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

ARCHIVES OF BIOCHEMISTRY Vol. 249, No. 2, September,

AND BIOPHYSICS pp. 306-3151986

Metabolism of Monoterpenes: Demonstration that (+)-cis-lsopulegone, Not Piperitenone, Is the Key Intermediate in the Conversion of (-)-lsopiperitenone to (+)-Pulegone in Peppermint (Mentha pipeMa)‘-* RODNEY Institute

CROTEAU3

of Biological Washington

K. V. VENKATACHALAM

AND

Chemistry and the Graduate Program State University, Pullman, Washingtmz Received

March

in Plant

Physiology,

99164-6340

10, 1986

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. o 1986 Academic press, lnc. The pathway for the biosynthesis of menthol stereoisomers in peppermint

(Mentha piperita L.) has been the object of experimental interest and speculation for over 60 years (1). Numerous studies based on the co-occurrence of metabolites (2-7), in vim tracer investigations (8-ll), and genetic analyses (12,13) have allowed major segments of the pathway to be elucidated. This task has been complicated by the fact that the first intermediate of the pathway to accumulate in appreciable quantity in peppermint oil is the late metabolite (+)-pulegone (Fig. 1). Loomis was the first to demonstrate the NADPH-dependent reduction of the isopropylidene double bond (A4p8) of (+)-pulegone to yield (-)-menthone and (+)-isomenthone in a cell-free extract of peppermint leaves (14), thereby confirming the results of earlier, but less definitive, studies supporting the intermediacy of this metabolite (8). Reduction of the carbonyl functions of (-)-

i This is Scientific Paper No. 7396, Project 0268, from the Washington State University Agricultural Research Center, Pullman, Wash. This investigation was supported in part by Department of Energy Contract DE-AM06-76RL02221, Agreement DE-ATOG82ER12027, and by grants from the Washington Mint Commission and Mint Industry Research Council. The Hewlett-Packard 5840A-5985B GLC/MS system used in this work was purchased through the assistance of the National Science Foundation (PCM 8100068). a Although the systematic names for the monoterpenes described here are based on substituted cyclohexanols and cyclohexanones, we have utilized the more common nomenclature based on numbering of the pmenthane system (i.e., (+)-cis-isopulegone = pmentha-1,2;8,9-dien-a-one) in which the methyl-substituted carbon is 1R and the isopropenyl-substituted carbon is 4R. ’ To whom correspondence should be addressed.

0003-9861/86 Copyright All rights

$3.00 0 1986 by Academic Press, Inc. of reproduction in any form reserved.

306

BIOSYNTHESIS

OF

ISOPULEGONE

A\

:;1 YPT 9

Piperitenone

10 8\943o--L (-)-lsopiperitenons

Q

A

y

9 O (+I-Pulsgone

0

(+)-cis&-lsopulegons

FIG. 1. Alternate pathways from (-)-isopiperitenone to (+)-pulegone involving Ai.’ reduction (R) and A*,‘: Aa,* isomerization (I).

AND

PULEGONE

307

inary communication not withstanding (21), neither this metabolic sequence nor the intermediacy of piperitenone itself in the pathway has been conclusively demonstrated. The alternative possibility, that reduction of the endocyclic double bond of (-)-isopiperitenone to yield (+)-cis-isopulegone precedes the double bond migration (Fig. l), has been essentially ignored, most likely because cis-isopulegone has neither been reported to occur in peppermint oil (7) nor is this metabolite measurably labeled from early precursors in the pathway (8, 11, 18). However, cis-isopulegone is difficult to separate from other peppermint oil constituents, and low levels of this metabolite may easily go unnoticed. Recently we isolated and characterized a 3-keto-p-menthene-AB~g:A4*‘-isomerase from peppermint leaves (19). The evidence suggested that the same enzyme was capable of isomerizing (at comparable rates) both (-)-isopiperitenone and (+)-cis-isopulegone to the respective a&unsaturated ketones, piperitenone, and (+)-pulegone. Thus, these results did not provide any clear distinction between the alternate routes to (+)-pulegone. In this communication, we describe a series of in vivo and in vitro experiments with peppermint which conclusively demonstrate that (+)cis-isopulegone, and not piperitenone, is the key intermediate in the conversion of (-)isopiperitenone to (+)-pulegone.

menthone and (+)-isomenthone to afford (-)-menthol and (+)-neomenthol, and (+)isomenthol and (+)-neoisomenthol, respectively, has been demonstrated in vivo (9, 15, 16), and the two stereospecific dehydrogenases responsible have been isolated from peppermint leaves and characterized (17). More recently, it was shown that the olefin (-)-limonene (derived by the cyclization of geranyl pyrophosphate) was the first cyclic intermediate in the biosynthesis of C3-oxygenated p-menthane monoterpenes in peppermint, and that this olefin was hydroxylated to (-)-trans-isopiperitenol, which was in turn oxidized to (-)-isopiperitenone (18). Each of the requisite transformations was demonstrated in vivo and several were confirmed in cellfree enzyme systems (18,19). Thus, all steps in the conversion of the universal acyclic EXPERIMENTAL PROCEDURES precursor geranyl pyrophosphate to (-)isopiperitenone, and in the transformation Plant materials, substrates, and reagents. Pepperof (+)-pulegone to the various menthone mint (M. pipcrita L. cv. Black Mitcham) plants, propand menthol stereoisomers, are now well agated from single-node cuttings of etiolated rhiestablished. zomes, were grown in sand:Perlite:peat moss (l:l:l, Most investigators have assumed that v/v/v) in a growth room with a 14-h photoperiod (9700 piperitenone is the key intermediate be- + 1100 lux, fluorescent/incandescent), a 29°C day/25”C tween (-)-isopiperitenone and (+)-pulenight temperature cycle and a relative humidity of 62 gone because piperitenone has been ob- * 12%. Plants were watered as needed and fertilized served as a minor constituent in the essen- weekly with a complete fertilizer (N:P:K, 20:20:20) plus tial oil of peppermint (7, 12), and because iron chelate and trace elements. Unless otherwise specified, mid and upper stem leaves of actively growthis metabolite is detectably labeled from ing plants (8- to 12-leaf stage) were used in all ex‘*CO2 (8,11,20). This scheme (Fig. 1) posits periments. Steam distillation of several samples of the isomerization of the isopropenyl functhis tissue followed by GLC/mass spectrometric tion to the isopropylidene group (A”~g:A4V8 analysis of the resulting oils indicated the presence isomerization) prior to the reduction of the of (-)-menthone (40%), (-)-menthol (20%), (+)-isoendocyclic (A’,‘) double bond; yet, a prelimmenthone (16%),1,8-cineole (5%),(+)-pulegone (3%),

308

CROTEAU

AND

(+)-neoisomenthol (3%), (-)-menthyl acetate (2%), and more than 20 minor components (-14% of total). The sources and purification (to 296% chemical and optical purity) of authentic samples of (-)-isopiperitenone, piperitenone, (+)-pulegone, (-)-piperitone, (-)-menthone, (+)-isomenthone, (+)-iso(iso)pulegol, (-)-menthol, and other menthol stereoisomers have been described elsewhere (18, 19). (+)-Piperitone (>97%)was a gift from W. D. Loomis of Oregon State University. (+)-Iso(iso)pulegol was oxidized to (+)cis-isopulegone with Cr03 (22) and the product purified (>97% by capillary GLC) by argentation TLC. A mixture of (+)-cis-isopulegone and (+)-tram-isopulegone (-3:2 by capillary GLC) was obtained by the NaBH, reduction of (-)-isopiperitenone in ethanol containing CoCI, * 6HaO (23), and the mixture separated from the coproduct (-)-piperitone by TLC on silica gel G. (+)-[aH]Isopiperitenone and [3H]piperitenone were prepared by exchange-labeling of (-)-isopiperitenone in 3HZ0. Thus, 1 mmol isopiperitenone, 100 ~1 dioxane, and 20 ~1 1,8-diazabicyclo[5.4.0]undec-7-ene were placed in a vial containing 50 ~1 aHa0 (90 Ci/mol) and the mixture was allowed to stand at room temperature for 2 h. Diethyl ether and 5% aqueous (NH&SO, were then added to the mixture, the ether layer was removed and dried by passage through a small column of MgS04, and the products contained therein were separated by TLC on silica gel G impregnated with 16% AgN03 [with hexanes:ethyl acetate (4:1, v/v) as developing solvent]. The identities and radiochemical purities of the products (>97%)were verified by radioGLC. The specific activity of both products was -2.5 Ci/mol and they were either used at this specific activity or were diluted with authentic carriers to 1 Ci/ mol. (+)-[3H]Pulegone (0.5 Ci/mol) was similarly prepared by exchange-labeling (+)-pulegone in ‘HzO, and was purified from minor co-products (to >97% by radio-GLC) by argentation TLC as above. [1,2-aH]Isopulegone [mixture of (+)-cisand (+)truns isomers] and (-)-[8,9-3H]piperitone were prepared by reduction of the double bonds of (-)-isopiperitenone (23). Thus, 0.1 mmol (-)-isopiperitenone was mixed with 0.25 ml ethanol containing 50 mmol CoClz * 6Hz0, and the mixture was cooled on ice, flushed with Nz, and then transferred to a vial containing 25 mCi NaBaH (360 Ci/mol). The vial was again flushed with Nz, sealed, and the reduction carried out at O4°C for 2 h. The reaction products were partitioned between ether and water, and the ether phase was dried over MgSO,. Radio-GLC was employed to verify the identity of the radioactive products in the ether extract and to determine the quantity of each labeled ketone present. The appropriate authentic standards were added to reduce the specific activity of each product to 15 Ci/mol, and the (-)-[aH]piperitone was separated from cis- and trun.s-[3H]isopulegone by TLC on silica gel G by using hexanes:ethyl acetate as above. The recovered [‘Hlisopulegone was then separated by

VENKATACHALAM TLC on silica gel G impregnated with 10% AgNOa by using hexanes:ethyl acetate (4:1, v/v) to afford the cis (60%) and trans (40%) isomers. Experiments with leaf discs. Thirty discs (1 cm diameter) were cut from the leaves with a cork borer and incubated with the appropriate labeled substrate (1.7 &i) in 0.5 ml water containing 20 pg Tween 20 in a 25-ml Erlenmeyer flask loosely sealed with foil. Following incubation for 8 h at 30-33°C with slow agitation, unincorporated substrate was washed from the discs with water, and the tissue was homogenized in a Ten-Broeck homogenizer with 5 ml each of water and diethyl ether. Carrier standards (-20 pg each) were added to the mixture and the ether layer was removed, decolorized with activated charcoal, dried over MgSO,, and concentrated to 50 ~1 at 0-4°C (-95% of the products were recovered by this procedure). A ~-PI aliquot was taken for determination of ‘H content and the remaining sample analyzed by TLC or radio-GLC. Control experiments with steam-inactivated tissue were run in all cases to evaluate nonenzymatic transformations and the possible influence of microbial contamination. For this purpose, the tissue was first incubated for 1 h with 0.5 ml of water containing 20 fig of Tween 20. This aqueous extract was withdrawn and the discs were heated with steam for 15 min. Following cooling to room temperature, the aqueous extract (presumed to be an appropriate microbial inoculum) and the substrate were added to the tissue and the mixture was incubated for the appropriate time. The tissue was then directly extracted with ether and water, and the ether-soluble products analyzed as above. Preparation and assay of cell-free systems. Preparation of the crude, soluble enzyme fraction (105,OOOg supernatant) from intact peppermint leaves has been described (19). Mechanical separation of epidermal fragments from the mesophyll and the preparation of the soluble enzyme fraction from each tissue have also been described previously (24), as has the largescale automated procedure for the preparation of leaf epidermis enzyme extracts [for use with mint leaves, glass beads are substituted for stainless steel beads in the published procedure (25)]. Since early studies (1319) had indicated that the entire sequence of metabolic reactions of interest, with the exception of limonene hydroxylation, was catalyzed by operationally soluble enzymes, particulate fractions were not examined. In all cases, the soluble enzyme fraction was concentrated by ultrafiltration (Amicon PM-30) and the extraction buffer removed by elution of the concentrate through a column (2 X 32 cm) of Sephadex G50 which was equilibrated and eluted with 50 mM sodium phosphate buffer (pH 7.0) or 50 mM glycineNaOH buffer (pH 8.0), in both cases containing 10% sorbitol (w/v) and 1 mM dithioerythritol. The Se-

BIOSYNTHESIS

OF

ISOPIJLEGONE

phadex G-50 desalting step, along with treatment with porous polystyrene (Amberlite XAD-4) during the enzyme extraction sequence (19, 26), additionally served to remove endogenous monoterpenes and permitted the use of assays with unlabeled substrates based on sensitive capillary GLC detection methods. The soluble enzyme preparations were either utilized directly at this stage, or were applied to a Sephacryl S-200 column (2.4 X 120 cm) previously equilibrated and eluted with either the phosphate or glycine buffer system. Proteins were eluted (36 ml/h, 4.5-m] fractions) with the same buffer while the column effluent was monitored at 280 nm. Fractions containing the appropriate double bond reductase and isomerase activities were separately pooled and concentrated by ultrafiltration if necessary. The assay for double bond reduction was typically run in 2 ml of the described buffer containing an amount of protein equivalent to that of 0.5 g of fresh whole leaf tissue. Reduced pyridine nucleotide (1 mM) was added to the mixture contained in a screw-capped vial, and the reaction was initiated by the addition of 0.5 mM of the unsaturated monoterpene ketone (1 prnol in 5 ~1 pentane). After incubation for 90 min at room temperature, the vial was chilled in ice and 3 ml of ether was added followed by vigorous shaking and centrifugation to separate the phases. The ether layer was removed and dried by passage through a 0.5 X 2cm column of anhydrous Na2S04 in a Pasteur pipet, and to this was added the internal standard [0.02 mg (131 nmol) of (+)-camphor in 20 al ether]. The mixture was concentrated to -0.1 ml under a stream of N2 and a l-~1 aliquot was analyzed by capillary GLC on a 30-m fused silica column coated with SE-30 and run isothermally at 80 or 90°C with a Hz flow rate of 4 ml/min (injector temperature, 185°C; detector temperature, 220°C). The flame ionization detector output was electronically integrated and the quantity of product(s) was determined via the internal standard, using an average response factor of 1.0. The assay for double bond isomerization was run in an identical manner in the absence of pyridine nucleotide, and in this instance the reaction was initiated by the addition of 0.5 mM of the p,y-unsaturated monoterpene ketone (1 rmol in 5 ~1 pentane). Products were extracted with ether and analyzed as before. The identities of the products generated in this and the above assay were confirmed by comparison of retention indices on the SE-30 column and on a similar capillary column coated with Carbowax 20 M, and by GLC/mass spectrometric analysis. Boiled controls (and controls for enzyme alone and substrate alone) were included in each experiment, and appropriate individual controls were used in those experiments in which different control values were possible (response to pH, time, etc.). In all cases, nonenzymatic product formation and the level of endogenous monoterpenes in the extracts were negligible.

AND

309

PULEGONE

Analytical methods. TLC with the developing solvents was done on I.O-mm layers of silica gel G (with or without impregnation with AgNOa) activated at 110°C for 2 h. The developed chromatograms were sprayed with a 0.2% ethanolic solution of 2,7-dichlorofluorescein to locate (under uv light) the appropriate components, which were eluted from the gel with diethyl ether. Radio-GLC was performed on a Gow-Mac 550P gas chromatograph (thermal conductivity detector) attached to a Nuclear Chicago radioactivity monitor (calibrated externally with [3H]toluene, with integration of radio output). The chromatography column was 12 ft X 0.125 in. o.d. stainless steel containing 10% Superox 20M on 80000 mesh Chromosorb WHP, and was programmed from 150°C (5 min) to 220°C at G”C/min with a helium flow rate of 60 ml/min. Capillary GLC analyses were performed on a Perkin-Elmer Sigma 3B chromatograph (with FID and No. 2 injection splitter at -15O:l) by using the aforementioned 30-m fused silica column coated with SE30 (or Carbowax 20 M) under the conditions previously described. GLC/mass spectrometric analyses were performed on a tandem Hewlett-Packard 5840A-5985B system by using a 25-m fused silica capillary column coated with SE-30 and programmed from 80 to 150°C at 5”C/ min. Spectra were obtained at 70 eV with an accelerating voltage of 2000 V, and in all cases were compared to those of authentic reference standards. Radioactivity in liquid samples and TLC isolates was determined in a counting solution (15 ml) consisting of 0.3% (w/v) Omnifluor (New England Nuclear) dissolved in 30% ethanol in toluene. The counting efficiency for aH was 27% and all samples were counted to ~1% probable error.

RESULTS

AND

DISCUSSION

Experiments with peppermint leaf discs. Earlier investigations had established that [U-‘4C]sucrose and [9-3H]limonene were efficiently incorporated into the monoterpenes of peppermint leaf discs, notably (+)pulegone and (-)-menthone, but that none of the known, more immediate, precursors of (+)-pulegone, such as (-)-trans-isopiperitenol and (-)-isopiperitenone, were detectably labeled under these conditions (18) (see Fig. 3 for structures). These results were consistent with the composition of peppermint oil in confirming that (+)pulegone was the first cyclic intermediate of the monoterpene pathway to accumulate to any appreciable extent in this plant, and

310

CROTEAU

AND

they thus indicated that the biosynthetic steps leading to (+)-pulegone were relatively fast compared to the reactions by which (+)-pulegone was metabolized. In spite of the lack of direct evidence, most investigators have considered piperitenone to be the likely immediate precursor of (+)pulegone (7,8,11,12,20); however, preliminary isotopic dilution analyses, in which the incorporation of [U-‘4C]sucrose and [3H]limonene in leaf discs was examined in the presence of a lo-fold molar excess of unlabeled piperitenone, failed to demonstrate either trapping of labeled piperitenone or a decrease in labeling of (+)-pulegone. To examine more directly the origin of (+)-pulegone [and its derived products (-)menthone and (+)-isomenthone] studies with peppermint leaf discs were carried out with various 3H-labeled ketones as exogenous precursors. Sets of leaf discs obtained from the middle and upper stem were incubated for 8 h with 1.7 &i of 3H-labeled piperitenone, (-)-isopiperitenone, (+)-cisisopulegone, (+)-pulegone, or (-)-piperitone, and the radioactive ether-soluble products from each (representing from 20 to 30% incorporation of 3H in most cases) were analyzed by radio-GLC after the addition of appropriate internal standards. Boiled tissue controls, and controls for microbial contamination, were included in all experiments, and in all cases nonenzymatic product formation (e.g., photochemical conversions) and microbial transformations were negligible. When incubated with [3H]piperitenone (Fig. 2A), leaf discs produced only piperitone (78% of incorporated label; 18% of applied label), which was presumed to be the (+) enantiomer previously reported to be an inert metabolite in peppermint (21,27). Leaf discs from mature, fully expanded leaves and shoot tips gave similar results. Independent examination of ( -)-[3H]piperitone as a substrate with leaf discs showed this stereoisomer to be inert also, since no metabolites were detected. Under the identical in vivo conditions employed for piperitenone and piperitone as substrates, ( +)-[3H]isopiperitenone was efficiently incorporated into a variety of me-

VENKATACHALAM

A

~

d h

c 8

16

8 Time

16 (min)

FIG. 2. Radio-gas-liquid chromatograms of the ether-soluble products obtained when rH]piperitenone (A), (f)-rH]isopiperitenone (B), (+)-cis-ljH]isopulegone (C), and (+)-[aH]pulegone (D) were incubated with peppermint leaf discs. The smooth bottom tracing(E) represents the response of the flame-ionization detector to coinjected standards of (-)-menthone (a); (+)-isomenthone (b); (+)-&-isopulegone/(+)-neomenthol (c); (-)-menthol (d); (+)-pulegone (e); (k)piperitone (f); (p)-isopiperitenone (g); and piperitenone (h). See text for details of the analyses on the Superox 20M column, and for incorporation rates and distribution of radioactivity into the various products.

tabolites (Fig. 2B), including (+)-pulegone (11% of incorporated label), (-)-menthone (5%), piperitone (5%), (+)-isomenthone (3%),piperitenone (2%),(-)-menthol(2%), and a new metabolite (28% ), not previously observed in the earlier in vivo studies, which eluted from the packed column with a retention time coincident with that of isopulegone and neomenthol. Since neither

BIOSYNTHESIS

OF

ISOPULEGONE

cis- or trans-isopulegone nor neomenthol was sufficiently resolved by the radio-GLC procedure to make a structural assignment, the products were separated by TLC and the radioactivity was shown to reside only in cis-isopulegone (verified by reanalysis of the purified product by radio GLC on an SE-30 column). Although too little material was available to permit determination of absolute configuration, the facts that the configuration at C-4 of the precursor was R and that all monoterpene metabolites in peppermint bore the methyl-substituted carbon in the 1R configuration (see Fig. 3) strongly suggested that the product was (+)-(lR,4R)-cis-isopulegone. When (+)-cis[3H]isopulegone was administered to leaf discs, this precursor was efficiently incorporated into (+)-pulegone (19% of incorporated label), (-)-menthone (7%), and (+)-isomenthone (24%) (Fig. ZC), strongly suggesting that this metabolite was the key intermediate in the biosynthesis of (+)pulegone and the menthones. (+)-[3H]Pulegone, when administered to leaf discs, gave rise mainly to (-j-menthone (49% of incorporated label), (-)-menthol (14.5% ), and (-)-isomenthone (
AND

PULEGONE

311

substrate accessibility to the mesophyll, and therefore irrelevant to monoterpene metabolism occurring in the epidermal oil glands (see below). It should be additionally noted here that (+)-[4,6,C-3-methyl“Hlisopiperitenone was prepared by exchange labeling in 3H,0; the C-4-3H isotope effect (19) slows the A8?A4,* isomerization of isopiperitenone and its metabolites and results in an underestimate of these rates in the above iu viz30experiments. The summary of the above results provides clear evidence that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone (Fig. 3). No evidence was obtained for the involvement of piperitenone in the formation of pulegone as implied from earlier, but less di: rect, studies (7, 8, 11, 12, 20). Experincenh with cell-free enzyme systems. In order to confirm the observations made with leaf disc experiments, a series of in vitro studies was performed by using a crude, soluble enzyme preparation (105,OOOgsupernatant at neutral pH) obtained from a whole leaf homogenate. Incubation of the enzyme preparation (0.5 g tissue equivalent) with (p)-isopiperitenone (0.5 mM, 1 pmol) and NADPH (1 mM) afforded a series of products identified by combination of capillary GLC and GLC/ mass spectrometric analysis as (+)-cis-isopulegone (106 nmol with reference to the internal standard), (+)-pulegone (-104 nmolj, piperitenone (27 nmol), (-j-piperitone (18 nmol), (-)-menthone (15 nmol) and (+)-isomenthone (20 nmol) (Fig. 4A). Under the identical conditions, piperitenone gave rise only to piperitone [-20 nmol, probably the (+)-isomer]; no pulegone was detected (Fig. 4B). (k)-Piperitone in the presence of NADPH was shown to be essentially inactive in this enzyme system, yielding only low levels of (+)-isomenthone (~15 nmolj presumably derived from the (-)-enantiomer. Incubation of the enzyme system with 0.5 mM (+)-cis-isopulegone and 1 mM NADPH afforded primarily (+)pulegone (I68 nmol), (+)-isomenthone (40 nmolj, (-)--menthone (30 nmolj, and (-jmenthol (<:15 nmol) (Fig. 4C); (+)-transisopulegone was inert as a substrate under

312

CROTEAU

AND

VENKATACHALAM

~=$++~~Q~Q~~~@H -w’ (-)-Limonene

wmphosphate

C-)-mlsopiperitend

(-blsopiperiie”O”e

c+>-&Ie3pul~gone

(+MM,me

(-)-Menthone

(+Menthol

\ 0 (+)-

FIG. 3. Proposed

pathways

for the biosynthesis

the same conditions. As expected, (+)pulegone in the presence of NADPH yielded (-)-menthone (60 nmol), (+)-isomenthone (22 nmol), and (-)-menthol (27 nmol) (Fig. 4D). Repetition of the above experiments at pH values from 6.0 (phosphate) to 8.0 (glycine-NaOH) afforded only quantitative, but no qualitative, changes in the products formed. Nor was product distribution markedly altered by the presence or absence of dithioerythritol. As had been observed in the leaf disc experiments, the proportion of (+)-isomenthone generated from (+)-cis-isopulegone, and the proportion of (-)-piperitone generated from (-)-isopiperitenone, were higher than would have been expected based on the composition of peppermint oil, suggestive of the operation of an isopropenyl double bond (A*,‘) reductase activity. The epidermal oil glands are considered to be the primary sites of monoterpene biosynthesis in mint and related species (20,24,28-30). To more accurately determine the metabolic capability of this specialized tissue and, at the same time, to determine the possible contribution of mesophyll-derived enzymes to the activity of the above whole leaf preparations, soluble enzyme extracts were prepared from man-

of C-3-oxygenated

s?-P lsmenthone

monoterpenes

\

OH (+)-lsomenthol

in peppermint.

ually separated peppermint leaf epidermis and compared with those from mesophyll tissue. Incubation of the mesophyll preparation with (-)-isopiperitenone plus NADPH as above yielded appreciable levels of (-)-piperitone (102 nmol) with low levels of (+)-cis-isopulegone (20 nmol) and (+)-isomenthone (15 nmol), whereas incubation of the epidermis preparation under the same conditions afforded primarily (+)-cis-isopulegone (113 nmol) and (+)pulegone (112 nmol), with lower levels of piperitenone (28 nmol), (-)-menthone (22 nmol), and (+)-isomenthone (10 nmol), and no piperitone was detected. Similarly, incubation of the mesophyll preparation with (+)-cis-isopulegone plus NADPH gave rise to approximately twice as much (+)-isomenthone (65 nmol) as (-)-menthone (25 nmol) in addition to (+)-pulegone (~10 nmol), whereas as with the epidermis preparation the ratio of (+)-isomenthone (20 nmol) to (-)-menthone (38 nmol) was nearly reversed with a reasonably high level of (+)-pulegone (98 nmol). Although there was likely some cross-contamination between tissue types in these preparations, it was obvious that the bulk of the relevant A’,‘-reductase activity and A8,g:A4~s-isomerase activity resides in the epidermis

BIOSYNTHESIS

OF

ISOPULEGONE

d

h

C

a

A

b

i A

a

-

0

5

Time

10

(mini

FIG. 4. Capillary gas-liquid chromatograms of the ether-soluble products obtained from the incubation of the crude, soluble enzyme preparation from peppermint leaves with 1 ttIM NADPH and 0.5 ItIM of (-)isopiperitenone (A); piperitenone (B); (+)-cis-isopulegone (C); and (+)-pulegone (D). The components identified are (+)-camphor (internal standard at 131 nmol in the extract) (a); (-)-menthone (b); (+)-isomenthone (c); (+)-cis-isopulegone (d); (-)-menthol(e); (+)-pulegone (f); piperitone (g); (-)-isopiperitenone (h); and piperitenone (i). See text for details of the analyses on the SE-30 column, and for conversion rates into the various products. (which constitutes -10% of the total leaf tissue) whereas the bulk of the apparent A’,‘-(isopropenyl) reductase activity resides in the mesophyll and is thus irrelevant to monoterpene metabolism occurring within the epidermal oil glands. All subsequent studies were carried out using a large-scale automated procedure for the preparation of leaf epidermis enzyme extracts (25) and each of the monoterpene ketones was retested as a substrate with this system (Table I). These results provided strong confirmatory evidence that (+)-cis-isopulegone was the intermediate in the conversion of (-)-isopiperitenone to

AND

PULEGONE

313

(+)-pulegone (i.e., A’*” reduction precedes As*g:A4*8isomerization). Since piperitenone was not converted to (+)-pulegone, and since (+)-piperitone was shown to be essentially inactive in this system, it was clear from the results (Table I) that, under the conditions of analysis employed, the rate of A’p2-double bond reduction of isopiperitenone to isopulegone (and its metabolites) exceeded the rate of AsVg:A4,s isomerization to piperitenone by a factor of at least 7, thus providing an underlying rationale for the reaction course observed [i.e., (-)-isopiperitenone + (+)-cis-isopulegone + (+)-pulegone]. Boiled controls were included in each of the above experiments, and in all cases no nonenzymatic product formation was observed. NADH (at 1 mM) was a less effective substitute for NADPH, affording rates of A1r2, A4v8,and AErgreduction of 20 to 30% of those observed with NADPH as cofactor. In the absence of pyridine nucleotide, only As,g:A4,8isomerization activity was observed with (-)-isopiperitenone and (+)-cis-isopulegone as substrates (Table I). Interestingly, at neutral pH the rate of isomerization of (+)-cis-isopulegone (at 0.5 mM) to (+)-pulegone exceeded the rate of isomerization of (-)-isopiperitenone (at 0.5 mM) to piperitenone by roughly a factor of 2. Earlier studies with this isomerase (19) had been carried out at the pH optimum of 8.0, and under these conditions the rate of isomerization of (-)-isopiperitenone to piperitenone was somewhat higher than that of (+)-cis-isopulegone to (+)-pulegone (Table I). The activity responsible for the reduction of the endocyclic (Alt2) double bond of (-)isopiperitenone was partially purified by gel filtration on Sephacryl S-200 and separated from the isomerase activity, which eluted somewhat later. The elution volume of the reductase was between that of bovine serum albumin (Mr 66,000) and the isomerase (Mr 54,000) (19), indicating a molecular weight of approximately 60,000. The partially purified enzyme, when incubated with (-)-isopiperitenone and NADPH, yielded (+)-cis-isopulegone as the sole product. Activities for the isomerization of (-)-isopiperitenone and (+)-cis-isopule-

314

CROTEAU

7521

*I

I I I I I

I I I**1

I

AND

VENKATACHALAM

gone were coincident on the S-200 column, suggesting that a single enzyme may catalyze both isomerizations. Comparison of relevant rates of the partially purified enzymes at pH 7.0 and on a common basis (0.5 g tissue equivalent) indicated the reduction of (-)-isopiperitenone to occur at 380 nmol/h, considerably faster than the isomerization of (+)-cis-isopulegone to (+)pulegone (160 nmol/h) or of (-)-isopiperitenone to piperitenone (60 nmol/h). The rate of reduction of (+)-pulegone (at saturation) to menthone and isomenthone was estimated to be about 70 nmol/h, somewhat slower than the isomerization and probably accounting for the accumulation of pulegone observed in vivo. From the summation of results obtained from in vivo and in vitro studies, it was concluded that (+)-cis-isopulegone is the immediate precursor of (+)pulegone, and that this intermediate was derived directly from (-)-isopiperitenone by reduction of the endocyclic (A’*“) double bond. The pathway observed for the conversion of (-)-isopiperitenone to (+)-pulegone and the menthones in peppermint can be readily rationalized on the basis of the relative rates of A’,” reduction versus As,g:A4,8 isomerization. However, it is also clear that the A’,” reductase is a highly specific enzyme, since neither piperitenone (C-4-isopropylidene) nor piperitone (C-4-isopropyl) were effective substrates for reduction. This observation not only indicates that the remote C-4-isopropenyl function is an essential feature of the natural substrate, isopiperitenone, but it also serves to confirm that piperitenone and piperitone play no significant role in pulegone or menthone biosynthesis in peppermint, in spite of various earlier proposals to the contrary (3, 6-8, 11, 12, 20). The A’,‘-keto functional grouping of isopiperitenone also appears to be an essential feature for enzyme recognition, since the A”‘-keto isomer [(-)carvone] was not a substrate for A’!” reduction. ACKNOWLEDGMENTS We thank G. Wichelns R. Hopp and R. Carrington monoterpene standards.

for raising the plants, and for the generous gifts of

BIOSYNTHESIS

OF

ISOPULEGONE

REFERENCES 1. KREMERS, R. E. (1922) J. Biol. Chem. 50,31-34. 2. REITSEMA, R. H., CRAMER, F. J., AND FASS, W. E. (1957) J. Agric. Food Chem. 5,779-‘780. 3. REITSEMA, R. H. (1958) J. Amer. Pharm. Assoc. Sci. Ed 47,267-269. 4. FUJITA, Y. (1961) Kol-yo 61,43-46. 5. SHIMIZU, S. (1963) Koryo 71,9-18. 6. KATSUHARA, J. (1966) Koryo 83,51-62. 7. LAWRENCE, B. M. (1981) in Essential Oils (Mookherjee, B. D., and Mussinan, C. J., eds.), pp. l81, Allured, Wheaton, Ill. 8. BATTAILE, J., AND LOOMIS, W. D. (1961) Biochim. Biophys. Acta 51,545-552. 9. REITSEMA, R. H., CRAMER, F. J., SCULLY, N. J., AND CHORNEY, W. (1961) J. Pharm. Sci. 50,18-21. 10. BATTU, R. G., AND YOUNGKEN, H. W., JR. (1966) Lloydia 29,360-367. 11. HEFENDEHL, F. W., UNDERHILL, E. W., AND VON RUDLOFF, E. (1967) Phytochemistry 6,823-835. 12. HEFENDEHL, F. W., AND MURRAY, M. J. (1976) Lloydia 39,39-52. 13. MURRAY, M. J., LINCOLN, D. E., AND HEFENDEHL, F. W. (1980) Phytochemistry 19,2103-2110. 14. BATTAILE, J., BURBO~, A. J., AND LOOMIS, W. D. (1968) Phytochemistry 7,1159-1163. 15. MARTINKUS, C., AND CROTEAU, R. (1981) Plant Physiol 68,99-106. 16. CROTEAU, R., AND MARTINKUS, C. (1979) Plant Physiol. 64,169-175. 17. KJONAAS, R., MARTINKUS-TAYLOR, C., AND CROTEAU, R. (1982) Plant Physiol. 69.1013-1017.

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18. KJONAAS, R., AND CROTEAU, R. (1983) Arch. B&hem. Biophys. 220,79-89. 19. KJONAAS, R. B., VENKATACHALAM, K. V., AND CROTEAU, R. (1985) Arch. Biochem. Biophys. 238, 49-60. 20. LOOMIS, W. D. (1967) in Terpenoids in Plants (Pridham, J. B., ed.), pp. 59-82, Academic Press, New York. 21. BURBOTT, A. J., AND LOOMIS, W. D. (1980) Plant Physiol. (Suppl) 65,96 (Abstr. 522). 22. BROWN, H. C., AND GARG, C. P. (1961) J. Amer. Chem. Sot. 83.2952-2953. 23. CHUNG, S.-K. (1979) J. Org. Chem. 44,1014-1016. 24. CROTEAU, R., AND WINTERS, J. N. (1982) Plant Physiol. 69,975-977. 25. CROTEAU, R., AND CANE, D. E. (1985) in Methods in Enzymology (Law, J. H., and Rilling, H. C., eds.), Vol. 110, pp. 383-405, Academic Press, New York. 26. LOOMIS, W. D., LILE, J. D., SANDSTROM, R. P., AND BURBOTT, A. J. (1979) Phytochemistry 18,10491054. 27. BURBOTT, A. J., HENNESSEY, J. P., JR., JOHNSON, W. C., AND LOOMIS, W. D. (1983) Phytoch,emistry 22,2227-2230. 28. CROTEAU, R. (1977) Plant PhysioL 59,519-520. 29. CROTEAU, R. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., eds.), Vol. 1, pp. 225-282, Wiley, New York. 30. CROTEAU, R., AND JOHNSON, M. A. (1984) in Biology and Chemistry of Plant Trichomes (Rodriguez, E., Healy, P. L., and Mehta, I., eds.), pp. 133185, Plenum, New York.