Phytochem Rev (2006) 5:373–383 DOI 10.1007/s11101-006-9008-2

Cytochrome P450 oxygenases of monoterpene metabolism Christopher J. D. Mau Æ Rodney Croteau

Received: 1 February 2006 / Accepted: 13 June 2006 / Published online: 28 October 2006
Springer Science+Business Media B.V. 2006

Abstract The cytochrome P450 monoterpene oxygenases are largely responsible for imparting structural and functional diversity to this family of natural products. In most cases, cytochrome P450mediated allylic hydroxylation of a parental monoterpene olefin leads to a series of redox transformations and conjugation reactions which yield a family of structurally related derivatives and isomers. An overview is provided of the extant monoterpene oxygenases, with examples mainly from the mint (Lamiaceae) family of essential oil plants, and, where possible, information on the structure, mechanism, localization and regulation of these enzymes is described. The review concludes with a brief assessment of biotechnological applications and a view to future research in this area. Keywords

Hydroxylase Æ Lamiaceae

Introduction Monoterpenoids are the major components of most plant essential oils (Guenther 1950). More than 3400 monoterpenes, which fall into several C. J. D. Mau Æ R. Croteau (&) Institute of Biological Chemistry, Washington State University, P.O. Box 646340, Pullman, WA 99164-6340, USA e-mail: [email protected]

basic cyclic carbon skeletons, including the thujanes, pinanes, p-menthanes, bornanes, caranes, camphanes and fenchanes, as well as a few acyclic types, have been described (Buckingham 2004). The generation of diverse oxygenated derivatives of these basic skeletal types is usually initiated by cytochrome P50 hydroxylases. Hydroxylation of an allylic carbon of the parental monoterpene olefin is often followed by dehydrogenation to the corresponding ketone, followed by reduction of the conjugated double bond and the carbonyl to produce a family of related derivatives and stereoisomers (Fig. 1) (Karp and Croteau 1992). Additional modification of the alcohol by methylation, acylation, or glycosylation can further diversify the product set and impart quite different physical and biological properties. The regiospecificity of hydroxylation of the parental olefin can have profound influence on the biological properties, including the organoleptic perception, of the resulting monoterpene derivatives. For example, a gamma-irradiation induced mutant of Scotch spearmint (Horner and Melouk 1977), which normally produces C6-oxygenated derivatives of limonene (see Fig. 2a), was shown to produce C3-oxygenated limonene derivatives with peppermint-like organoleptic properties (Croteau et al. 1991). Mutations that uncover previously silent oxygenases (Bertea et al. 2003) or alter the regio- or stereospecificity of monoterpene hydroxylation reactions may

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Fig. 1 Generation of structural and functional diversity by allylic hydroxylation of a monoterpene olefin, followed by redox transformations (and often conjugation reactions) to yield a family of derivatives and isomers with the same oxygenation patterns [adapted from Karp and Croteau (1992)]

similarly influence biological properties and thus natural selection processes by altering monoterpene-based defensive and reproductive strategies (Dudareva et al. 2004). In this review, we summarize information about cytochrome P450-mediated oxygenations of plant monoterpenoids based on studies with both native and recombinant enzymes. Where possible, organizational and regulatory aspects of these cytochrome P450 hydroxylases are reviewed, structure–function relationships are described, and biotechnological applications are addressed. Hydroxylases involved in monoterpene iridoid alkaloid biosynthesis are not covered here.

Monoterpenoid biosynthesis Several cytochrome P450 monoterpene hydroxylases of the mint (Lamiaceae) family have been cloned to permit detailed characterization and assessment of the roles of these catalysts in monoterpene metabolism; these studies form the principal focus of this review. The monoterpenoids of mint and of other plant species are derived from the universal precursor geranyl diphosphate (Fig. 2a) which is produced by the action of geranyl diphosphate synthase (Burke et al. 1999) using isoprenoid precursors from the plastidial methyl erythritol phosphate pathway (Rohmer 1999). Geranyl diphosphate is initially ionized, and the resulting carbocation can give

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rise to simple acyclic products (Fig. 2b) or undergo a series of internal additions and rearrangements followed by deprotonation to yield the parent cyclic olefins of the various structural types (Fig. 2a). These parental olefins are the substrates for the allylic hydroxylation steps of the ‘‘allylic oxidation-conjugate reduction’’ pathways (Fig. 1) that give rise to most monoterpene derivatives. The p-menthane monoterpene limonene [the (+)-enantiomer is found in caraway and citrus species, and the (–)-enantiomer is produced in mints] is the simplest of the cyclic monoterpenes, and the hydroxylation of this compound Fig. 2 Pathways for the biosynthesis of oxygenatedc monoterpenes of the cyclic (a) and acyclic (b) type that are initiated by plant cytochrome P450 hydroxylases and epoxidases. Dashed arrows represent multiple enzymatic steps. In peppermint, (–)-trans-isopiperitenol leads to the fully reduced monoterpenol (–)-menthol, and the intermediate (+)-pulegone is converted to (+)-menthofuran in a side reaction; in spearmint, (–)-trans-carveol is converted to the corresponding ketone (–)-carvone; in perilla, (–)perillyl alcohol is oxidized to perillyl aldehyde; in sage, (+)-cis-sabinol is transformed into sabinone and the reduced ketones, thujone and isothujone, and in senescing sage cell suspension cultures, 6-exo-hydroxycamphor eventually is transformed into 2-hydroxycampholonic acid; in hyssop, (+)-trans-pinocarveol gives rise to pinocarvone and the reduced ketones, pinocamphone and isopinocamphone, while (–)-myrtenol is the terminal product unlike the case of wild strawberry in which (–)-myrtenol is either glycosylated or further acylated to myrtenyl acetate; (+)trans-carveol is oxidized to (+)-carvone in caraway. For the acyclic monoterpenoids, 10-hydroxycitronellol is transformed into nepetalactones in catmint

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provides the most illustrative example of the function of cytochrome P450 hydroxylases in monoterpene metabolism.

Functionally assigned hydroxylase genes In peppermint (Mentha · piperita), spearmint (Mentha spicata) and perilla (Perilla frutescens), (–)-limonene is regiospecifically hydroxylated at the C3, C6 or C7 positions, respectively, to yield (–)-trans-isopiperitenol, (–)-trans-carveol or (–)perillyl alcohol (Fig. 2a) (Karp et al. 1990). These highly specific cytochrome P450-mediated reactions set the oxygenation pattern of all subsequent monoterpene derivatives produced by these three mint species (Koezuka et al. 1986; Kjonaas et al. 1985). In mints, the essential oil is produced in glandular trichomes (Gershenzon et al. 1989; Turner et al. 1999; Turner and Croteau 2004), which can be readily isolated as an enriched source of monoterpene biosynthetic enzymes and their corresponding transcripts, a feature which has greatly assisted in pathway elucidation, enzyme characterization and defining the underlying molecular genetics of monoterpene production (McCaskill et al. 1992; Gershenzon et al. 1992; Lange et al. 2000; Ringer et al. 2003; Davis et al. 2005; Ringer et al. 2005). cDNAs encoding the regiospecific limonene-3hydroxylases (CYP71D13 and CYP71D15) from peppermint and the regiospecific limonene-6hydroxylase (CYP71D18) from spearmint have been cloned (Lupien et al. 1999), and isolation of the cDNA encoding limonene-7-hydroxylase from Perilla frutescens is in progress. The spearmint and peppermint enzymes are 70% identical and 85% similar at the deduced amino acid level (Lupien et al. 1999). The limonene-3-hydroxylases from M. spicata L. ‘Crispa’ (Lu¨cker et al. 2004) and M. gracilis mutant 643 (Bertea et al. 2003) are about 90% identical to the limonene-3hydroxylases of peppermint and about 70% identical to the limonene-6-hydroxylase of spearmint, illustrating the similarity of these limonene hydroxylases from Mentha species. The apparent Km values for the native (–)limonene hydroxylases are in the 18 – 21 lM range (Karp et al. 1990), and the kd values for the

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recombinant forms (expressed in E. coli) are roughly ten-fold lower with turnover numbers in the range of 0.2–1.1 min–1 (Haudenschild et al. 2000). The products of C3- and C6-hydroxylation (trans-isopiperitenol and trans-carveol, respectively) do not appreciably accumulate in the essential oils of peppermint and spearmint (McCaskill et al. 1992), indicating that subsequent redox steps of the respective pathways were very efficient during monoterpenoid production in these mint species (Turner and Croteau 2004). Another cloned monoterpene hydroxylase from peppermint is (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase) (Fig. 2a) (Bertea et al. 2001). This enzyme also catalyzes an allylic hydroxylation in the conversion of (+)pulegone to (+)-menthofuran; because of its strong odor, menthofuran is an undesirable component of peppermint essential oil when the levels exceed about 5%. Alteration of the expression of this gene has been the target of metabolic engineering in transgenic plants (see below). Menthofuran synthase exhibits 35–38% sequence identity with the limonene-3- and –6-hydroxylases, and is expressed late in leaf development (Gershenzon et al. 2000) and probably under physiological stress (Burbott and Loomis 1967; Clark and Menary 1980). Recent investigation of fragrances emitted by strawberry fruit (Fragaria spp) (Aharoni et al. 2004) led to the acquisition of a cytochrome P450 cDNA clone that, when expressed in yeast, catalyzed the allylic hydroxylation of a-pinene (at the C10 position) to myrtenol (Fig. 2a); the recombinant enzyme could also hydroxylate limonene to yield perillyl alcohol. Wild strawberry fruit emit myrtenol acetate as a prevalent component but not perillyl alcohol because wild strawberry fruit utilizes geranyl diphosphate to produce only linalool, a-pinene, b-myrcene and b-phellandrene, but not limonene as precursors. The strawberry hydroxylase gene is a member of the CYP71 family which also includes other known monoterpene hydroxylases, such as the Mentha limonene hydroxylases and a geraniol hydroxylase from Catharanthus roseus. The first plant cytochrome P450 gene to be described (CYP71A1) was isolated from a cDNA

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library prepared from ripening avocado (Persea americana) fruit, and encoded a microsomal protein previously purified from this source (O’Keefe and Leto 1989; Bozak et al. 1990). The recombinant enzyme, expressed in yeast, converted the acyclic monoterpenols geraniol and nerol to their corresponding 2,3- and 6,7-epoxides (Fig. 2b) (Hallahan et al. 1994); however, neither geraniol nor nerol, nor the epoxides, were detected in avocado fruit. Thus, these monoterpenols are unlikely to be the physiological substrates for the CYP71A1 enzyme, and the function of this cytochrome P450 gene remains unknown.

Other monoterpene hydroxylases A range of native monoterpene hydroxylases, many from other members of the Lamiaceae, have been described in the literature (Bouwmeester et al. 1999; Karp et al. 1987; Karp and Croteau 1992; Funk and Croteau 1993; Hallahan et al. 1994). The lack of suitable tissue isolation methods for enriching in transcripts for monoterpene metabolism and, perhaps, the lower economic value of these essential oils, relative to that of commercial mint species, has slowed efforts to clone the responsible cytochrome P450 genes. Two cytochrome P450-mediated monoterpene transformations have been described in common sage (Salvia officinalis) (Karp et al. 1987; Funk and Croteau 1993) which produces an essential oil containing the parent olefin (+)-sabinene, along with the oxygenated derivatives (–)-3-isothujone and (+)-3-thujone. The biosynthesis of these thujyl ketones proceeds through the intermediate (+)-cis-sabinol, which is formed by the allylic (C3)-hydroxylation of (+)-sabinene (Karp et al. 1987) (Fig. 2a). The microsomal (+)-sabinene-3hydroxylase prepared from immature leaves is inhibited by CO, and inhibition is partially reversed by illumination with blue light. The COdifference spectrum shows a peak around 450 nm, and with sabinene as a substrate yields a type I binding spectrum with maximum at 398 nm and minimum at 416 nm. The addition of FAD and FMN enhances activity, whereas, cytochrome c, which acts as an alternate acceptor of electrons

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from the P450 reductase, inhibits sabinene hydroxylation. All of these features are typical of cytochrome P450 hydroxylases (Omura and Sato 1964; Estabrook et al. 1963; Narasimhulu et al. 1965; Testa and Jenner 1981). A second cytochrome P450 hydroxylase from sage has been shown to be involved in the catabolism of camphor, another major component of the essential oil of this species (Funk and Croteau 1993). In the few cases that have been studied, catabolism of monoterpenes occurs during senescence and involves the eventual conversion of the resulting monoterpenol into a glucoside or glucose ester [summarized in Croteau (1987)]; these conjugated cyclic forms subsequently undergo lactonization (probably cytochrome P450-mediated) as a means of ring opening and further degradation. The microsomal camphor-6-exo-hydroxylase (Fig. 2a) was isolated from sage cell-suspension cultures undergoing late-logarithmic growth, and was inducible by metal ions (Funk and Croteau 1993). The membranous enzyme has an apparent Km for (+)camphor of 34 lM and is inhibited by cytochrome c and clotrimazole, typical characteristics of a cytochrome P450 oxygenase. The camphor-6-exohydroxylase is the only documented example thus far of a P450-mediated reaction involved in the less well studied area of plant monoterpene catabolism. Unlike the situation in species of the Lamiaceae, in which the essential oil is formed in glandular trichomes, the essential oil of caraway (Carum carvi, Apiaceae) is formed in oil ducts within the fruit. This essential oil contains (+)-carvone and (+)-limonene as major monoterpenoid components, and the biosynthesis of (+)-carvone from (+)-limonene has been shown to be analogous to the pathway used to form (–)carvone in spearmint. The initial cyclization yields the parent olefin (+)-limonene, which undergoes cytochrome P450-catalyzed hydroxylation at C6 to give (+)-trans-carveol followed by dehydrogenation of the alcohol to yield the ketone (+)-carvone (Bouwmeester et al. 1998). The microsomal (+)-limonene-6-hydroxylase isolated from caraway fruit possesses all of the characteristics of a cytochrome P450 oxygenase (Bouwmeester et al. 1999), although the pattern of

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inhibition by substituted imidazoles differs from that of the spearmint (–)-limonene-6-hydroxylase, and this enzyme appears to be rate-limiting in the production of (+)-carvone [i.e., the precursor limonene is abundant, and the following activity catalyzed by (+)-carveol dehydrogenase is present in excess (Bouwmeester et al. 1998)]. The essential oil of hyssop (Hyssopus officinalis), another member of the Lamiaceae, contains the bicyclic monoterpenoids pinocamphone and isopinocamphone (derived from (+)-trans-pinocarveol) along with minor amounts of myrtenol derivatives (Fig. 2a) (Karp and Croteau 1992). (–)-Pinocamphone and (–)-isopinocamphone arise via allylic hydroxylation of (–)-b-pinene to (+)-trans-pinocarveol followed by dehydrogenation and subsequent double bond reduction in a reaction sequence analogous to that for the formation of the thujone ketones from sabinene via cis-sabinol, whereas the (–)-myrtenol originates by allylic hydroxylation of (–)-a-pinene (Fig. 2a) in a manner analogous to the allylic hydroxylation of limonene to perillyl alcohol in perilla. Evidence for the participation of a cytochrome P450 hydroxylase in the conversion of (–)-b-pinene to (+)-trans-pinocarveol derives from characterization of this microsomal enzyme (i.e., requirement for molecular oxygen and NADPH, stimulation by flavins, inhibition by CO and blue light reversal, and inhibition by clotrimazole, miconazole and cytochrome c) (Karp and Croteau 1992). Catmint (Nepeta racemosa, Lamiaceae) produces an essential oil containing nepetalactones (Regnier et al. 1967; Clark et al. 1997b); the biosynthesis of these iridoid monoterpenoids is initiated by the allylic hydroxylation of citronellol at C10 (Bellesia et al. 1984). Microsomal preparations from N. racemosa leaves catalyze the C10hydroxylation of nerol and geraniol (Hallahan et al. 1994) and both enantiomers of citronellol (Hallahan and West 1996) (Fig. 2b). Two cDNAs, CYP71A5 and CYP71A6, have been isolated from a catmint leaf cDNA library, and a survey of expression patterns showed that CYP71A5 is expressed in leaf glandular trichomes whereas CYP71A6 transcripts are found in the leaf blade (Clark et al. 1997a). The functions of these Nepeta P450s have not yet been confirmed by heterologous expression.

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Localization and regulation of expression of monoterpenoid hydroxylases In essential oil plants, monoterpene biosynthesis is generally restricted to highly specialized secretory structures (Fahn 1979). For example, the essential oil of mint species is produced and accumulated specifically within the glandular trichomes on the aerial parts of the plants (Gershenzon et al. 1989). Peltate glandular trichomes consist of a basal cell surmounted by a stalk cell that is topped by a disk of eight secretory cells covered by a common subcuticular storage cavity (Fahn 1979). The biosynthesis of monoterpenes in these non-photosynthetic structures is polarized in that fixed carbon precursors are transported through the basal and stalk cells into the secretory cell sites of synthesis and the resulting products are then secreted into the subcuticular space (Turner et al. 2000). In the case of the mint limonene hydroxylases, immunocytochemical localization has shown that these enzymes specifically reside in the secretory cells of the glandular trichomes, and are absent from the basal and stalk cells and from mesophyll parenchyma cells (Turner and Croteau 2004). Within the secretory cells, antibodies recognizing limonene-6-hydroxylase are associated with the smooth endoplasmic reticulum, consistent with the microsomal location of the native enzyme [and the majority of eukaryotic P450s (Omura 1982)] and with the presence of an Nterminal membrane insertion sequence encoded by the corresponding cDNA (see below). Transcripts of the limonene-3-hydroxylase gene of peppermint reach a maximum steady-state level slightly before peak hydroxylase activity in the oil glands (at about 15 days of leaf expansion) which correlates with the maximum rate of monoterpene biosynthesis as measured by 14CO2 incorporation (McConkey et al. 2000). This developmental pattern matches that of most other monoterpene biosynthetic enzymes of peppermint oil glands; (–)-limonene-3-hydroxylase transcripts are quite abundant (6.1%) in the peppermint oil gland EST library, whereas those for menthofuran synthase are only moderately abundant (0.8%) (Lange et al. 2000). In the case of the strawberry pinene hydroxylase, transcripts were found at very low levels in

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the leaf and stem, at high levels in the wild fruit (but not the fruit from the cultivated species) and at even higher levels in the root (Aharoni et al. 2004). Myrtenol, the product of this enzyme, is known to be the precursor to one of the contributing components of wild strawberry aroma, so accumulation of hydroxylase transcripts in the wild fruit is to be expected; the role of myrtenol and the corresponding glycoside, which are detected in the root, is yet to be defined but the occurrence in this unexpected locale may portend a new biological function for these monoterpenoids in the rhizosphere.

Structure and mechanism of monoterpene hydroxylases The most detailed studies of structure–function relationships and mechanism of action have been conducted with the limonene hydroxylases of mint. Because the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase are 70% identical at the primary structural level and utilize the same olefin substrate, these two catalysts provide a unique opportunity for comparative study of the determinants of regiospecificity of hydroxylation. The cDNAs for both hydroxylases encode N-terminal membrane insertion sequences consistent with the intracellular localization to the endoplasmic reticulum, and both contain all of the conserved structural elements expected for a cytochrome P450 hydroxylase, including the sequences required for heme binding, oxygen binding, and docking of the P450 reductase. The two sequences differ most markedly in the presumptive substrate recognition sites (SRS) (Gotoh 1992). A single amino acid residue (F363), located within the SRS-5 region in the loop between the K helix and the b-sheet 1–4, is solely responsible for controlling the regiospecificity of the 6-hydroxylase because substitution at this position (F363I) converts the 6-hydroxylase to a kinetically competent, and regiospecific, limonene-3-hydroxylase (Schalk and Croteau 2000). The corresponding conversion of the limonene-3-hydroxylase into a 6-hydroxylase (by the reciprocal I364F mutation) confirmed the importance of this residue in controlling regioselectivity.

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Active site interactions of the limonene hydroxylases were also investigated using substrate analogues (Wu¨st et al. 2001; Wu¨st and Croteau 2002). Incubating the recombinant 3-hydroxylase with (+)-limonene (instead of the normal (–)limonene) yielded (+)-trans-isopiperitenol as the only product, whereas the 6-hydroxylase generated primarily (+)-cis-carveol, along with four other side-products (Wu¨st et al. 2001). Because both enzymes have virtually identical binding constants for both limonene enantiomers, these results suggested that the 3-hydroxylase has a much more constrained active site. Measurements of the kinetic isotope effects using deuterated substrates with the 6-hydroxylase also supported this proposal and suggested a nondissociative kinetic mechanism (Wu¨st and Croteau 2002). In the case of the cytochrome P450 limonene hydroxylases, complementarity of fit between the shape of the active site cavity and the shape of the bound substrate is a major factor in hydroxylation specificity, and only a few side chain residues actually determine regiospecificity of oxygenation of the small monoterpene substrate. Harnessing the regio- and stereospecificity of P450-mediated hydroxylation reactions is of major interest for synthetic and biotechnological applications. More detailed studies on the structure–activity relationships of the limonene hydroxylases could be of value in these areas.

Biotechnological applications of monoterpenoid hydroxylases Because cytochrome P450 monoterpene hydroxylases generally catalyze the committed steps in the production of biologically active oxygenated derivatives, these genes provide good targets for pathway redirection and, potentially, for flux alteration. This is especially true for pathways that diverge from a single terpene olefin parent and involve subsequent redox transformations and conjugation reactions. A recent review covers the latest attempts to alter terpenoid production in plants (Aharoni et al. 2005). In peppermint, expression of an additional copy of the limonene-3-hydroxylase cDNA under the regulation of CaMV 35S promoter yielded a

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few transgenic plants with a marginal increase in hydroxylase activity but no significant effect on monoterpene composition or yield (Mahmoud et al. 2004). The hydroxylation step is presumed to be relatively fast because (–)-limonene does not appreciably accumulate in the essential oil at maximum yield; neither does the product of the reaction, (–)-trans-isopiperitenol, accumulate at any but trace levels indicating that the immediate downstream dehydrogenation step is also very fast. Of considerable interest were peppermint transformants in which the limonene hydroxylase was cosuppressed. In these plants, essential oil yield was unaffected but only low levels of oxygenated monoterpenes were formed, and limonene was the principal oil component (~80%) (Mahmoud et al. 2004). These experiments indicated that the oil glands are capable of trafficking and secreting, without adverse influence, a monoterpene olefin with very different physical properties than the normal monoterpene ketones and alcohols produced. In another set of experiments, constitutive expression of an antisense copy of the menthofuran synthase cDNA resulted in decreased levels (by 50%) of the undesirable oil component menthofuran with no effect on the final oil yield (Mahmoud and Croteau 2001). Interestingly, plants with low menthofuran content also contained, by way of an unusual regulatory effect (Mahmoud and Croteau 2003), lower than normal levels of pulegone, another less desirable monoterpene, to afford a new peppermint line of commercial value. Tobacco has been transformed with the Citrus limon genes for (+)-limonene synthase, c-terpinene synthase and (–)-b-pinene synthase, and the limonene-3-hydroxylase gene from M. spicata L. ‘Crispa’ (Lu¨cker et al. 2004). The most highly expressing transgenic plant produces both (+)limonene and (+)-trans-isopiperitenol. This is an example of introducing a new monoterpenoid biosynthetic pathway into a heterologous host which may lead to the production of novel scents or modification of emitted volatiles that may be useful in protecting the plant from herbivorous insects (Degenhardt et al. 2003; Kappers et al. 2005). The production of terpenes in microbial hosts has also garnered much interest (Barkovich and

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Liao 2001). An attempt to synthesize (–)-carvone by introducing the entire pathway (i.e., isopentenyl diphosphate isomerase, geranyl diphosphate synthase, limonene synthase, limonene-6-hydroxylase and carveol dehydrogenase) into E. coli failed to produce significant amounts of the desired product (Carter et al. 2003). Although all of the enzymes were confirmed to be functionally expressed in the microbial host, the host cytoplasmic machinery was apparently unable to transfer efficiently the olefin product of the soluble synthase to the membranous hydroxylase, and instead excreted the bulk of the (–)-limonene intermediate into the medium. More recently, the successful engineering of the mevalonic acid pathway in E. coli has resulted in a 20-fold increase in production of terpenoid precursors to the antimalarial sesquiterpenoid drug artemisinin (Martin et al. 2003). Genetic enhancements of the deoxyxylulose phosphate pathway supplying isoprenoid precursors have also led to increased terpenoid production (Reiling et al. 2004). While these successes are heartening, differences between the relatively simple prokaryotic cytoplasm and the complex compartments of eukaryotic cells may ultimately limit the amount of product made via multistep enzymatic processes without the introduction of structured metabolons, for example, or of the metabolite trafficking apparatuses of eukaryotes [for a recent discussion, see Jorgensen et al. (2005)]. The ability of plant and microbial cytochrome P450s to oxygenate monoterpenes, such as limonene (Duetz et al. 2003), as the initial biotransformational step supports the suggestion that these enzymes could be engineered to degrade persistent small molecule xenobiotics in the soil, such as herbicides (Werck-Reichhart et al. 2000) and industrial pollutants (Kellner et al. 1997). High-throughput methods for directed evolution of cytochrome P450s will accelerate the functional screening of enzymes that bind and functionalize the compounds of interest (Otey et al. 2004).

Conclusions While much progress has been made in identifying plant cytochrome P450 genes in recent genome

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and EST projects, a major difficulty remains in elucidating the function of the encoded enzymes. Most progress in defining the roles, structures and mechanisms of the subset of cytochrome P450 monoterpene oxygenases has derived from studies of essential oil plants in which monoterpene production is restricted to specialized structures, such as oil glands or ducts, for which both metabolic context and a means for enrichment of the relevant enzymes and transcripts are available. In other instances, tissue-specific, developmental and inducible expression patterns have provided the context for isolation and definition of monoterpene oxygenases that play roles in scent production and plant defense. Undoubtedly, many monoterpene oxygenases remain to be defined, and novel biological functions for these genes and enzymes are almost certain to be discovered. The present inability to predict function from sequence information alone poses a serious limitation; however, recent innovations, such as combining computational docking of putative substrates with modeled P450 sequences [for a recent summary, see Schuler and Werck-Reichhart (2003)] and laser-capture microdissection to harvest specialized biosynthetic cells [see references in (Simone et al. 1998; Day et al. 2005)], could permit the elucidation of catalytic capability in the absence of any other context, and this would greatly assist in defining the biological functions of this most interesting family of enzymes. References Aharoni A, Giri AP, Verstappen FWA, Bertea CM, Sevenier R, Sun Z, Jongsma MA, Schwab W, Bouwmeester HJ (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell 16:3110–3131 Aharoni A, Jongsma MA, Bouwmeester HJ (2005) Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci 10:594–602 Barkovich R, Liao JC (2001) Metabolic engineering of isoprenoids. Metab Eng 3:27–39 Bellesia F, Grandi R, Pagnoni UM, Pinetti A, Trave R (1984) Biosynthesis of nepetalactone in Nepeta cataria. Phytochemistry 23:83–87 Bertea C, Schalk M, Mau CJD, Karp F, Wildung MR, Croteau R (2003) Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis. Phytochemistry 64:1203–1211

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