Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase.

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site.

NMR spectroscopic search module for Spektraris, an online resource for plant natural product identification--Taxane diterpenoids from Taxus × media cell suspension cultures as a case study.

Development and testing of Spektraris-NMR, an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMR allows users to search with multiple spectra at once and returns a table with a list of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of (1)H NMR and (13)C NMR spectroscopic peaks (query versus database entry) is provided.

Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies.

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation.

Improving peppermint essential oil yield and composition by metabolic engineering.

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway.

Synthesis and In Vitro Evaluation of Taxol oxetane ring D precursors.

A series of potential taxoid substrates was prepared in radiolabelled form to probe in vitro for the oxirane formation step and subsequent ring expansion step to the oxetane (ring D) presumably involved in the biosynthesis of the anticancer agent Taxol. None of the taxoid test substrates underwent transformation in cell-free systems from Taxus suggesting that these surrogates bore substitution patterns inappropriate for recognition or catalysis by the target enzymes, or that taxoid oxiranes and oxetanes arise by independent biosynthetic pathways.

A candidate cDNA clone for (-)-limonene-7-hydroxylase from Perilla frutescens.

Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (-)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (-)-limonene.

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production.

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5alpha,13alpha-diacetoxy-9alpha,10beta-diol to generate taxadien-5alpha,10beta,13alpha-tri-acetoxy-9alpha-ol and taxadien-5alpha,9alpha,13alpha-triacetoxy-10beta-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5alpha- or the 13alpha-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin.

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis.

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of beta-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2'-deoxytaxol, followed by hydroxylation on the side chain at the C2'-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2'-deoxytaxol [K. Walker, R.

A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint.

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha x piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran.

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (+/-)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase.

Structure of limonene synthase, a simple model for terpenoid cyclase catalysis.

The crystal structure of (4S)-limonene synthase from Mentha spic ata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-A; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction.

Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures.

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2.

Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway.

Administering Taxus suspension cells with labeled 5alpha-hydroxytaxadiene and 5alpha,10beta-dihydroxytaxadiene, and the corresponding 5alpha-acetate esters, demonstrated that acetylation at C5 of the monool precursor promotes the formation of 14beta-hydroxy taxoids, such as taxuyunnanine C, at the expense of 13alpha-hydroxy taxoids, including Taxol and its congeners, but that the major bifurcation in taxoid biosynthesis, toward 13alpha- or 14beta-hydroxy taxoids, occurs after 10beta-hydroxylation of the taxane core.

(-)-Menthol biosynthesis and molecular genetics.

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant.

Genetic engineering of peppermint for improved essential oil composition and yield.

The biochemistry, organization, and regulation of essential oil metabolism in the epidermal oil glands of peppermint have been defined, and most of the genes encoding enzymes of the eight-step pathway to the principal monoterpene component (-)-menthol have been isolated. Using these tools for pathway engineering, two genes and two expression strategies have been employed to create transgenic peppermint plants with improved oil composition and yield. These experiments, along with related studies on other pathway genes, have led to a systematic, stepwise approach for the creation of a 'super' peppermint.

Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae.

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size.

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis.

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity).

Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase.

(+)-Pulegone is a central intermediate in the biosynthesis of (-)-menthol, the most significant component of peppermint essential oil. Depending on environmental conditions, this branch point metabolite may be reduced to (-)-menthone en route to menthol, by pulegone reductase (PR), or oxidized to (+)-menthofuran, by menthofuran synthase (MFS). To elucidate regulation of pulegone metabolism, we modified the expression of mfs under control of the CaMV 35S promoter in transformed peppermint plants.

Alternative termination chemistries utilized by monoterpene cyclases: chimeric analysis of bornyl diphosphate, 1,8-cineole, and sabinene synthases.

Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile.

Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations.

Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site.

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures.

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv.

Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase.

The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-A resolution. Each monomer contains two alpha-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis.

Strategies for transgenic manipulation of monoterpene biosynthesis in plants.

Monoterpenes, the C(10) isoprenoids, are a large family of natural products that are best known as constituents of the essential oils and defensive oleoresins of aromatic plants. In addition to ecological roles in pollinator attraction, allelopathy and plant defense, monoterpenes are used extensively in the food, cosmetic and pharmaceutical industries. The importance of these plant products has prompted the definition of many monoterpene biosynthetic pathways, the cloning of the relevant genes and the development of genetic transformation techniques for agronomically significant monoterpene-producing plants.