inhibitor. Thermally denatured limonene synthase and synthase that had been inactivated with the histidine-directed reagent diethylpyrocarbonate or the cysteine-directed reagent p-hydroxymercuribenzoate (two reagents known to modify the active site of the enzyme and inhibit catalysis) were not labeled when treated with the [1-3H]-analogue, indicating that the functional enzyme was necessary to effect complex formation. All of the evidence is consistent with the analogue serving as a mechanism-based inactivator that must undergo both ionization-dependent isomerization and cyclization steps to reveal an allylic cation which alkylates the protein. In addition to furnishing supporting evidence for the electrophilic reaction sequence, this mechanism-based inactivator provides a powerful new approach for the examination of cyclase active sites.

Intramolecular Lyases↗

PubMed1992

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-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 (4R)- and the (4S)-thia and -aza analogs of the alpha-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 (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-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 discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.

Aza Compounds↗

PubMed1992

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.

Binding Sites↗

PubMed1992

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 alpha- and beta-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 cross-react 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.

Antibodies↗

PubMed1992

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.

Chromatography, Liquid↗

PubMed1992

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 (-)-alpha-pinene (40%) and (-)-beta-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 Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). 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.

Cations, Divalent↗

PubMed1992

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.

Cell-Free System↗

PubMed1992

Catabolism of camphor in tissue cultures and leaf disks of common sage (Salvia officinalis).

(+)-Camphor constitutes nearly 30% of the monoterpenes accumulated in the leaves of common sage (Salvia officinalis), and as the plant approaches maturity the content of this monoterpene ketone decreases by roughly half. Although the ability to catabolize camphor has been demonstrated previously in sage leaf disks, tissue cultures proved to be a more suitable system for examining the responsible degradative pathway. Cell suspension cultures were shown to convert (+)-[3-3H2]camphor, in sequence, to 6-hydroxycamphor, 6-oxocamphor, alpha-campholonic acid, and 2-hydroxy-alpha-campholonic acid, and each intermediate of the pathway was identified by chromatographic and spectroscopic means. This oxidative ring opening sequence resembles the pathway for camphor degradation by the soil diphtheroid, Mycobacterium rhodochrous, that ultimately leads to isoketocamphoric as the last defined metabolite that contains all 10 carbons of the original bicyclic nucleus. Studies with both cell cultures and leaf disks also demonstrated that the catabolism of camphor via 1,2-campholide, a metabolite in sage leaves previously described, was a minor degradative pathway. The first step in the metabolism of camphor was demonstrated in cell-free extracts of the cultured sage cells, and several lines of evidence indicated that this microsomal (+)-camphor-6-exo-hydroxylase is a cytochrome P-450-dependent monooxygenase.

Camphor↗

PubMed1992

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

(4S)-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 (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-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 Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, 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.

Chromatography, DEAE-Cellulose↗

PubMed1992

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.

Journal Article↗