Metabolism of Monoterpenes: Conversion of l-Menthone to 1-Menthol and d-Neomenthol by Stereospecific Dehydrogenasesfrom Peppermint (Mentha piperita) Leaves" 2
Received for publication September 1, 1981 and in revised form December 14, 1981
ROBERT KJONAAS, CHARLOTT MARTINKUS-TAYLOR, AND RODNEY CROTEAU3Institute of Biological Chemistry, and Biochemistry/Biophysics Program, Washington State University,Pullman, Washington 99164
ABSTRACT
The monoterpene ketone l-menthone is specifically converted to I-men-thol and I-menthyl acetate and to d-neomenthol and d-neomenthyl-f8-n-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham)leaves. The selectivity of product formation results from compartmentationof the menthol dehydrogenase with the acetyl transferase and that of theneomenthol dehydrogenase with the glucosyl transferase. Soluble enzymepreparations, but not particulate preparations, from mature peppermintleaves catalyzed the NADPH-dependent reduction of 1-menthone to bothepimeric alcohols, and the two dehydrogenases responsible for thesestereospecific transformations were resolved by affinity chromatographyon Mitrex Gel Red A. Both enzymes have a molecular weight of approxi-mately 35,000, possess a Km for NADPH of about 2 x 10' M, are verysensitive to inhibition by thiol-directed reagents, and are not readilyreversible. The menthol dehydrogenase showed a pH optimum at 7.5,exhibited a Km for 1-menthone of about 2.5 x 10' M, and also reduced d-isomenthone to d-neoisomenthol. The neomenthol dehydrogenase showeda pH optimum at 7.6, exhibited a K. for I-menthone of about 2.2 x 10' M,and also reduced d-isomenthone to d-isomenthol. These stereochemicallydistinct, but otherwise similar, enzymes are of key importance in determin-ing the metabolic fate of menthone in peppermint, and they are probablytypical of the class of dehydrogenases thought to be responsible for themetabolism of monoterpene ketones during plant development.
appears to be a common feature of maturing essential oil plants.In peppermint, the metabolic disposition of the diastereomericreduction products of 1-menthone is highly specific in that only 1-menthyl acetate (with little d-neomenthyl acetate) and d-neomen-thyl-,f-D-glucoside (with little l-menthyl-fi-D-glucoside) areformed (6, 12). In vivo experiments, using labeled CO2, menthone,menthol and neomenthol as precursors, provided compelling evi-dence that compartmentation effects, and not the substrate speci-ficity of the transferases, were responsible for the selectivity ofpathways observed; this conclusion was confirmed by isolationand characterization of the acetyl CoA:monoterpenol acetyltrans-ferase and the UDP-glucose:monoterpenol glucosyltransferase in-volved (5, 18). The nature of the terpenyl moiety of the acetateand ,B-D-glucoside is, thus, determined at the menthone reductionstep by compartmentation of these stereospecific reactions withthe appropriate, relatively nonselective, transferase. Consistentwith chemical considerations (15) and with earlier genetic studies(13, 19), such a metabolic scheme requires the presence in pep-permint of two stereochemically distinct dehydrogenases. TheNADPH-dependent conversion of d-pulegone to menthol (pre-sumably via menthone) by extracts of peppermint leaves has beendetected by Loomis and coworkers (3), suggesting the existence ofone of the necessary enzymes. In this communication, we describein detail the separation and characterization of the two dehydro-genases which catalyze the NADPH-dependent reduction of 1-menthone to i-menthol and d-neomenthol, respectively.
MATERIALS AND METHODS
The metabolism of 1-menthone in maturing peppermint leaveshas been shown to involve the reduction of this monoterpeneketone to i-menthol, some of which is subsequently acetylated,and to d-neomenthol, most of which is glucosylated and trans-ported to the rhizome (Fig. 1) (6, 12, 18). Based on sequentialanalysis of the terpene composition of several other species (7, 8,10, 1 1, 20, 21), such reductive metabolism ofmonoterpene ketones
'Supported in part by National Science Foundation Grant PCM 78-19417 and by grants from the Washington Mint Commission and MintIndustry Research Council. Scientific Paper 6021, Project 0268, College ofAgriculture Research Center, Washington State University, Pullman, WA99164.
2Although the systematic name for 1-menthone is (5R,2S)-trans-5-methyl-2-(1-methylethyl)cyclohexanone, we have utilized here the morecommon nomenclature based on numbering ofthep-menthane system (i.e.menthone = p-menthan-3-one), in which the methyl-substituted carbon isI R and the isopropyl-substituted carbon is 4S.'To whom inquiries should be made.
Plant Material, Substrates, and Reagents. Peppermint (Menthapiperita L. cv. Black Mitcham) plants were grown from stolonsunder controlled conditions described previously (6). Leaves fromthe midstem to apex of flowering plants were used for all experi-ments.
l-[G-3H]Menthone was prepared by Cr03 oxidation of l-[G-3H]menthol (obtained by 3H2 exposure) and was purified as describedin detail elsewhere (6). For use as a substrate, the l-[G-3H]men-thone was diluted with authentic material to a specific radioactiv-ity of 50 Ci/mol or 15 Ci/mol and was dispersed in water with theaid of Tween-20 (10 ,ug/,umol) and sonication (Biosonik III). d-[G-3H]Isomenthone (50 Ci/mol) was prepared by equilibration ofl-[G-3H]menthone in refluxing sodium ethoxide (5%)-ethanol.After the addition of water and ether extraction the isomenthone(RF = 0.47) was separated from residual menthone (RF = 0.53) byTLC (Silica Gel G, with hexane:ethyl acetate (85:15, v/v) asdeveloping solvent [system A]). The purity of the [3H]isomenthonewas verified by radio-GLC and by preparation of the crystallineoxime (as was also done for [3H]menthone), and this material wasdispersed in H20 with Tween-20, as before.
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1-Menthone, d-isomenthone, I-menthol, d-neomenthol, d-iso-menthol, d-neoisomenthol, d-pulegone, d-piperitone, and the an-alogs described in the text (all 99+% pure) were provided throughthe generosity of K. Bauer and G. K. Lange of Haarmann andReimer GmbH (Holzminden, West Germany) and R. Carringtonof I. P. Callison and Sons (Chehalis, WA). Insoluble PVP (GAFCorporation} and Amberlite XAD-4 resin (Rohm and Haas Cor-poration) were purified by standard procedures for use as adsorb-ents (16, 17). Soluble PVP (GAF) was pharmaceutical grade.Unless otherwise specified, all other reagents and biochemicalswere obtained from Sigma Chemical Co. or Aldrich Chemical Co.
Preparation of Derivatives. Ketone oximes and the phenylure-thane derivatives of monoterpenols were prepared by literaturetechniques (23). After work-up of the reaction mixtures, thederivatives were purified by preparative TLC (Silica Gel G,with hexane:ethyl acetate (70:30 v/v) as developing solvent [systemB]) before crystallization (oximes from aqueous ethanol, pheny-lurethanes from petroleum ether).Enzyme Preparation and Purification. Peppermint leaves (5-10
g) were collected, washed with distilled H20, and ground with anequal tissue weight of insoluble PVPP4 (Polyclar AT) in a Ten-Broeck homogenizer with cold 0.1 M Na-phosphate buffer (pH6.7) containing 0.25 M sucrose, 50 mt Na-ascorbate, 50 mMNa2S205, 1 mM dithioerythritol, and 1 mm EDTA. The homoge-nate was then slurried with an equal tissue weight of hydratedAmberlite XAD-4 polystyrene resin for 5 min at 0 to 4°C, filteredthrough several layers of cheesecloth, and centrifuged at 27,000gfor 15 min (pellet discarded) and then at 105,000g for 90 min toprovide the soluble supematant used as the enzyme source. Whenutilized at this stage of purification, the preparation was dialyzedto standard assay conditions.
Finely powdered (NH4)2SO4 was added to the 105,000g super-natant (at 0-4°C) over a period of 30 min until 30%o saturation(0.176 g/ml) was obtained. Precipitated protein was removed bycentrifugation at 27,000g (10 min), and the supernatant wasbrought to 80%1o saturation with (NH4)2SO4 (0.561 g/ml), as before.Precipitated protein was collected by centrifugation (27,000g, 10min) and resuspended in 4 to 5 ml of 50 mm Na-phosphate buffer(pH 7.5) containing 5% (w/v) sorbitol, 50 mm Na2S205, 15 mmmercaptoethanol, and 10 mm Na-ascorbate. The suspended ma-terial, after dialysis against the same buffer for several h, wasrecentrifuged (27,000g, 10 min) to remove denatured protein, andthen it was applied to a Sephadex G-100 column (2.5 x 100 cm)previously equilibrated with 50 mm Na-phosphate buffer (pH 7.5)containing sorbitol and mercaptoethanol, as above. Proteins wereeluted (-0.9 ml/min, 5.4-ml fractions) while monitoring the col-umn effluent at 280 nm. Fractions containing dehydrogenaseactivity (menthol-specific and neomenthol-specific activities wereessentially coincident) were combined and concentrated by ultra-filtration (Amicon PM-10), and, when utilized at this stage of
Acetyltransferose
Z_tonHGlcolAc
t I-Menthol l~~~~~~~~-Menthyl acetate
I-Menthone A GluctrnsfeosyleH " Gluc
d-Neomenthol d-Neomenthyl glucosideFIG. 1. Metabolic transformations of 1-menthone in peppermint.
4Abbreviation: PVP, polyvinyl-polypyrrolidone
purification (e.g. determination of pH optimum), the preparationwas brought to assay conditions by dialysis.To resolve the two dehydrogenase activities, the Sephadex-
purified preparation (in 50 mm Na-phosphate buffer [pH 7.5]containing sorbitol and mercaptoethanol, and to which 0.5 mmNADPH had been added) was applied to an 8-mm x 8-cm columnof Mitrex Gel Red A (Amicon Corporation), which had beenequilibrated with the same buffer. Protein was allowed to equili-brate in the gel for 1 h, before step gradient elution with 2.5 voidvolumes each of starting buffer, starting buffer containing 0.1 MKCI, and starting buffer containing 2 M KCI. For initial studies,individual column fractions were collected and dialyzed to assayconditions before determination of activity. For routine separa-tions, only the starting buffer eluant and the 2 M KCI gradientfraction were collected, separately concentrated by ultrafiltration(Amicon PM-10), and brought to assay conditions by dialysis. Therecovery of enzymic activity of the affinity column was routinely40 to 60%.
For preparing particulate fractions, the same extraction bufferwas used, except that soluble PVPP (Plasdone K-90; mol wt,400,000) was substituted for the insoluble polymer and the XAD-4 treatment and preliminary filtration were omitted. Particulatefractions, prepared by differential centrifugation, were washedbefore the assay (by suspension and recentrifugation) with 50 mMNa-phosphate buffer (pH 7.5) containing 5% (w/v) sorbitol and15 mm mercaptoethanol.Enzyme Assay. The standard reaction mixture, containing 20 to
90 ,ug protein, 0.5 mm l-[G-3H]menthone, 0.6 mM NADPH, and aregenerating system consisting of 6 mM glucose-6-P and 1 unit ofglucose-6-P dehydrogenase (Sigma Type XI), in a total volume of1 ml of 50 mM Na-phosphate buffer (pH 7.5) containing 5% (w/v)sorbitol and 15 mm mercaptoethanol, was incubated in a Teflon-sealed screw cap vial at 30°C for 90 min. The reaction mixturewas then chilled on ice and extracted with 1.5 ml diethyl ether.Appropriate internal standards (10 mg each of the ketone andcorresponding diastereomeric alcohols) were added to the etherextract, which was dried by passage through a short column ofanhydrous Na2SO4, concentrated at 0°C under a stream on N2and subjected to TLC analysis (system A). The i-menthol (RF =0.28), d-neomenthol (RF = 0.39), and 1-menthone (RF = 0.53) wereisolated for determination of 3H content by liquid scintillationspectrometry. The assay conditions were the same when using d-[G-3HJisomenthone as the substrate, and the appropriate internalstandards (d-isomenthol, RF = 0.26; d-neoisomenthol, RF = 0.37;d-isomenthone, RF = 0.47) were added for TLC analysis (systemA). Appropriate boiled controls were included in each experiment,and protein was determined by the Bio-Rad Assay (BSA asstandard) based on the dye-binding principle (4).Chromatography and Determination of Radioactivty. TLC was
carried out on 1 mm layers of Silica Gel G (EM Laboratories,Elmsford, NY) activated at 110°C for 4 h. Developing solventsare indicated elsewhere in the text. After development, the chro-matograms were sprayed with an ethanolic solution of 2,7-dich-lorofluorescein (0.2% w/v) and viewed under UV light to locatethe appropriate products. Radioactivity in TLC fractions wasdetermined either by eluting the material from the gel with diethylether or by scraping the gel directly into a counting vial andadding 15 ml of a solution consisting of 0.4% (w/v) Omnifluor(New England Nuclear) dissolved in 30%o ethanol in toluene (35%efficiency for 3H). All assays were done with a SD of less than 3%.Radio-GLC was performed on a Varian chromatograph at-
tached to a Model 7357 Nuclear Chicago radioactivity monitorwhich was calibrated with [3H]toluene. The stainless steel columnused (3-m x 3-mm o.d.) was packed with 12% Carbowax 4000 on80/100 mesh Gas-chrome Q, and it was operated at 160°C withan argon flow rate of 120 cm3/min.
Demonstration of Dehydrogenase Activities. Previous in vivostudies have provided strong evidence that two distinct, stereospe-cific dehydrogenases must be present in maturing peppermint toaccount for the conversion of 1-menthone, the major monoterpenecomponent of this species, to the diastereomeric alcohols i-mentholand d-neomenthol (6, 12, 18). To examine this possibility directly,a homogenate of mature peppermint leaves was incubated with 1-[G-3H]menthone (1 mM) in the presence of both NADH andNADPH (each at 0.5 mM). Examination of the ether-solublereaction products by radio-TLC revealed the presence of only twocomponents derived from the ketone, and they were chromato-graphically coincident with i-menthol and d-neomenthol, respec-tively. Differential centrifugation of the homogenate showed thatapproximately 95% of the dehydrogenase activity (for both alcoholproducts) resided in the 105,000g supematant, which was used asthe enzyme source in subsequent experiments. The residual 5% ofthe activity of the homogenate was scattered throughout thevarious particulate fractions, and this activity was not examinedfurther.
Product Identification. To confirm the identification of theproducts derived from l-[G-3H]menthone by the soluble enzymesystem, the putative [3H]menthol and [3H]neomenthol (-6 x 105dpm, each), generated by large-scale incubation, were individuallyisolated by TLC. Radio-GLC under the conditions described in"Materials and Methods" indicated the presence in each fractionof a single radioactive component chromatographically coincidentwith the corresponding alcohol standard. The tentatively identi-fied [3H]neomenthol was diluted to a specific radioactivity ofabout 0.186 ,uCi/mmol with pure d-neomenthol and the phenylurethane derivative was prepared and crystallized, with negligibleloss, to a constant specific radioactivity of 0.185 ,uCi/mmol (m.p.,107-108°C; literature m.p., 107-108°C [2]), thereby confirmingthe identification.The [3Hlmenthol was similarly diluted with authentic material
to a specific radioactivity of about 0.156 ,uCi/mmol, and thephenylurethane derivative was prepared and crystallized to aconstant specific radioactivity of 0.095 ,ICi/mmol (m.p.,111-1 12°C; literature m.p., 112°C [2]). These results confirmedthe identification of the starting material as i-menthol; however,because of the decrease in specific activity from the initial dilution,they also suggested the presence of a labeled contaminant in theoriginal TLC fraction. That an impurity did comigrate withmenthol on TLC was subsequently verified by studies on theeffect of pyridine nucleotides on the various enzymic activities.
Response to Cofactors. In the absence of reduced pyridinenucleotides, the enzymic conversion of 1-[3H]menthone to d-[3H]neomenthol was negligible, while, in the presence of NADPH (0.6mM), the rate of neomenthol formation by the 105,000g superna-tant preparation approached 12 nmol/h. NADH (0.6 mM) was afar less effective reductant, affording a rate of neomenthol for-mation of about 1.7 nmol/h under identical assay conditions.Similar analysis of menthol formation in the absence of pyridinenucleotide showed that radioactivity was present in the mentholregion on TLC assay (equivalent to 3.5 nmol/h). However, radio-GLC of this fraction showed that only a small quantity of authen-tic [3H]menthol was actually present (equal to 1.1 nmol/h) andthat the bulk of the radioactivity of this fraction did not elute fromthe column under the conditions of analysis. As the polarity ofthis unknown material was similar to that of menthol on TLC(silica gel), a higher mol wt contaminating species was indicated.In the presence ofNADPH (0.6 mM), the rate of menthol synthesisfrom the labeled ketone increased to 16 nmol/h, while, with thesame concentration of NADH, a rate of 2 nmol/h was noted (ineach case the TLC-isolated menthol fraction was subjected toradio-GLC, which was externally calibrated to provide quantita-tive analysis of [3H]menthol production). By this means, the
conversion of menthone to menthol and neomenthol was dem-onstrated to be NADPH-dependent, while formation of the un-known product(s) did not require pyridine nucleotides and wasnot influenced by the presence of these cofactors. Boiled controlswere incapable of synthesizing any of the aforementioned prod-ucts.The quantity of the contaminating substance formed by the
soluble enzyme fraction varied from preparation to preparationand with reaction conditions, and we were unable to separate thisunidentified material from the products of interest by TLC tech-niques or simple derivatization procedures. Additionally, we couldnot fully resolve the responsible activity from the enzyme ofinterest by the protein fractionation techniques employed. There-fore, it was necessary to run the appropriate controls (withoutNADPH), with additional radio-GLC verification ofthe authenticproduct, and to apply the needed corrections to each assay formenthol synthesis. Attempts to circumvent the problem for routine
0
xE
a-
CL
010
4
Fraction NumberFIG. 2. Sephadex G-100 gel filtration of the protein concentrate of the
105,OOOg supernatant preparation from peppermint leaves. A at 280 nmand NADPH-dependent dehydrogenase activity (neomenthol formation[0- - -0] and menthol formation [@-]) are plotted. Chromatographyand assay procedures are described in "Materials and Methods." VO wasat fraction 6. Assays were run at subsaturating concentrations of menthone(0.1 mM), which, in part,-accounts for the relative rates observed.
2.0w
I0x
E0.0
0
a-0-
5 10 15 20
0
Fraction NumberFIG. 3. Affinity chromatography of the dehydrogenase preparation on
Matrex Gel Red A. The menthol-specific activity (@-), neomenthol-specific activity (0- - -0), and KCI gradient (- - -) are plotted. Chroma-tography and assay procedures are described in "Materials and Methods."
assays, by measurement of the reverse reaction, were unsuccessful,as, even under optimum conditions (pH 6.0, menthol and NADPconcentration at 1 mm each), the rate of menthol oxidation wasless than 7% of menthone reduction. A similar lack of reversibilitywas noted with neomenthol as substrate.
Resolution of the Dehydrogenases. The protein concentrate(30-80% (NH4)2SO4 precipitate) obtained from the 105,000g su-pernatant of a peppermint leaf homogenate was subjected topreliminary fractionation by gel permeation chromatography onSephadex G-100. Figure 2 illustrates the elution pattern of thedehydrogenase activities measured by the reduction of 1-[3HJmen-thone in the presence of NADPH, and it indicates that the twoactivities for the synthesis of diastereomeric alcohols were coinci-dent. Calibration of the column with protein standards provideda mol wt for the dehydrogenases of about 35,000 (i.e. activityeluted slightly after ,B-lactoglobulin). Although the gel permeationstep did not resolve the dehydrogenases, this procedure did removea portion of the activity responsible for the formation of thementhol-like contaminant (some 20-30%o of this activity was re-moved; however, corrections for menthol synthesis were still re-quired). The pooled dehydrogenase fractions (Nos. 13-22) fromthe Sephadex G- 100 step (in 50 mm Na-phosphate buffer [pH 7.5]containing 5% [w/v] sorbitol and 15 mm mercaptoethanol) were,therefore, concentrated and applied to a column of Matrex GelRed A (Amicon) that was equilibrated and eluted with the samebuffer containing 0.5 mm NADPH. Following elution of the voidvolume, which afforded both the menthol-specific dehydrogenaseand contaminating activity, and an intervening concentration of0.1 M KC1, buffer containing 2 M KCl was applied to the columnto remove the neomenthol-specific dehydrogenase (Fig. 3). Thisstep-gradient technique was adapted from a linear gradient elutionprocedure in which the menthol-specific dehydrogenase, essen-tially free of the neomenthol-specific dehydrogenase, and theneomenthol-specific dehydrogenase, essentially free of menthol-specific dehydrogenase, could be obtained. However, this proce-dure was abandoned in favor of the step-gradient method, becausethe time involved in running the linear gradient and desaltingeach fraction before assay resulted in considerable loss of dehy-drogenase activity. The menthol-specific dehydrogenase was par-ticularly labile, suffering greater loss of activity on handling andstorage than did the neomenthol-specific dehydrogenase, a prob-lem which precluded further attempts to separate this enzymefrom contaminating activities. While the affmity chromatographicseparation was not without difficulty (40-60% loss of activity), thistechnique clearly demonstrated that two distinct, stereoselectivedehydrogenases, which utilized 1-menthone as substrate, werepresent in the peppermint leaf extract. All subsequent studies werecarried out with the Sephadex G-100 purified preparation, or,when necessary, with the separate enzymes obtained by affinitychromatography.
Effect of Time, Protein Concentration, and pH. The rate ofconversion of 1-menthone to i-menthol or d-neomenthol increasedlinearly with time up to at least 90 min at the 100lg/ml proteinlevel for both Sephadex G-100- and affinity chromatography-purified preparations, and all measurements of enzyme activitywere made under these linear conditions.The neomenthol-specific dehydrogenase exhibited a pH opti-
mum at 7.6 (half maximal activities at pH 6.3 and 9.5) whenexamined in citrate-phosphate-borate buffer (50 mM) containing5% (w/v) sorbitol and 15 mm mercaptoethanol. In the same buffer,the menthol-specific dehydrogenase showed an optimum at 7.0,with half maximal activities at pH 5.6 and 8.0. In 50 mm Na-phosphate buffer (same additions as above), the pH optimum ofthe neomenthol-specific enzyme remained unchanged, while thatof the menthol-specific enzyme shifted to pH 7.5 with considerablebroadening of the response curve. For convenience, all assays ofdehydrogenase activity were carried out using the Na-phosphate
buffer (pH 7.5), in which maximum enzyme stability and nearlymaximum activity were obtained.
Effect of Inhibitors. To examine the effect of thiol-directedreagents on enzyme activity, the Sephadex-purified preparationwas first thoroughly dialyzed to remove mercaptoethanol (andresidual dithioerythritol). This procedure resulted in the loss of 80to 90%o of both activities (dialysis against mercaptoethanol for anequal length of time decreased the activities by 10-50%). Activityof the neomenthol-specific enzyme could be restored to roughly50% of the original level by readdition of 15 mm mercaptoethanol,but the activity of the menthol-specific enzyme was irreversiblylost. The residual activity of both enzymes, remaining after re-moval of mercaptoethanol, was almost completely abolished by a50 iLM level ofp-hydroxymercuribenzoate and by 250 /LM levels ofHgCl2, N-ethylmaleimide, and iodoacetamide. Thus, both en-zymes were extremely sensitive to the absence of thiol-protectingreagents and to the presence of thiol-directed reagents.
Divalent cations had relatively little effect on the dehydrogen-ases; MnCl2 and CaCl2 (at 10 mM) exhibited a consistent, butminor (<10%), inhibition of the neomenthol-specific enzyme,while CaCl2 at the 1 mm level showed slight (-10%) stimulationof the menthol-specific enzyme.
Kinetic Properties and Substrate Specificity. As the concentra-tion of 1-menthone in the assay medium was increased, the rate offormation of i-menthol and d-neomenthol also increased, givingrise in each case to a typical Michaelis-Menten hyperbolic satu-ration curve. The double reciprocal plots were linear (Fig. 4), fromwhich Km values of 2.2 x 10-5 M (neomenthol-specific) and 2.5X l0-4 M (menthol-specific) were determined. Under identicalconditions (i.e. the Sephadex G-100-purified preparation), and ata saturating level ofNADPH (0.6 mM), the relative velocities were15 and 20 nmol/h *mg protein, respectively (Fig. 4). Increasing theconcentration of NADPH in the presence of a saturating level of1-menthone (1 mM) provided a pair of similar hyperbolic saturationcurves, affording linear double reciprocal plots, from which Kmvalues for NADPH were determined to be 2.2 x 10-5 M (neomen-thol-specific) and 1.5 x 10-5 M (menthol-specific). Inasmuch as[3HJmenthone was dispersed with Tween-20 and was not in a true
0.0 0.04 0.08
[Menthone eLM]FIG. 4. Plot of reciprocal of reaction velocity for menthol (@-)
and neomenthol (0- - -0) synthesis versus reciprocal of concentration of1-menthone. Each reaction mixture, containing 45 ,g protein, 0.6 mmNADPH (with regenerating system), and the appropriate amount of /-IG-3H]menthone, in a total volume of 1 ml of 50 mm Na-phosphate buffer(pH 7.5) containing 5% (w/v) sorbitol and 15 mm mercaptoethanol, was
solution, the observed values should be taken with the usualprecautions.The ketone d-isomenthone (1R:4R, epimeric with 1-menthone
at the isopropyl-substituted carbon) is also a component of pep-permint oil (3.5%, compared to -25% menthone in a commercialoil sample [14]). The reduction products of d-isomenthone are d-isomenthol ([1 R:3 S:4R], epimeric with d-neomenthol [1 R:3 S:4S]at the isopropyl-substituted carbon) and d-neoisomenthol([l R:3R:4RJ, epimeric with i-menthol [I R:3R:4S] at the isopro-pyl-substituted carbon) (Fig. 1), which are minor constituents ofpeppermint oil (22). Because it seemed likely that the presentdehydrogenases could also account for the presence of isomentholand neoisomenthol in peppermint oil, d-[G-3Hlisomenthone wasprepared and tested as a substrate with the enzymes separated byMatrex Gel Red chromatography. The menthol-specific enzymereduced d-isomenthone to d-neoisomenthol, while the neomen-thol-specific enzyme reduced d-isomenthone to d-isomenthol.Thus, as anticipated on the basis of chemical (15) adfd geneticconsiderations (13, 19), the ketone carbonyl was consistently re-duced to the 3R alcohol configuration in one instance and to the3 S configuration in the other. Rates of isomenthone reductionwere comparable to those of menthone reduction in both cases(within 15% under saturating conditions), indicating that theorientation of the isopropyl substituent had little influence on thereactions.To examine the question of substrate specificities in greater
detail, the effect of various substrate analogs on the reduction ofl-[G-3H]menthone was examined. At twice the concentration of 1-menthone, the following compounds inhibited i-menthol and d-neomenthol synthesis, respectively, by the indicated amounts: djl-4-methyl-2-isopropylcyclohexanone(diequatorial), 9%lo and 14%;d,l-5-methyl-2-propylcyclohexanone(diequatorial), 65% and 61%;d,l-5-methyl-3-isopropylcyclohexanone(diequatorial), 32% and63%; d-pulegone, 34% and 48%; d-piperitone, 13% and 45%; and1-carvone, 20o and 6%. Although significant differences in inhi-bition between the two enzymes were noted, the data do suggestreasonably broad specificities for substituted cyclohexanones andcyclohexenones. Thus, the enzymes described here, while clearlyresponsible for catalyzing the two key reductions in the metabo-lism of l-menthone in peppermint (Fig. 1), are also likely to beresponsible for the formation of menthol stereoisomers from d-isomenthone and, perhaps, of other minor alcohol constituents ofthe oil, such as isopulegol and carveol (9, 22).The distinguishing feature of these dehydrogenases is their
stereospecificity. In other characteristics, they are rather similar,and they are probably typical of enzymes likely to be present inother species where the reduction of monoterpene ketones is acharacteristic feature of development (7, 8, 10, 11, 20, 21). Al-though the enzymes reported here are theoretically dehydrogen-ases (i.e. oxidoreductases), their observed apparent equilibria favorreduction. In this and in virtually all other properties (pH opti-mum, cofactor preference, mol wt, response to thiol-direct re-agents, etc.), they resemble the general class of cytoplasmic ketoreductases that are very widespread in nature (1).With the present work and the earlier studies (5, 18), all of the
key enzymes of l-menthone metabolism in peppermint leaf havebeen demonstrated, partially purified, and characterized. All ofthese enzymes are operationally soluble under the conditions ofour isolation procedures, which raises an obvious question regard-
ing the physical basis for the compartmentation of pathways thatis necessarily operative in the metabolism of menthone in pepper-mint leaves (6, 18). This important issue, which has only recentlybeen resolved, will be addressed in a subsequent communication.
Acknowledgments-We thank R. Hamlin and C. Whitney, for raising the plants,and K. Bauer, G. K. Lange, and R. Carrington, for the generous gifts ofmonoterpenestandards.
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