Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase.

The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.     

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. The perilla cDNA was a partial clone of 1550 bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively.     

Effect of Mentha x piperita essential oil and monoterpenes on cucumber root membrane potential.

Peppermint (Mentha piperita L.) essential oil and its main components were assessed for their ability to interfere with plant plasma membrane potentials. Tests were conducted on root segments isolated from etiolated seedlings of cucumber (Cucumis sativus L.). Increasing the concentration of peppermint essential oil from 5 to 50 ppm caused a decrease in membrane potential (Vm) hyperpolarization of 10-3 mV, whereas concentrations from 100 up to 900 ppm caused an increasing depolarization of Vm (from 5 to 110 mV). When tested at 300 ppm, (+)-menthyl acetate, (-)-limonene and 1,8-cineole did not exert any significant effect on V(m), whereas (+)-menthofuran (73 mV), (+)-pulegone (85 mV), (+)-neomenthol (96 mV), (-)-menthol (105 mV) and (-)-menthone (111 mV) showed increased ability to depolarize V(m). A plot of log of octanol-water partition coefficient (K(ow)) against their depolarizing effect showed a significant negative correlation, suggesting that among all monoterpenoids increased membrane depolarization depends on lower K(ow). However, among monoterpene ketones, alcohols and furans, increased membrane depolarization is associated with a decline in water solubility. The possible effect of monoterpenoids on membrane ion fluxes is also discussed, since changes in the bioelectric potential of cells imply changes in the flux of ions across the plasma membrane     

Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and saccharomyces cerevisiae

The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In spearmint (M. spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and thence (-)-carvone, whereas in peppermint (M. x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol. cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181-192, 1999). Although both hydroxylase genes were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detailed study of the structure-function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases. Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined.     

Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis)

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.     

Volatile Terpenoids of Endophyte-free and Infected Peppermint (<Emphasis Type="Italic">Mentha piperita</Emphasis> L.): Chemical Partitioning of a Symbiosis

The study reports the effects on volatiles of an endophytic fungus inhabiting asymptomatically the leaves of peppermint. By means of headspace solid-phase microextraction (HS-SPME) and gaschromatography-mass spectrometry (GC-MS) terpenoids were sampled in time course from the head space of peppermint leaves and roots. After removal of the mycelium from peppermint tissues, fungal volatiles were analyzed and compared with those of pure fungal cultures. In the presence of the endophyte, the relative amount of all main compounds increased in leaves. Starting from the first 14 d of culture, (−)-menthone and (+)-neomenthol were consistently higher than in control plants. On the contrary, (+)-menthofuran increased only by 28 d of culture. Root volatiles were also dramatically altered by the presence of the fungus, with (+)-pulegone accounting for at least 44% of the total volatile emission. (+)-Pulegone was also the main compound of PGP-HSF mycelium isolated from peppermint roots. The sesquiterpenoid cuparene was found as a novel compound of peppermint leaf headspace and was a main volatile of ex planta and pure culture mycelia. The chemical spectrum of terpenoids and their distribution among peppermint roots, leaves, and mycelia are likely to account for a fine regulation of the mutualism in planta and for the acquisition by the fungus of novel metabolic competences. This work is dedicated to the memory of Prof. Silvano Scannerini.     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Effect of (+)-pulegone and other oil components of Mentha x Piperita on cucumber respiration

Peppermint (Mentha x piperita L.) essential oil and main components were assessed for their ability to interfere with plant respiratory functions. Tests were conducted on both root segments and mitochondria isolated by etiolated seedlings of cucumber (Cucumis sativus L.). Total essential oil inhibited 50% of root and mitochondrial respiration (IC50) when used at 324 and 593 ppm, respectively. (+)-Pulegone was the most toxic compound, with a 0.08 and 0.12 mM IC50 for root and mitochondrial respiration, respectively. (-)-Menthone. followed (+)-pulegone in its inhibitory action (IC50 values of 1.11 and 2.30 mM for root and mitochondrial respiration respectively), whereas (-)-menthol was the less inhibitory compound (IC50 values of 1.85 and 3.80 mM respectively). A positive correlation was found for (+)-pulegone, (-)-menthone and (-)-menthol between water solubility and respiratory inhibition. The uncoupling agent. carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), lowered (-)-menthol and (-)menthone inhibition and annulled (+)-pulegone inhibition of mitochondrial respiration, whereas salicyl-hydroxamic acid (SHAM) 2-hydroxybenzohydroxamic acid, the alternative oxidase (AO) inhibitor, increased (-)-menthone inhibition and annulled both (+)-pulegone and (-)-menthol inhibitory activity. The possible interaction of (-)-pulegone and (-)-menthol with AO and the mechanism of action of(+)-pulegone, (-)-menthone and (-)-menthol on mitochondrial respiration are discussed.     

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.     

Influence of phosfon D and cycocel on growth and essential oil content of sage and peppermint

Foliar application of Phosfon D at 50–100 ppm stimulates the growth of Salvia officinalis (sage) and moderately retards the growth of Mentha piperita (peppermint), while increasing the essential oil yield of both species by 50–70 % Phosfon D increases the proportions of (−)-3-isothujone and (+)-3-thujone in sage oil and decreases the level of (−)-β-pinene and (+)-camphor, whereas this growth retardant increases the proportions of (+)-isomenthone and (+)-neoisomenthol in peppermint oil and decreases the level of(−)-menthone and (−)-menthoL Foliar application of Cycocel at 250–500 ppm slightly stimulates growth and essential oil formation in peppermint, and retards growth of sage with little effect on oil yield. The influence of Cycocel on sage oil composition was the opposite of that of Phosfon, with a tendency to increase the level of (−)-β-pinene and decrease the level of (−)-3-isothujone under severe stunting. The effect of Cycocel on the composition of peppermint varied with concentration. The influence of growth retardants on essential oil composition and yield are most readily explained by alterations in the levels or activities of the relevant enzymes.     

Mathematical Modeling-Guided Evaluation of Biochemical,Developmental, Environmental,and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

We have previously reported the use of a combination of computational simulations and targeted experiments to build a first generation mathematical model of peppermint (Mentha × piperita) essential oil biosynthesis. Here, we report on the expansion of this approach to identify the key factors controlling monoterpenoid essential oil biosynthesis under adverse environmental conditions. We also investigated determinants of essential oil biosynthesis in transgenic peppermint lines with modulated essential oil profiles. A computational perturbation analysis, which was implemented to identify the variables that exert prominent control over the outputs of the model, indicated that the essential oil composition should be highly dependent on certain biosynthetic enzyme concentrations [(+)-pulegone reductase and (+)-menthofuran synthase], whereas oil yield should be particularly sensitive to the density and/or distribution of leaf glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. A microscopic evaluation of leaf surfaces demonstrated that the final mature size of glandular trichomes was the same across all experiments. However, as predicted by the perturbation analysis, differences in the size distribution and the total number of glandular trichomes strongly correlated with differences in monoterpenoid essential oil yield. Building on various experimental data sets, appropriate mathematical functions were selected to approximate the dynamics of glandular trichome distribution/density and enzyme concentrations in our kinetic model. Based on a χ² statistical analysis, simulated and measured essential oil profiles were in very good agreement, indicating that modeling is a valuable tool for guiding metabolic engineering efforts aimed at improving essential oil quality and quantity.The essential oil distilled from peppermint (Mentha × piperita) leaves is used in numerous consumer products (e.g. chewing gum, toothpaste, and mouthwash), as a flavor in the confectionary and pharmaceutical industries, and as a source of active ingredients for aromatherapy. Peppermint oil consists primarily of p-menthane-type monoterpenes, with smaller amounts of other monoterpenes and very minor quantities of sesquiterpenes (Rohloff, 1999). The essential oil is synthesized and accumulated in specialized anatomical structures called peltate glandular trichomes (Gershenzon et al., 1989; McCaskill et al., 1992). These trichomes contain secretory cells, arranged in an eight-celled disc, which are responsible for the synthesis of the oil. Nascent essential oil is secreted into an emerging cavity formed by the separation of a preformed layer of cuticular material (Amelunxen, 1965). Over the last two decades, the entire complement of genes and enzymes involved in the peppermint monoterpenoid essential oil biosynthetic pathway has been characterized (for review, see Croteau et al., 2005).Transgenic peppermint plants have been generated in efforts aimed at modulating essential oil yield and composition. Mahmoud and Croteau (2001) reported that, by overexpressing the gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), oil yield increases (compared with wild-type plants) of up to 50% were observed. Antisense suppression of the (+)-menthofuran synthase (MFS) gene led to a dramatic decrease in the amounts of the undesirable side product (+)-menthofuran (elite transgenic line designated MFS7a; Mahmoud and Croteau, 2001). A slight increase in overall monoterpene yields was reported for transgenic plants with increased expression levels of the gene encoding (−)-limonene synthase (LS; Diemer et al., 2001), whereas only negligible effects on yield were detected in an independent study (Krasnyansky et al., 1999). Transgenic plants overexpressing the gene coding for (−)-limonene 3-hydroxylase (L3H) did not accumulate increased levels of the recombinant protein, and the composition and yield of the essential oils were the same as in wild-type controls; however, cosuppression of the L3H gene resulted in a vastly increased accumulation of the intermediate (−)-limonene, without notable effects on oil yield (elite transgenic line designed L3H20; Mahmoud et al., 2004).Mathematical modeling can be a powerful tool to support metabolic engineering efforts, including those performed with peppermint. Stoichiometric modeling only requires knowledge of the topology of reactions in the pathway and inputs/outputs. This is a particularly useful approach to determine flux distributions and the systemic characteristics of metabolic networks (for review, see Llaneras and Picó, 2008). When experimental designs supporting metabolic and isotopic steady state are employed, isotope labeling data can be utilized for the development of quantitative flux maps of metabolic pathways (for review, see Libourel and Shachar-Hill, 2008). For dynamic systems, kinetic modeling is regarded as the generally most suitable method (McNeil et al., 2000; Poolman et al., 2004; Bruggeman and Westerhoff, 2006; Rios-Estepa and Lange, 2007; Mendes et al., 2009). Building on the rich body of published data on the enzymology and physiology of the peppermint monoterpene pathway (for review, see Croteau et al., 2005), we recently developed a first generation kinetic model to simulate the dynamics of peppermint monoterpene composition (Rios-Estepa et al., 2008). Modeling indicated that the monoterpene profiles observed in leaves of plants grown under low-light conditions could be explained if one assumed that (+)-menthofuran, a dead-end side product, acted as a heretofore unknown competitive inhibitor against (+)-pulegone, the primary substrate of the branch point enzyme (+)-pulegone reductase (PR; Fig. 1). Follow-up biochemical studies established that this prediction was correct (Rios-Estepa et al., 2008), thus illustrating the utility of an approach that integrates mathematical modeling with experimental testing.Open in a separate windowFigure 1.Outline of p-menthane monoterpene biosynthesis in peppermint glandular trichomes. The following enzymes are involved in this pathway: 1, 1-deoxy-d-xylulose 5-phosphate synthase; 2, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; 3, 2C-methyl-d-erythritol 4-phosphate cytidyltransferase; 4, 4-(cytidine 5′-diphospho)-2C-methyl-d-erythritol kinase; 5, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; 6, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase; 7, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; 8, isopentenyl diphosphate isomerase; 9, geranyl diphosphate synthase; 10, (−)-limonene synthase; 11, (−)-limonene 3-hydroxylase; 12, (−)-trans-isopiperitenol dehydrogenase; 13, (−)-trans-isopiperitenone reductase; 14, (+)-cis-isopulegone isomerase; 15, (+)-menthofuran synthase; 16a, (+)-pulegone reductase [(−)-menthone-forming activity]; 16b, (+)-pulegone reductase [(+)-isomenthone-forming activity]; 17a, (−)-menthone:(−)-menthol reductase [(−)-menthol-forming activity]; 17b, (−)-menthone:(−)-menthol reductase [(+)-neoisomenthol-forming activity]; 18a, (−)-menthone:(+)-neomenthol reductase [(+)-neomenthol-forming activity]; 18b, (−)-menthone:(+)-neomenthol reductase [(+)-isomenthol-forming activity]. The subcellular compartmentation of p-menthane metabolic enzymes is color coded as follows: Cyt (blue), cytosol; ER (orange), endoplasmic reticulum; Lpl (green), leucoplasts; Mit (red), mitochondria. The inhibitory effects of (+)-menthofuran on (+)-pulegone reductase and geranyl diphosphate on isopentenyl diphosphate isomerase are indicated by red arcs with orthogonal red lines. Names of selected metabolites are shown in the colors that are used to indicate the corresponding profiles in Figures 2 to 5​5​​.As part of this study, a computational perturbation analysis was used to predict factors with the potentially greatest impacts on peppermint essential oil yield and composition (specific biosynthetic enzymes and the density of oil-synthesizing trichomes). To test these modeling predictions experimentally, we first acquired biometric data with peppermint plants grown under several environmental conditions known to adversely affect oil accumulation (Burbott and Loomis, 1967; Clark and Menary, 1980) and the transgenic line MFS7a, for which an altered essential oil profile had been reported earlier (Mahmoud and Croteau, 2001). Building on these experimental data sets, we then developed a second generation model that accounts for biochemical, developmental, environmental, and genotypic factors of essential oil formation. This updated model was then used to simulate monoterpenoid essential oil profiles for the transgenic line MFS7a grown under low-light environmental stress conditions and the transgenic line L3H20, which had previously been shown to have vastly reduced expression levels of the gene encoding L3H. In both cases, simulated and measured monoterpene patterns were very similar, indicating that mathematical modeling has great potential for guiding efforts aimed at developing peppermint lines with high oil yields and favorable composition, even under adverse environmental conditions.     

Metabolism of monoterpenes: demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita)

Piperitenone is commonly considered to be the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint; however, [3H]piperitenone gave rise only to the inert metabolite (+)-piperitone when incubated with peppermint leaf discs. Under identical conditions, (-)-[3H]isopiperitenone was efficiently incorporated into (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and yielded an additional metabolite identified as (+)-cis-isopulegone; piperitenone was poorly labeled. Moreover, (+)-cis-[3H]isopulegone was rapidly converted to (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and the reduction of (+)-[3H]pulegone to (-)-menthone and (+)-isomenthone was similarly documented. Each step of the pathway was demonstrated in a crude soluble preparation from peppermint leaf epidermis and each of the relevant enzymes was partially purified in order to compare relative rates of catalysis. The results of these studies indicate that the endocyclic double bond of (-)-isopiperitenone is reduced to yield (+)-cis-isopulegone, which is isomerized to (+)-pulegone as the immediate precursor of (-)-menthone and (+)-isomenthone, and they rule out piperitenone as an intermediate of the pathway.     

Biochemical characterization of a spearmint mutant that resembles peppermint in monoterpene content

A radiation-induced mutant of Scotch spearmint (Mentha × gracilis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the C6-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and C6-oxygenated families of monoterpenes from the common precursor (−)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (−)-limonene hydroxylase; the C6-hydroxylase producing (−)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (−)-trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the α,β-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3- and C6-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha.     

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL/6 mouse liver

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.     

Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.