Metabolism of monoterpenes: Oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves

ARCHIVES

Vol.

OF BIOCHEMISTRY

238, No. 1, April,

AND

BIOPHYSICS

pp. 49-60,

1985

Metabolism of Monoterpenes: Oxidation of lsopiperitenol to lsopiperitenone, and Subsequent lsomerization to Piperitenone by Soluble Enzyme Preparations from Peppermint (Mentha piperita) Leaves’ ROBERT

B. KJONAAS; Institute

of

K. V. VENKATACHALAM,

Biological Washington

Received

Chemistry and the Graduate Program State University, Pullman, Washington

September

19, 1984, and in revised

RODNEY

AND

form

CROTEAU3

in Plant Physiology, 99X.&6340

December

6, 1984

Soluble enzyme extracts from peppermint leaves, when treated with polystyrene resin to remove endogenous monoterpenes and assayed with unlabeled substrates coupled with capillary gas-liquid chromatographic/mass spectrometric detection methods, were shown to oxidize isopiperitenol to isopiperitenone, and to isomerize isopiperitenone to piperitenone. The enzymes responsible for the monoterpenol dehydrogenation and the subsequent allylic isomerization were separated and partially purified by chromatography on Sephadex G-150, and were shown to have molecular weights of approximately 66,000 and 54,000, respectively. The general properties of the NAD-dependent dehydrogenase were examined, and specificity studies indicated that a double bond adjacent to the carbinol carbon was a required structural feature of the monoterpenol substrate. General properties of the isomerase were also determined, and it was demonstrated that the double bond migration catalyzed by this enzyme involved an intramolecular 1,3-hydrogen transfer. These enzymatic transformations represent two key steps in the metabolic pathway for the conversion of the initially formed cyclic olefin, (f)-limonene, to (-)-menthol and related monoterpenes characteristic of peppermint. Some stereochemical features of these reactions, and of the overall biogenetic scheme, are described. Q 1985 Academic press, IW.

The major pathway for the conversion of the acyclic precursor, geranyl pyrophosphate, to the various menthol isomers (Fig. 1) in peppermint (Me&a piper&) has been deduced largely through time-

course studies and direct feeding experiments with labeled precursors (l-6). The overall pathway for the metabolism of limonene, the initial cyclic product, is similar to the transformation of other monoterpene olefins to their oxygenated derivatives, and involves an allylic hydroxylation, oxidation to the corresponding a&unsaturated carbonyl compound and then reduction of the conjugated double bond (5-7). The pathway in peppermint represents an interesting variation of this general scheme in that it involves the intermediacy of a symmetrical, conjugated dienone, piperitenone, thought to arise by a double bond migration, and proceeds with the eventual reduction of both double bonds and the carbonyl moiety of this intermediate to give rise to four of the

r This is Scientific Paper No. 6936, Project 0268, College of Agriculture Research Center, Washington State University, Pullman, Wash. 99164. This investigation was supported in part by Department of Energy Contract DE-AM06-76RL02221, Agreement DE-AT06-82ER12027, and by grants from the Washington Mint Commission and Mint Industry Research Council. The Hewlett-Packard 5840A-5985B GLCMS system used in this work as purchased through the assistance of the National Science Foundation (PCM 8100068). ’ Present address: AGRACETUS, Middleton, Wise. 53562. 3 To whom correspondence should be addressed. 49

0003-9861/85 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

KJONAAS,

Gemnyi pymhaphote

I-Limonk

l-j&lsopiperitenol

VENKATACHALAM,

I-lsopiperitenon.

AND

Piperitenone

CROTEAU

I - hbthone

d -Pulqcne

1 -Menthol

\ 0 P-5? d-lsamenthone

OH d-Ismenthol

\ OH & d-Neoisomenthol

FIG. 1. Pathway in peppermint.

for

the conversion

of geranyl

eight possible menthol stereoisomers with (-)-menthol predominating. The possible role of piperitone (a minor constituent of mint presumed to originate by reduction of the exocyclic double bond of piperitenone) in the overall biosynthetic scheme is presently uncertain (4, 8, 9). All of the steps in the conversion of piperitenone, via pulegone, menthone, and isomenthone, to the menthol stereoisomers have been demonstrated in cell-free preparations from peppermint leaves, and each of the NADPH-dependent reductases has been subjected to preliminary characterization (10-13). Several of the enzymes of this metabolic sequence are located primarily in the leaf epidermis (14), and are thought to reside specifically in the epidermal oil glands which are considered to be the primary sites of monoterpene biosynthesis in this tissue (1, 15, 16). The cylization of geranyl pyrophosphate to a mixture of (+)- and (-)-limonene [-80% of the (-) enantiomer] in crude soluble enzyme extracts of peppermint leaves has been described (5), and it has recently been shown that membranous preparations from this tissue catalyze the NADPH-dependent hydroxylation of (+)-

pyrophosphate

to C3-oxygenated

monoterpenes

[9-31XJlimonene to trans-isopiperitenol (unpublished results). In this communication we report on the isolation and properties of the isopiperitenol dehydrogenase and isopiperitenone isomerase, and provide evidence that an intramolecular 1,3 hydrogen shift occurs in the allylic isomerization step which affords piperitenone. EXPERIMENTAL

PROCEDURES

Plant materials, substrates, and reagents. Peppermint (1M. piper& L. cv. Black Mitcham) plants were grown from stolons in sand:Perlite:peat moss (l:l:l, v/v/v) in a growth chamber maintained at 24°C day temperature and 16°C night temperature during a regular 24-h cycle with 14-h days under 15,609~lux light intensity. Plants were fertilized weekly with Peters soluble mixture (20:20:20, N:P:K) plus ironchelate and trace elements. Shoot-tips and the first leaf pair of actively growing plants (8- to 12-leaf stage) were used in all experiments. Isopiperitenone was prepared by allylic oxidation of limonene with CrOz-pyridine complex (17). (-)Limonene (84% optically pure), (+)-limonene (93% optically pure), and (+)-limonene (dipentene) were obtained from Aldrich Chemical Company and were used without purification for the synthesis of the corresponding isopiperitenones. In each case, the reaction mixture was poured onto a column of Florisil (J. T. Baker) followed by elution with ether. Evaporation of solvents provided an oil that was separated

BIOSYNTHESIS

OF

ISOPIPERITENONE

by dry bed column chromatography on silica [with hexane:ethyl acetate (41, v/v)] to afford isopiperitenone (20% yield, Rf = 0.41) and the coproduct carvone (Rf= 0.58). A mixture of c& and truns-isopiperitenol (-4:l) was obtained by NaBH, reduction of isopiperitenone in the presence of CeCh to minimize conjugate reduction (18). The mixture was purified by TLC (R, = 0.3 on silica gel G with hexane: ethyl acetate [4:1, v/v (system A)] and an aliquot was subjected to capillary GLC on a 30-m SE-30 column for accurate determination of isomer content. For use in enzyme assays, the purified isopiperitenone, isopiperitenol, and other monoterpenes were dissolved in redistilled pentane at a concentration such that the appropriate amount of substrate could be added in 5 ~1 or less of this solvent. Authentic samples of (-)-isopiperitenone, piperitenone, (+)-pulegone, (-)-piperitone, (+)-tram-piperitol, (+)-trots-pulegol, (+)-iso(iso)pulegol, (+)menthol, (+)-neomenthol, and other menthol stereoisomers were obtained from R. Hopp of Haarmann and Reimer GmbH, Holzminden, West Germany, and R. Carrington of I. P. Callison and Sons, Chehalis, Washington. Isopulegol was obtained from K & K Laboratories and was oxidized to isopulegone with CrOB (19). Each sample was obtained at, or purified by argentation TLC to, greater than 96% purity as determined by capillary GLC. zI-I,O (99.8% *H atom) was obtained from KOR Isotopes. Polyvinylpolypyrrolidone (GAF Corp.) and Amberlite XAD-4 polystyrene resin (Rohm and Haas Corp.) were purified by standard procedures for use as adsorbents (20, 21). Unless otherwise specified, all other reagents and biochemicals were obtained from Sigma Chemical Company or Aldrich Chemical Company and were used without further purification. Enzyme prepurutim Based on many preliminary experiments and observations that high levels of reducing agents, polymeric adsorbents, and bovine serum albumin increased the level of extractable activity or the stability of the enzymes, the following procedure was adopted for the routine preparation of isopiperitenol dehydrogenase and isopiperitenone isomerase from peppermint leaves. The tissue, after washing with distilled water, was homogenized in a Ten-Broeck homogenizer with 100 mM sodium phosphate buffer, pH 6.6 (5 ml/g tissue), containing 20% sorbitol, 50 mM sodium ascorbate, 50 mM NazSz05, 1 mM dithioerythritol, and 0.5% bovine serum albumin, in the presence of an equal tissue weight of insoluble polyvinylpolypyrrolidone. The homogenate was then slurried with an equal tissue weight of hydrated Amberlite XAD-4 polystyrene resin for 15 min at O4”C, filtered through several layers of cheesecloth, and centrifuged at 27,OOOg for 20 min (pellet discarded) and then at 105,OOOg for 90 min (pellet discarded) to provide the soluble supernatant used as the enzyme source.

AND

PIPERITENONE

51

This soluble enzyme preparation was concentrated either by ultrafiltration (Amicon PM-30) or by slurrying with dry Sephadex G-25-399 and centrifugation, and the concentrate was either exhaustively dialyzed to the appropriate assay conditions or applied to a Sephadex G-150 column (2.5 X 120 cm) previously equilibrated with 50 mM glycine buffer, pH 8.0, containing 5% sorbitol and 1 mM dithioerythritol. Proteins were eluted (30 ml/h, 5.5-ml fractions) with the same buffer while monitoring the column effluent at 280 nm. Fractions containing the isopiperitenol dehydrogenase and isopiperitenone isomerase, respectively, were pooled, concentrated by ultrafiltration (Amicon PM-30), and dialyzed to assay conditions as necessary. In preparing membranous fractions, the same extraction buffer was employed, with soluble polyvinylpyrrolidone (Type NP-K90) being substituted for the insoluble polymer. The homogenate was filtered through several layers of cheesecloth before centrifugation at 3000g to remove whole cells. Particulate fractions were collected at 27,OOOg and 105,oOOg, and the pellets were washed before the assay by resuspension in, and recentrifugation from, the assay buffer described below. Assay for i.sopiperitenol dehydrogenme. The assay was typically run in 2 ml of 50 mM glycine buffer, pH 10.5, containing 5% sorbitol (w/v), 1 mM dithioerythritol, and an amount of protein equivalent to that of 0.2 g of fresh tissue. NAD (1 mM) was added to the mixture contained in a screw-capped vial, and the reaction was initiated by the addition of 0.6 mM isopiperitenol (1.2 Gmol in 5 pl pentane). After incubation for 90 min at room temperature, the vial was chilled in ice for 10 min and then 1.4 ml of distilled diethyl ether was added, followed by the addition of -1 mg of activated charcoal (previously washed with CHCl,:MeOH (21, v/v)) and another 1.4-ml portion of ether. The vial was resealed, shaken vigorously, and then centrifuged to separate the phases. The ether layer was removed and dried by passage through a 0.5 X 2-cm column of anhydrous NazSO, in a Pasteur pipet, and to this was added the internal standard [22.5 pg (144 nmol) of (+)neomenthol in 0.1 ml ether]. The mixture was concentrated to -0.25 ml under a stream of NP and a 0.87~1 aliquot was analyzed by capillary GLC on a 30-m fused silica solumn coated with SE-30 at 110°C with a Nz flow rate of 2 cm3/min (injector temperature, 185°C; detector temperature, 220°C). The FID output was electronically integrated and the quantity of isopiperitenone produced was determined via the internal standard with a response factor of 0.97. 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 (pH optimum, response to time and

52

KJONAAS,

VENKATACHALAM,

protein concentration, etc.). In all cases, nonenzymatic formation of isopiperitenone was negligible. Protein was determined by the Bio-Rad dye-binding method (22), and alcohol (ethanol) dehydrogenase was assayed spectrophotometrically (340 nm) according to the procedure of Vallee and Hoch (23). Assay for tiqwt$eritenone isomerase. The assay was typically run in 2 ml of 50 mM glycine buffer, pH 8.0, containing 5% sorbitol (w/v), 1 mM dithioerythritol, and an amount of protein equivalent to that of 0.5 g of fresh tissue. The reaction was initiated by the addition of 0.5 mM isopiperitenone (1 pmol in 5 ~1 pentane) followed by incubation for 90 min at room temperature, at which time the piperitenone was extracted and the amount present was determined by capillary GLC in a manner identical to that described above for the assay of isopiperitenol dehydrogenase. A complete set of controls was run as before, with the addition of controls run with and without light to examine the possible involvement of photochemical isomerization processes. In studies with inhibitors, the enzyme was preincubated with the inhibitor for 15 min before initiating the reaction by substrate addition. Controls were similarly treated. Experiments with stable isotopes. In order to run assays in s,O, l-ml aliquots of the partially purified isomerase were frozen at -40°C and lyophilized. The residue was dissolved in 1 ml ‘Hz0 (99.8% ‘H atom), substrate was added, and the assay was run as before. Such lyophilized preparations retained 8085% of the original activity when assayed following the addition of 1 ml H,O. Product mixtures extracted from assays run in both HZ0 and ‘Hz0 were analyzed by capillary GLC and by combined GLC/MS. [2H]Isopiperitenone was prepared by treating (+)isopiperitenone (25 mg) with 5% KOH in a 10% solution of dioxane in ‘Hz0 (99.8% ‘H atom) at room temperature (2 ml). The reaction mixture was extracted with pentane and the PHlisopiperitenone was purified (from coproduced [2H]piperitenone) by TLC (silica gel G with 8% AgN03, and solvent system A). A portion of the purified material was subjected to GLC/MS4 analysis for the determination of total ‘H content by comparison to an authentic reference standard. In order to determine the specific ‘H enrichment at C4 of isopiperitenone, a 5-mg sample was hydrogenated in hexane over Pd to afford a mixture of menthone and isomenthone. GLC/MS of this sample was followed by exchange in excess 5% KOH in 10% dioxane in Hz0 and repeated GLC/MS analysis, the difference in ‘H content giving an estimate of enrichment specifically at C4. The [2H]isopiperitenone was then utilized as

4 Abbreviations used: MS, maas spectrometry; N-[2-hydroxy-1,1-bis(hydroxymethyl)ethylklycine.

Tricine,

AND

CROTEAU

the substrate in large-scale incubations with the partially purified isomerase. Anulgtical methods. TLC with the developing solvents indicated in the text was done on l.O-mm layers of silica gel G (with or without impregnation with 8% AgNO,) 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 eiuted from the gel with diethyl ether. Capillary GLC was performed on a Perkin-Elmer Sigma 3B chromatograph (with FID and No. 2 injection splitter at -15O:l) using the aforementioned 30-m fused silica column coated with SE-30 under the conditions previously described. A 30-m Carbowax 20 M column was also employed for determination of retention indices. GLC/MS analyses were performed on a tandem Hewlett-Packard 5840A-5985B system using a 25-m fused silica capillary column coated with OV-I and programmed from 80 to 150°C at 5”C/min. Spectra were obtained at ‘70 eV with an accelerating voltage of 2000 V. In all cases, spectra were compared to those of authentic reference standards. For deuterated products, the chromatographic traces were scanned at 0.04-min intervals, the intensities of the appropriate parent and [P + ‘HXl+ ions were summed and corrected for natural abundances, and the ratios were calculated. RESULTS

Demonstration of isopiperitenol dehydrogenase and isopiperitenone isomerase activity in cell-free extracts. Improved methods for plant enzyme isolation (20), particularly the treatment of extracts with porous polystyrene (Amberlite XAD-4) to remove endogenous monoterpenes (21), and very sensitive capillary GLC analytical techniques have made it possible to study metabolic reactions without using radiolabeled substrates. This approach was adapted to search for isopiperitenol dehydrogenase and isopiperitenone isomerase activities in peppermint leaf extracts. A soluble enzyme preparation (105,0009 supernatant) from a leaf homogenate was dialyzed to the 50 mM glytine, pH 8.0, buffer system, and aliquots equivalent to 0.2 g of the original tissue were incubated with 0.6 mM (+)-isopiperitenol (cis/trans mixture) plus 1 mM NAD, or with 0.5 mM (f)-isopiperitenone. Following incubation the products were extracted and analyzed by capillary GLC

BIOSYNTHESIS

OF

ISOPIPERITENONE

(Figs. 2A and B). Both activities sought were readily detected, giving rise to some 50 nmol of product and representing about 5% conversion of substrate. Verification of the respective reaction products as isopiperitenone (Fig. 2A) and piperitenone (Fig. 2B) was provided by comparison, with authentic standards, of both retention indices on 30-m SE-30 and Carbowax 20 M columns and mass spectra by GLC/ MS analysis. Boiled controls exhibited negligible product formation (~1 nmol) and endogenous product was not observed in the enzyme extract or substrate preparation, thus confirming the enzymatic nature of the reaction. Assays run under fluorescent lights or in the dark showed no measurable difference, thereby eliminating the possible involvement of photochemical processes. Membranous preparations consisting of the 27,000g and 105,OOOg pellets showed little dehydrogenase or isomerase activities when assayed under identical conditions (as soluble extracts). More than 10% of the activity level of whole leaf extracts

,Bbc,d

I

04804

,a

c.de 0

TIME (min.)

FIG. 2. Capillary gas-liquid chromatograms of the ether-soluble reaction products obtained from the incubation of the soluble enzyme preparation from peppermint leaves with 0.6 mM (+)-isopiperitenol (cis/truns mixture) plus 1 mM NAD (A) or with 0.5 mM (f)-isopiperitenone (B). The components identified are (a) neomenthol (internal standard at 144 nmol in the extract), (b) isopiperitenol (irons followed by cis isomer), (c) isopiperitenone, (d) solvent impurities, and (e) piperitenone. Samples were analyzed on the 30-m SE-30 capillary column using the conditions described under Experimental Procedures.

AND

PIPERITENONE

53

was recovered in soluble supernatants prepared from manually isolated leaf epidermis (14). Since the epidermis comprises considerably less than 10% of the leaf tissue, this observation suggests that the epidermis (presumably the epidermal oil glands) is enriched in these enzymes of monoterpene metabolism. Partial pwi$cation of the isopiperitenol dehydrogenase. Gel-permeation chroma-

tography of the soluble enzyme preparation on Sephadex G-150 revealed the presence of one fairly sharp peak of isopiperitenol dehydrogenase activity (Fig. 3). Calibration of the column with a series of protein standards demonstrated that the dehydrogenase eluted in a position essentially identical to that of bovine serum albumin (Sigma), thus indicating a molecular weight of -66,000. Preliminary studies with crude preparations suggested that the presence of bovine serum albumin improved stability of the dehydrogenase. Therefore, this protein was routinely added to the extraction buffer; it also provided a convenient internal marker for gel filtration experiments (Fig. 3). The profile of isopiperitenol dehydrogenase activity indicated in Fig. 3 was unchanged in the absence of bovine serum albumin, and further testing showed the albumin to be without significant effect on enzyme stability or the activity of partially purified preparations. Additional studies (see below) showed the dehydrogenase to possess a pH optimum near pH 10.5; thus, the gel-filtration step was routinely carried out in 50 mM glycine, pH 10.5, to provide an appropriate medium for the partially purified preparation used as the enzyme source in most subsequent experiments. Since certain plant-derived alcohol (ethanol) dehydrogenases possess the ability to oxidize monoterpenols (24,25), the possible presence of ethanol dehydrogenase in the mint preparation was investigated. Examination of column fractions by techniques capable of detecting 0.03 unit of such activity revealed no measurable levels of this enzyme. This observation was surprising in that alcohol dehydrogenase was readily detectable in extracts of other

54

KJONAAS,

FRACTION

VENKATACHALAM,

NUMBER

FIG. 3. Sephadex G-150 column chromatography of the soluble-protein fraction obtained from a homogenate of peppermint leaves. Absorbance at 280 nm (-), (-)-trots-isopiperitenol dehydrogenase (D) activity (0), and (-)-isopiperitenone isomerase (I) activity (0) are plotted. The protein peak centered on fraction number 57 is bovine serum albumin which was added to the extraction buffer. Chromatography and assay procedures are described under Experimental Procedures. V, was at fraction 41.

essential oil plants (26-28). Because the mint extracts were exposed to rather alkaline pH for extended periods during gel filtration and assay, we examined the influence of the pH 10.5 glycine buffer system on yeast alcohol dehydrogenase as a model. Under these conditions the yeast enzyme was very unstable, exhibiting a half-life of less than 30 min. Thus, if the higher plant enzyme possessed comparable instability, less than 0.001% of the original activity would have survived the gel-filtration step and would therefore be unlikely to interfere or be confused with isopiperitenol dehydrogenase. The halflife for partially purified isopiperitenol dehydrogenase under the same conditions was about 4 days. With the Sephadex G-150-purified preparation, the rate of isopiperitenol oxidation increased linearly with protein concentration up to about 80 pg/ml (equivalent to 0.3 g tissue) for a 2-h assay in the absence of bovine serum albumin. Assays conducted in the linear range (-50 pg protein/ml for a 90-min assay) typically afforded 50-100 nmol product, representing up to 10% conversion of substrate. Properties of the isopiperitenol dehydm genuse. Response to pH was determined in 50 mM sodium phosphate (pH 6.5~8.2), Tricine (pH 7.4~8.8), and glycine-NaOH (pH 8.4-12.5) buffers, each containing 5% sorbitol and 1 mM dithioerythritol. The

AND

CROTEAU

enzyme exhibited a broad optimum, with maximal activity between pH 8.5 and 10.5 and half-maximal activities at pH 7.5 and 11.0. Since the enzyme was very active and apparently quite stable at pH 10.5, whereas ethanol dehydrogenase apparently was not, all further studies with the isopiperitenol dehydrogenase were carried out in 50 mM glycine, pH 10.5. The reaction was not readily reversible in the pH range 6.5 to 10.5 when measured in the presence of 0.6 mM isopiperitenone and 1 mM NADH (i.e., ~15% of the corresponding forward reaction). The dehydrogenase showed an absolute requirement for oxidized pyridine nucleotide, the absence of which afforded product levels equivalent to boiled controls. As the concentration of NAD increased, the rate of isopiperitenol oxidation increased, giving rise to a typical hyperbolic saturation curve. The double-reciprocal plot was linear, from which an apparent Km of 1.7 X 10m5 M was calculated. Similar determination for NADP afforded a K, of 2.5 X lop3 m and, at saturating levels of pyridine nucleotide, NADP produced a maximum oxidation rate less than 10% that of NAD. Flavin nucleotides were completely ineffective in the oxidation, and divalent cations were without significant effect on the NAD-dependent reaction. In the absence of dithioerythritol, dehydrogenase activity was reduced by about 30%, but readdition of the thiol reagent (1 mM) fully restored activity. The enzyme was very sensitive to mercurials; p-hydroxymercuribenzoate and HgClz afforded essentially complete inhibition at 25 and 100 PM, respectively. Other thiol-directed reagents were considerably less effective, with N-ethylmaleimide and iodoacetamide exhibiting only 10% inhibition at the 250 &M level. Chelators such as o-phenanthroline and a,cY-dipyridyl had no appreciable effect on the enzymatic oxidation of isopiperitenol. As the concentration of the substrate C(f)-isopiperitenol consisting of one part tram to four parts tis] increased, the rate of oxidation by the dehydrogenase increased, giving rise to a typical substrate

BIOSYNTHESIS

OF

ISOPIPERITENONE

saturation curve. A Km of 2 X 10e5 M for (+)- or (-)-trans-isopiperitenol and of 8 X 10e5 M for (+)- or (-)-cis-isopiperitenol were calculated from the double-reciprocal plot, which appeared to be linear. Since the substrate mixture consisted of all four isopiperitenol stereoisomers whose fate and solubility properties were uncertain, these values must be taken with considerable caution. However, the saturated monoterpenol, (-)-menthol, is reported to possess a solubility in excess of 2 mM (29) and so kinetic measurements with isopiperitenol were likely made in the appropriate solubility range. Additionally, although a considerable degree of uncertainty is introduced by the presence of all four isomers in the substrate mixture, the double-reciprocal plot was linear over eight data points in the range 2-80 PM, indicating that possible inhibition by the anomalous isomers was linear competitive (30,31) and that the Km values determined can be safely regarded as minimums. When the three available isopiperitenol mixtures, consisting of a (+)-cidtrans mix, (+)-&s/tram mix, and (-)-cidtrans mix, were compared at saturation, the relative rates of oxidation were 33, 57, and 100, respectively. As the substrate level was lowered in each case, it became apparent by GLC analysis of the residual substrate that the trarzs isomer was preferentially utilized. In fact, if the incubation period was extended, or the protein level sufficiently high, it could be shown that (-)trans-isopiperitenol was totally depleted from the (-)-&/tram mixture [Km for the (-)-trans-isopiperitenol determined with this substrate mixture was -3 X 10e5 M]. Thus, it was demonstrated that the dehydrogenase preferred (-)-trans-isopiperitenol as the substrate, but that the enzyme could also utilize (+)-trans-isopiperitenol (at roughly one-third the eficiency at saturation and with a Km approaching 10e4 M as determined with the (+)-b-s/tram mixture). This observed lack of absolute enantioselectivity is consistent with the facts that limonene is hydroxylated solely to trans-isopiperitenol in vitro, and that the cyclization of geranyl pyrophosphate in mint leads to a 4:l mix-

AND

55

PIPERITENONE

ture of (-)- and (+)-limonene. This product ratio persists in residual limonene throughout development and thus implies that both olefin isomers formed on cyclization are subsequently metabolized at roughly comparable rates in vivo. The saturated analogs of (-)-trane-isopiperitenol were very poor substrates for the dehydrogenase (Fig. 4). Of all eight stereoisomers of menthol examined, only (+)-neomenthol (Fig. 1) was oxidized at a significant rate [-5% relative to (-)trans-isopiperitenol]. (+)-Iso(iso)pulegol was also an inefficient substrate, whereas the oxidation of (+)-trans-pulegol was appreciable (Fig. 4). At equivalent concentrations, (+)-trans-piperitol was oxidized at rates comparable to (-)-trans-isopiperitenol, indicating the endocyclic allylic double bond at Cl to be an important structural feature of the substrate. Partial putificatim of the isopiperitenone isomerase. Gel-permeation chromatography of the soluble enzyme preparation on Sephadex G-150 revealed the presence of one fairly sharp peak of isopiperitenone isomerase activity (Fig. 3). Calibration of the column with a series of protein standards, as before, indicated that the isomerase eluted in a position

(-)-trans lsopiperitenol (100)

(+)-lso(iso) Pulegol

-

(+)-bansPiperitol

(+)-bans-

(95)

(19)

(+)-lsomenthol (<2)

Pulegol

(+)-Menthol (<2)

(<2)

FIG. 4. Substrate specificity of isopiperitenol dehydrogenase. Numbers within parentheses beneath each monoterpenol indicate the relative rate of oxidation to the corresponding ketone when dehydrogenase activity was assayed under standard conditions at a saturating level of NAD and at a 10e4 M concentration of alcohol.

56

KJONAAS,

VENKATACHALAM,

between that of bovine serum albumin and /3-lactoglobulin, consistent with a molecular weight of -54,000. The profile of the isomerase activity indicated in Fig. 3 was unchanged in the presence or absence of bovine serum albumin (or of P-lactoglobulin or ovalbumin, which were occasionally employed as internal gel-filtration marker proteins), and neither albumin nor globulin (at 0.5%, w/v, in the buffer) had significant influence on enzyme stability or activity of partially purified preparations. As further studies (see below) showed the isomerase to possess a pH optimum near pH 8.0, the gel-filtration step was routinely carried out in 50 mM glycine, pH 8.0, containing 5% sorbitol and 1 mM dithioerythritol, to provide the partially purified preparation used as the enzyme source in most subsequent experiments. Properties of the isopiperitenme isomerase. Response to pH was determined in 50 mM sodium citrate (pH 4.5-6.3), sodium phosphate (pH 6.3-7.5), and glycine-NaOH (pH 7.5-10.0) buffers, each containing 5% sorbitol and 1 mM dithioerythritol. The enzyme exhibited an optimum in the pH 7.5 to 8.5 range, with a sharp drop on either side and half-maximal activities at pH 7 and 9. All further assays of isomerase activity were conducted in the glytine buffer system at pH 8.0, and under linear conditions with respect to protein concentration and time (-100 pugprotein/ ml for a 90-min assay with 5-10% conversion of substrate). Isopiperitenone isomerase activity was insensitive to the presence of pyridine or flavin nucleotides in either reduced or oxidized form. Additionally, the preparation could be extensively dialyzed at pH 5.5 or 9.5 without loss of activity. Preparations so treated were still insensitive to pyridine or flavin nucleotides, suggesting that the isomerization was not a redox interconversion involving free (or bound) nucleotide cofactors. When NADPH was included in the isomerase assays, however, the additional formation of pulegone (via piperitenone) was noted, indicating the presence in these preparations of the pre-

AND

CROTEAU

viously reported (11, 12) piperitenone-A’reductase activity. When dithioerythritol was dialyzed from the preparation, isomerase activity was reduced by about 40%, and the readdition of the thiol reagent (1 mM) restored activity to only 70% of the original level. As might be expected from the above result, the isomerase was sensitive to the presence of thiol-directed reagents; activity was inhibited by 60% with 0.1 mM phydroxymercuribenzoate or 0.3 mM Nethylmaleimide. The enzyme was insensitive to iodoacetamide at 1.5 mM, and activity was unaffected by chelators such as cr,a-dipyridyl at the 1 mM level. Divalent cations (Me and Mn2+) at the 1 mM level were without effect. Preliminary studies with the isomerase had been carried out with racemic substrate, and when the three substrates, (-)-isopiperitenone, racemate, and (+) isomer, were compared at the 0.5 mM level, the relative rates of isomerization observed were 100, 78, and 12, respectively. As the concentration of (-)-isopiperitenone in the incubation medium increased, the rate of piperitenone formation increased, giving rise to a typical hyperbolic saturation curve. An apparent Km of 0.27 mM was determined from the linear double-reciprocal plot. A similar determination for (+)-isopiperitenone gave a Km in excess of 1 mM. Maximum observed rates of (-)-isopiperitenone isomerization occurred at the 2 mM level, a concentration probably close to the solubility limit of this substrate. At this substrate level, the rate of isomerization of the (+) isomer approached 30% of that of the (-) isomer. These results clearly indicate that (-)isopiperitenone is the preferred substrate for isomerization, yet the (+) isomer can also be isomerized to piperitenone at readily measurable rates. Thus, the isomerase, like the dehydrogenase, is not completely enantiospecific in substrate utilization. Studies outlined earlier suggest that the primary substrate likely to be available to the isomerase in viva is (-)isopiperitenone [derived originally from (-)-limonene].

BIOSYNTHESIS

OF

ISOPIPERITENONE

Mechanism of isomerixation. A number of enzymes catalyze allylic isomerizations of their unsaturated substrates (32, 33). The direct precedent for the present reaction is that catalyzed by the well-known ketosteroid isomerases, which carry out an intramolecular 1,3 proton shift and thus exhibit little or no incorporation of protons from the medium into the end product (34). To examine the possibility that the isomerization of isopiperitenone to piperitenone occurs with a similar internal 1,3 proton transfer, lyophilized enzyme preparations were assayed under the standard conditions in 2H20 (99.8% 2H atom), and the isolated substrate/ product mixture was analyzed directly by GLC/MS. Examination of the parent (m/e 150) and P + 1 (m/e 151) ions and comparison with the corresponding ion intensities of an unlabeled reference standard of piperitenone, which had been incubated with boiled enzyme in 2H20 and isolated and analyzed under identical conditions, established that the product derived enzymatically in 2H20 contained only 4.5 f 0.3% 2H atom. Nonenzymatic isomerization of (f)-isopiperitenone was negligible under the conditions of the analysis, and no evidence was obtained for the generation of multiply deuterated species with either active or boiled enzyme. Deuterated (?)-isopiperitenone was prepared by base-catalyzed exchange in 2H20-dioxane at 25°C and virtually all exchangeable positions were labeled (i.e., via piperitenone), affording species with 4% 2H5, 19% 2Hg, 61% 2HT, and 17% 2H8. Hydrogenation of an aliquot of the purified material yielded a mixture of menthone and isomenthone which, by GLC/MS analysis followed by vigorous exchange in HZ0 and repeated analysis, indicated that 16% of the deuterium of the mixture resided specifically at C4. The deuterated substrate underwent extremely slow rates of isomerization (i.e., ~5% of the rate of the normal substrate), presumably as the result of a significant primary deuterium isotope rate effect, thus suggesting that removal of the (Y hydrogen is the ratelimiting step of the reaction. Because the

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PIPERITENONE

57

rate of the enzymatic reaction was slow, the contribution of nonenzymatic isomerization to total product formation was significant, and thus precluded an accurate assessment of enzyme-catalyzed exchange (35). In spite of the failure of experiments with deuterated substrate, studies of the reaction in 2H20 did establish that the isomerization was carried out by intramolecular proton transfer from C4 to C9 and, thus, was consistent with a carbanion mechanism (32). Internal transfers exceeding 95% are not uncommon in enzymatic isomerizations of this type (33), and examples involving complete retention of hydrogen are known (36). The lack of proton exchange during the isomerization of isopiperitenone to piperitenone can be accounted for by a mechanism such as that shown in Fig. 5 in which the same basic group B of the enzyme is involved in deprotonation at C4 and reprotonation at C9. Direct evidence for base catalysis by an enzyme was first provided by Talalay for the ketosteroid isomerase (34),

FIG. 5. Mechanistic model for the (-)-isopiperitenone isomerase reaction involving a proton donor (HA) and acceptor (B), and an enolic intermediate.

58

KJONAAS,

VENKATACHALAM,

after which the mechanism shown in Fig. 5, including the intermediacy of the enol rather than enolate ion, is closely patterned. Photoinactivation of ketosteroid isomerase in the presence of methylene blue, and chemical inactivation with diethylpyrocarbonate, indicate that the active-site, monoprotonic base of this enzyme is the imidazole ring of a histidine residue (34). In the present instance, isopiperitenone isomerase was sensitive to the presence of methylene blue in the light (65% inhibition at 0.25 mM) and activity was inhibited by diethylpyrocarbonate (93% inhibition at 0.5 ITIM at pH 8.0). When the diethylpyrocarbonate and isopiperitenone were added simultaneously to the enzyme at the initiation of the assay, inhibition was reduced to lo%, indicating that the substrate afforded significant protection against the presumptive N-carbethoxylation of the histidine residue(s). As expected on the basis of the far greater resonance stability of the conjugated, a,@ unsaturated ketone product relative to the P,r unsaturated ketone substrate, no significant reversibility of the isomerization reaction could be demonstrated by incubation of the isomerase with pure piperitenone. (+)-cis-Isopulegone [cf. (+)-iso(iso)pulegol in Figure 41 at the 0.5 mM level was isomerized to (+)pulegone by the enzyme preparation at a rate approximately 66% that observed for the conversion of (-)-isopiperitenone to piperitenone. DISCUSSION

Results described in this communication document the presence in peppermint leaf extracts of soluble enzymes which catalyze the NAD-dependent oxidation of tramisopiperitenol to isopiperitenone and the isomerization of isopiperitenone to piperitenone, two important steps in the conversion of the initially formed cyclic olefin, limonene, to the CS-oxygenated p-menthane monoterpenes characteristic of peppermint. These results confirm, in cellfree systems, a key segment of the bio-

AND

CROTEAU

genetic scheme proposed earlier on the basis of in viva studies. In general properties, the isopiperitenol dehydrogenase resembles other monoterpenol dehydrogenases that have been described: molecular weight in the range 60,000 to 90,000; preference for NAD with a Km in the range 2-10 X 10m5M; sensitivity to thiol-directed reagents; and a narrow range of possible substrates (27, 28). A singular exception was the pH optimum, which was considerably higher for isopiperitenol dehydrogenase (pH -9.5) than for similar dehydrogenases (27,28) which utilize saturated monoterpenols as substrates (pH 8.0). While too few examples of the present enzyme type are known to permit speculation about this property, the available information suggests that plant-derived dehydrogenases which utilize (Y,B unsaturated alcohols operate optimally at a higher pH than that observed for either alcohol dehydrogenase (26, 37) or dehydrogenases which oxidize saturated monoterpenols (27, 28). The isopiperitenone isomerase from this higher plant resembles the well-known ketosteroid isomerase from Pseudommas (34) in both general properties and mechanism of action [little is known about mammalian ketosteroid isomerases, since they are membranous (34, 35)]. Both the plant and bacterial enzymes are operationally soluble, with molecular weights of 54,000 and 41,000, respectively. Both enzymes show a pH optimum near 8.0 (with the bacterial enzyme having the broader operating range) and exhibit a Km of -0.3 mM for their presumptive substrates. The transformation of isopiperitenone to piperitenone does not involve redox isomerization, but rather appears to proceed via the enol with internal 1,3 proton transfer as is the case with ketosteroid isomerase. No evidence was obtained for metal ion assistance in the presumptive enolization, and the overall reaction is essentially irreversible. Isopiperitenone isomerization is inhibited by methylene blue and by diethylpyrocarbonate, implying a role for a histidine residue in catalysis as has been demon-

BIOSYNTHESIS

OF

ISOPIPERITENONE

strated for the ketosteroid isomerase (34). The plant-derived isomerase is moderately inhibited by thiol-directed reagents; the bacterial isomerase is not since this enzyme contains no thiol functions. Substrate specificity studies indicate that (-)-trans-isopiperitenol and (-)-isopiperitenone are the preferred substrates for the dehydrogenase and isomerase, respectively. However, an interesting feature of both enzymes is the readily measurable utilization of the enantiomeric substrates derivable from (+)-limonene. Both (+)and (-)-limonene are synthesized by peppermint, and earlier indirect evidence had suggested that both enantiomers were subsequently, and nearly completely, metabolized (i.e., the stereochemical composition of the mixture of direct cyclization products [80% (-) to 20% (+)] is virtually identical to the small amount of residual, unmetabolized limonene which accumulates in the oil (5)). More direct evidence for the implied intermediacy of (&)&an.+isopiperitenol and (+-)-isopiperitenone could not be obtained in vivo since no intermediates between limonene and pulegone accumulate, and the two stereochemically discrete pathways (as in Fig. 1) would obviously converge at the symmetrical intermediate, piperitenone, thus masking the distinction. The stereochemical consequences of this scheme are of some interest, both because of the novelty of such a pathway and because in vivo studies with Mentha p&egium by Banthorpe and associates (38) have indicated, through the use of [4,10-‘4Cz]geraniol as an exogenous precursor, that the gemdimethyls of the subsequent metabolite of piperitenone, pulegone, were scrambled. Such scrambling of label in pulegone [also reported for carvone, a C2 oxygenated derivative of limonene in Menthu spicata (39)], may result from equilibration of the terminal methyls of the acyclic precursor at some step of the reaction sequence, as proposed by these investigators (38, 39). However, if the methylene groups of (+)and (-)-limonene are derived regioselectively from different carbons of the gemdimethyl substituent of the acyclic pre-

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59

cursor, as seems quite possible, then the observed “scrambling” may be a natural consequence of the utilization of both (+)and (-)-limonene as precursors of the C3 and C2 oxygenated compounds by pathways in which the stereochemical identities of the gem-methyls in passing from precursor to product are, in fact, preserved. Clarification of these possibilities must await further investigation of the earlier steps of the pathway, specifically those catalyzed by the limonene cyclase(s) and the limonene hydroxylation systems, and a more detailed examination of the isomerization of isopiperitenone, the enzymatic step at which the apparent scrambling would be most likely to occur. These studies are now in progress. ACKNOWLEDGMENTS We thank S. Combelic Hopp and R. Carrington monoterpene standards, the manuscript.

for for and

raising the plants, R. the generous gifts of Mary Bull for typing

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