Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase.

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.

Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites.

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.

Genomic organization of plant terpene synthases and molecular evolutionary implications.

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.

Peppermint (Mentha x piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.

Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast.

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme.

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.

Distribution of peltate glandular trichomes on developing leaves of peppermint.

The pattern of peltate glandular trichome initiation and ontogeny on expanding peppermint (Mentha x piperita) leaves was defined by surveying the populations of peltate glands in each of seven developmental stages within sampling areas of leaf apical, mid-, and basal zones for both abaxial and adaxial surfaces. It was shown that new peltate glands continue to form until leaf expansion ceases and that regions of active gland initiation are unevenly distributed. The distribution of gland initiation reflects the basipetal pattern of leaf maturation, with relatively immature regions at the leaf base continuing to produce oil glands long after gland production has stopped at the leaf apex. The proportion of glands in the secretory stage as a function of leaf development and the direct observations of living glands over a period of 33 h indicate that a period of only 20 to 30 h of secretory activity is required for filling of the gland storage compartment with essential oil. These findings are discussed in relation to earlier literature describing age-related changes in glandular essential oil content.

Development of peltate glandular trichomes of peppermint.

Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha x piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.

Regulation of monoterpene accumulation in leaves of peppermint.

Plants synthesize numerous classes of natural products that accumulate during development and are thought to function as constitutive defenses against herbivores and pathogens. However, little information is available about how the levels of such defenses are regulated. We measured the accumulation of monoterpenes, a model group of constitutive defenses, in peppermint (Mentha x piperita L.) leaves and investigated several physiological processes that could regulate their accumulation: the rate of biosynthesis, the rate of metabolic loss, and the rate of volatilization. Monoterpene accumulation was found to be restricted to leaves of 12 to 20 d of age, the period of maximal leaf expansion. The rate of monoterpene biosynthesis determined by (14)CO(2) incorporation was closely correlated with monoterpene accumulation, as determined by gas chromatographic analysis, and appeared to be the principal factor controlling the monoterpene level of peppermint leaves. No significant catabolic losses of monoterpenes were detected throughout leaf development, and monoterpene volatilization was found to occur at a very low rate, which, on a monthly basis, represented less than 1% of the total pool of stored monoterpenes. The composition of volatilized monoterpenes differed significantly from that of the total plant monoterpene pool, suggesting that these volatilized products may arise from a separate secretory system. With the demonstration that the rate of biosynthesis is the chief process that determines monoterpene accumulation in peppermint, efforts to improve production in this species can now focus on the genes, enzymes, and cell differentiation processes that regulate monoterpene biosynthesis.

Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint.

Monoterpene production in peppermint (Mentha x piperita L.) glandular trichomes is determined by the rate of biosynthesis, as determined by (14)CO(2) incorporation, and is restricted to leaves 12 to 20 d of age. Using oil glands isolated from peppermint leaves of different ages, in vitro assay of the eight sequential enzymes responsible for the biosynthesis of the principal monoterpene (-)-menthol indicated that all but one biosynthetic enzyme had a very similar developmental profile. Activities were highest in leaves 12 to 20 d of age, with a sharp peak centered at 15 d. The exception, (-)-menthone reductase, the last enzyme of the pathway, exhibited a later peak of activity, which was centered at approximately 21 d. The correlation between in vitro enzyme activity and the rate of biosynthesis measured in vivo suggests that monoterpene formation is controlled mainly by the coordinately regulated activity of the relevant biosynthetic enzymes. Developmental immunoblotting of limonene synthase, which catalyzes the committed step of the pathway, demonstrated a direct correlation between enzyme activity and enzyme protein, suggesting that the dynamic time course for the remaining pathway enzyme activities also reflects the corresponding protein levels. RNA-blot analyses indicated that the genes encoding enzymes of the early pathway steps are transcriptionally activated in a coordinated fashion, with a time course superimpossible with activity measurements and immunoblot data. These results demonstrating coincidental temporal changes in enzyme activities, enzyme protein level, and steady-state transcript abundances indicate that most of the monoterpene biosynthetic enzymes in peppermint are developmentally regulated at the level of gene expression.

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate.

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.

Method for radio-capillary gas chromatography employing a modified oxidation-reduction train and flow-through detector.

A conventional radioactivity monitor was modified by reducing the volume of the oxidation-reduction train and gas proportional counting tube to permit coupled radio-capillary gas chromatographic analysis of labeled isomeric metabolites. The utility of this radioactivity monitoring technique was demonstrated by separating 3H-labeled sesquiterpene olefins. The advantages and limitations of mass (thermal conductivity) and radioactivity detection by this method are discussed.

Applications of gas chromatography to the study of terpenoid metabolism.

Studies on the enzymology and mechanism of biosynthesis of the essential oil terpenes are often hampered by the need to resolve and detect trace levels of these metabolites, an analytical requirement for which gas chromatography is ideally suited. Essential principles in the application of gas chromatography to terpenoid metabolism are described, with particular emphasis on experimental strategies employing flame ionization, mass spectrometric and thermal conductivity-radiochemical detection methods. The general approaches described can be readily adapted to studies on the origin of other volatile natural products.

Resolution of monoterpene enantiomers by gas chromatography.

Enantiomeric monoterpenes can be resolved by gas chromatography on conventional capillary and packed columns following conversion to diastereomeric ketals of (2R,3R)-2,3-butanediol. Efficient methods are described for the derivatization and separation of sub-milligram quantities of the enantiomers of alpha-pinene, beta-pinene, camphene, sabinene, alpha-thujene, limonene and 3-carene, as well as of structurally related alcohols and ketones.

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.

Mechanism of the pyrophosphate migration in the enzymatic cyclization of geranyl and linalyl pyrophosphates to (+)- and (-)-bornyl pyrophosphates.

Soluble enzymes from sage (Salvia officinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of 2-bornyl pyrophosphate, were employed to evaluate mechanistic alternatives for the pyrophosphate migration in monoterpene cyclization reactions. Separate incubation of [1-3H2,alpha-32P]- and [1-3H2,beta- 32P]geranyl and (+/-)-linalyl pyrophosphates with partially purified preparations of each enantiomer-generating cyclase gave [3H, 32P]bornyl pyrophosphates, which were selectively hydrolyzed to the corresponding bornyl phosphates. Measurement of 3H:32P ratios of these monophosphate esters established that two ends of the pyrophosphate moiety retained their identifies in the cyclization of both precursors to both products and also indicated that there was no appreciable exchange with exogenous inorganic pyrophosphate in the reaction. Subsequent incubations of each cyclase with [8,9-14C,1-18O]geranyl pyrophosphate and with (1E)-(+/-)-[1-3H,3-18O]linalyl pyrophosphate gave the appropriate (+)- or (-)-bornyl pyrophosphates, which were hydrolyzed in situ to the corresponding borneols. Analysis of the derived benzoates by mass spectrometry demonstrated each of the product borneols to possess an 18O enrichment essentially identical with that of the respective acyclic precursor. The absence of P alpha-P beta interchange and the complete lack of positional 18O isotope exchange of the pyrophosphate moiety are compatible with tight ion pairing of intermediates in the coupled isomerization-cyclization of geranyl pyrophosphate and establish a remarkably tight restriction on the motion of the transiently generated pyrophosphate anion with respect to its cationic terpenyl reaction partner.