Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Characteristic flavonoids from Acacia burkittii and A. acuminata heartwoods and their differential cytotoxicity to normal and leukemia cells.

Bioassay-guided fractionation of extracts from Acacia burkittii and A. acuminata heartwoods against an L1210 (mouse lymphoma) cell line led to the isolation of two flavan-3,4-diols, melacacidin (1) and isomelacacidin (2), and three flavonoids, 3,7,8,3',4'-pentahydroxyflavone (3), 7,8,3',4'-tetrahydroxyflavanone (4) and 3,7,8,3',4'-pentahydroxyflavanone (5). HPLC analyses (280 nm) of the 70% acetone extracts of the two plants showed different profiles in terms of the relative concentration of the five identified compounds. When tested against L1210, compounds 1 and 2 were the most active, giving ID50 values of 2.5 and 4.5 microg/mL, respectively. The lower activity of the other isolated compounds indicated the importance of the 3,4-hydroxyl groups for the cytotoxic activity of these flavonoids. The isolated compounds were either non-toxic or had very low toxicities against the "normal" CV-1 cell line (green monkey kidney cells). The O-methyl and O-acetyl derivatives of these compounds were inactive. Ten commercially available phenolic compounds (6-15) were also tested for their activity against both CV-1 and L1210 cell lines. Flavan-3,4-diols 1 and 2 were more cytotoxic to L1210 cells than all tested compounds, including catechin and epicatechin, the most abundant flavan-3-ols in many fruits and vegetable.

Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates.

Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the alpha,beta-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate. HPO also catalyzes the equivalent regio-specific (C-2) hydroxylation of several eremophilane-type (decalin ring system) sesquiterpenes, such as with 5-epi-aristolochene. Moreover, HPO displays interesting comparisons to other sesquiterpene hydroxylases. 5-Epi-aristolochene di-hydroxylase (EAH) differs catalytically from HPO by introducing hydroxyl groups first at C-1, then C-3 of 5-epi-aristolochene. HPO and EAH also differ from one another by 91-amino acid differences, with four of these differences mapping to putative substrate recognition regions 5 and 6. These four positions were mutagenized alone and in various combinations in both HPO and EAH and the mutant enzymes were characterized for changes in substrate selectivity, reaction product specificity, and kinetic properties. These mutations did not alter the regio- or stereo-specificity of either HPO or EAH, but specific combinations of the mutations did improve the catalytic efficiencies 10-15-fold. Molecular models and comparisons between HPO and EAH provide insights into the catalytic properties of these enzymes of specialized metabolism in plants.

ent-Beyerane diterpenoids from the heartwood of Excoecaria parvifolia.

Chromatographic fractionations of the toluene extract of the heartwood of Excoecaria parvifolia collected in Australia resulted in the isolation of 12 beyerane diterpenes (1-12), and the triterpene, lupeol. Four of the isolated diterpenoids (5-7 and 12) have unusual structures: ent-3-oxa-beyer-15-en-2-one, (5); ent-15,16-epoxy-2-hydroxy-19-norbeyer-1,4-dien-3-one (6); methyl ent-2,4-seco-15,16-epoxy-4-oxo-3,19-dinorbeyer-15-en-2-oate (7); and ent-2,17-dihydroxy-19-norbeyer-1,4,15-trien-3-one (12). The structures were established by spectroscopic analyses, NMR data comparisons with similar diterpenes, and chemical correlations. All the diterpenes are assumed to have the same absolute configuration as the co-occurring (+)-stachenol (4). Diosphenol 2 and nor-lactone 5 exhibited significant potency in bioassays for cytotoxic activity against leukemia cells (L1210). Plausible biosynthetic pathways are proposed to explain the origin of the diterpene metabolites.

Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH4 hybrid cyclase.

Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography-mass spectrometry (GC-MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3-->CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25-4.64 for the CH3-->CH2 eliminations; (3) a significant intermolecular KIE (1.33+/-0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1beta position of epiaristolochene; (5) stereoselective eliminations of the 1beta and 9beta protons in formation of epiaristolochene and its delta(1(10)) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1alpha proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.

Stereochemistry of the macrocyclization and elimination steps in taxadiene biosynthesis through deuterium labeling.

Taxadiene synthase catalyzes the cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) to taxa-4(5),11(12)-diene (Scheme 1, 5 --> 2) as the first committed step of Taxol biosynthesis. Deuterated GGPPs labeled stereospecifically at C-1, C-4, and C-16 were synthesized and incubated with recombinant taxadiene synthase from Taxus brevifolia to elucidate the stereochemistry of the cyclization reaction at these positions. The deuterium-labeled taxadienes obtained from (R)-[1-(2H1)]-, (S)-[1-(2H1)]-, and [16,16,16-(2H3)]GGPPs (9, 10, and 23b) were established to have deuterium in the 2alpha and 2beta CH2 and 16CH3 positions, respectively, by high-field 1H NMR spectroscopy (eqs 1-3). Incubation of (R)-[4-(2H1)]GGPP (17) with the recombinant enzyme gave a 10:10:80 mixture of [5beta-(2H1)]taxa-3(4),11(12)-diene, [5beta-(2H1)]taxa-4(20),11(12)-diene, and unlabeled taxa-4(5),11(12)-diene according to GC/MS analyses of the products (eq 4). It follows that C-1 of GGPP underwent inversion of configuration, that the A ring cyclization occurs on the si face of C15, and that the terminating proton abstraction removes H5beta from the final taxenyl carbocation intermediate. Thus, the C1-C14 and C15-C10 bonds are formed on the opposite faces of the 14,15 double bond of the substrate, i.e., overall anti electrophilic addition. The implications of these findings for the mechanism of the cyclization and rearrangement are discussed.

Eremophilane sesquiterpenes from capsidiol.

A series of eremophilane sesquiterpene alcohols and hydrocarbons was prepared from the phytoalexin capsidiol (1) for mechanistic studies with epiaristolochene synthase and epiaristolochene dihydroxylase. Among them, 3-deoxycapsidiol (10) was obtained through selective derivatization and reductive cleavage of the equatorial 3 alpha hydroxyl group. Two novel isomers of aristolochene and eremophilene were accessed from the 1- and 3-deoxycapsidiol isomers. 4-Epieremophilene (17) was obtained by conjugate reduction of epiaristolochen-1-one tosylhydrazone with catecholborane followed by sulfinate elimination and diimide rearrangement. Epimerization of epiaristolochen-3-one (27a) at the C4 methyl followed by reductions led to the previously unknown aristolochene isomer, eremophila-9(10),11(12)-diene (30). Optical rotations and characteristic (1)H NMR data for the related eremophilenols and dienes are collected in Tables 1 and 2. Finally, bioassays were used to assess the antifungal potencies of capsidiol and its synthetic derivatives. The minimum inhibitory concentration for capsidiol (3-10 ng) was at least 1 order of magnitude lower than that of any of the derivatives and considerably lower than those previously reported for ketoconazole, nystatin, and propiconazole.

Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis.

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene. Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9beta-pimara-7,15-diene synthase (OsDTS2). To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity. Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals. Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds. Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4. The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region. These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation. Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways.

Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products.

Rice produces a number of phytoalexins, and at least one allelopathic agent, from syn-copalyl diphosphate (CPP), representing the only known metabolic fate for this compound. Thus, the class II terpene synthase that converts the universal diterpenoid precursor geranylgeranyl diphosphate to syn-CPP catalyzes the committed step in biosynthesis of these natural products. Here the extensive sequence information available for rice was coupled to recombinant expression and functional analysis to identify syn-copalyl diphosphate synthase (OsCPSsyn). In addition, OsCPSsyn mRNA was found to be specifically induced in leaves by conditions that stimulate phytoalexin biosynthesis. Therefore, transcription of OsCPSsyn seems to be an important regulatory point for controlling the production of these defensive compounds. Finally, alignments carried out with OsCPSsyn revealed that class II terpene synthases exhibit a sequence conservation pattern substantially different from that of the prototypical class I enzymes. One particularly notable feature is the specific conservation of the functionally cryptic 'insertional' sequence element in class II terpene synthases, indicating that this region is important for the corresponding cyclization reaction.

Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates.

Abietadiene synthase (AS) catalyzes the complex cyclization-rearrangement of (E,E,E)-geranylgeranyl diphosphate (8, GGPP) to a mixture of abietadiene (1a), double bond isomers 2a-4a and pimaradienes 5a-7a as a key step in the biosynthesis of the abietane resin acid constituents (1b-4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a-c) were synthesized and evaluated as alternative substrates and/or inhibitors for recombinant AS from grand fir. The stereospecific cyclization of 8 alpha-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (K(i) = 0.1 nM) both suggest pimarenyl intermediates having the 13 beta methyl configuration and 8,14-double bond corresponding to sandaracopimaradiene (5a). The 2000-fold stimulation of inhibition by 26a in the presence of inorganic pyrophosphate indicates an important role for carbocation/OPP anion stabilization of the secondary sandaracopimaren-15-yl(+) ion. The failure of 8 beta-hydroxy-17-nor CPP (19b) to undergo enzymatic cyclization was taken as evidence that 9 is bound with a "coplanar" side chain conformation and that the S(N)' cyclization occurs on the 17 alpha face. The routing of the sandarcopimara-15-en-8-yl carbocation toward various diterpenes in biogenetic schemes is attributed to differing conformations of ring C and/or orientations of the C13 vinyl group in the active sites of the corresponding diterpene cyclases.