Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew.

The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa-4(20),11(12)-diene were tested as possible olefinic intermediates in taxa-4(5),11(12)-diene formation by a series of inhibition, trapping, and direct conversion experiments; no evidence was obtained that these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.

Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the committed step of taxol biosynthesis in Pacific yew.

The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol.

Biosynthesis of monoterpenes: partial purification, characterization, and mechanism of action of 1,8-cineole synthase.

Geranyl pyrophosphate: 1,8-cineole cyclase (cineole synthase) catalyzes the conversion of geranyl pyrophosphate to the symmetrical monoterpene ether 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) by a process thought to involve the initial isomerization of the substrate to the tertiary allylic isomer, linalyl pyrophosphate, and cyclization of this bound intermediate to the alpha-terpinyl carbocation that is subsequently captured by water and undergoes heterocyclization to the remaining double bond. The enzyme was isolated from the secretory cells of the glandular trichomes of Salvia officinalis (garden sage) and partially purified, and the properties of this monoterpene cyclase, previously determined in crude cell-free extracts, were reexamined. These properties (pH optimum, divalent metal ion requirement, molecular weight, pI) were similar to those determined previously with the exception of substrate utilization; geranyl pyrophosphate was shown to be a more efficient substrate than the cis-isomer, neryl pyrophosphate, in the absence of competing phosphatase activity that contaminated earlier preparations of this enzyme. As with other monoterpene cyclases of herbaceous species, cineole synthase was inhibited by cysteine- and histidine-directed reagents, and protection against inactivation was provided by the substrate-metal ion complex. Studies with 18O-labeled acyclic precursors and H(2)18O, followed by mass spectrometric analysis of the product, confirmed that water was the sole source of the ether oxygen atom of 1,8-cineole. The electrophilic nature of the coupled isomerization-cyclization reaction was examined with a series of substrate and intermediate analogues. The overall stereochemistry of the cyclization of geranyl pyrophosphate to the symmetrical monoterpene was established by determining the enantioselectivity for (3R)- or (3S)-linalyl pyrophosphate as an alternative substrate and by oxidation of [3-3H]1,8-cineole, derived from [1-3H]geranyl pyrophosphate, to (+/-)-3-keto-1,8-cineole and radio-GLC separation of diastereomeric ketal derivatives to determine the labeled enantiomer.

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.

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.