Characterization and mechanism of (4S)-limonene synthase, A monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita)

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 296, No. 1, July, pp. 49-57, 1992

Characterization and Mechanism of (4S)-Limonene Synthase, A Monoterpene Cyclase from the Glandular Trichomes of Peppermint (/Ventha X piperita)’ Jean I. M. Rajaonarivony,2 Institute

Jonathan

Gershenzon, and Rodney Croteau3

of Biological Chemistry and Plant Physiology Program, Washington State University,

Received December 23,1991, and in revised form February

Washington

99164-6340

18, 1992

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha X piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (>94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The K,,, value measured for geranyl pyrophosphate was 1.8 pM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence. 0 1992 Academic Press, Inc.

’ This investigation was supported by a grant from the U.S. Department of Energy (DE-FG06-88ER13869) and by Project 0268 of the Agricultural Research Center, Washington State University. * Recipient of an AFGRAD III Fellowship from the African-American Institute. 3 To whom correspondence should be addressed.

cao3-9861/9‘2 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

Pullman,

Monoterpenes constitute a large class of isoprenoid natural products that arise from the acetate-mevalonate pathway via the acyclic, Cl0 intermediate geranyl pyrophosphate. The cyclization of geranyl pyrophosphate to monoterpenes is catalyzed by enzymes referred to as monoterpene cyclases (1). These enzymes have been the subject of continuing investigation because they direct the formation of the basic skeletal types of monoterpenes and they catalyze unusual, multistep reaction sequences involving the isomerization, cyclization, and deprotonation of carbocationic intermediates (l-3). Similar electrophilic reaction mechanisms are believed to be characteristic of many enzymes of isoprenoid metabolism, including the prenyltransferases that catalyze the synthesis of geranyl pyrophosphate and homologous prenyl pyrophosphates (4, 5). In a previous paper (6), the purification of the monoterpene cyclase, (4S)limonene synthase, from peppermint (Me&ha X piper&u) and spearmint (M. spicatu) was described. This enzyme catalyzes the conversion of gerany1 pyrophosphate to (4S)-limonene (Scheme I), the first cyclic intermediate in the biosynthesis ofp-menthane (lmethyl-4-isopropylcyclohexane) monoterpenes in Menthu species (7). Like other monoterpene cyclases, (4S)-limonene synthase is thought to mediate the cyclization of geranyl pyrophosphate via a series of carbocationic intermediates (1). According to the postulated mechanism (Scheme I), geranyl pyrophosphate first ionizes (1) and then isomerizes to linalyl pyrophosphate, which remains bound to the enzyme. Such an isomerization is topologically necessary because the configuration of the Cl-C2 double bond of geranyl pyrophosphate prevents direct closure to form the cyclohexene ring. After rotation from the transoid to the cisoid conformer, linalyl pyrophosphate is itself ionized (2) and then cyclized to the cu-terpinyl cation (3), that is subsequently deprotonated to give limonene. Both the isomerization and cyclization steps 49

Inc. reserved.

50

RAJAONARIVONY,

GERSHENZON,

AND

CROTEAU

\/ 6

geranyl pyrophosphate

(-)-a-pinene [l s.ss]

SCHEME I. Proposed mechanism for the enzymatic limonene synthase. OPP indicates the pyrophosphate intermediates.

cyclization of geranyl pyrophosphate to (-)-(4S)-limonene and other products by (4s) moiety. Linalyl pyrophosphate and the numbered carbocations are all enzyme-bound

of the reaction sequence are considered to take place at the same active site (3, 8, 9). In this paper, we describe several properties of (4S)-limonene synthase and examine key features of the reaction mechanism based on the results of experiments with a number of substrate analogs and inhibitors. EXPERIMENTAL

PROCEDURES

Plant materials, substrates, and reagents. Peppermint (Men&z X piperita L. cv. Black Mitcham) plants were grown under controlled conditions as previously described (6). Apical buds and newly emerged, rapidly expanding leaves (5-10 mm long) of vegetative stems (3-5 weeks old) were harvested for isolation of the glandular trichome cells used as the enzyme source. The preparations of [l-3H]geranyl pyrophosphate (5.55 GBq/mmol; Fig. 1; 6, lo), [l-3H]neryl pyrophosphate (11.2 GBq/mmol; lo), (3R)(lZ)-[1-3H]linalyl pyrophosphate (984 MBq/mmol; ll), (3S)-(lZ)-[l3H]linalyl pyrophosphate (1.49 GBq/mmol; ll), and the sulfonium ion analog of the linalyl cation, (I&S)-methyl-(4-methylpent-3-en-lyl)vinylsulfonium perchlorate (12) have all been previously described. (4R,S)-[3-3H]a-Terpinyl pyrophosphate was synthesized by solvolysis of [l-3H]neryl pyrophosphate in 1 M HCl with a pentane overlay at 30°C for 2 h (10). After argentation TLC of the pentane-soluble products, the isolated [3-3H]cu-terpineol was pyrophosphorylated according to the modified method of Cramer and Bohm (10, 13). [8,9-2HB,1-3H]Gerany1 pyrophosphate (‘Ho, 1.6%; ‘Hs, 3.2%; *Hs, 95.2%; 2.52 GBq/mmol) was a gift from R. M. Coates, University of Illinois. Polystyrene resin (Amberlite XAD-4, Rohm and Haas) was prepared for use by standard procedures (14). Monoterpene standards were from our own collection. All other reagents were purchased from Research Organics, Aldrich Chemical Co., or Sigma Chemical Co., unless otherwise specified. Enzyme isolation and partial purification. Limonene synthase was extracted from a purified preparation of glandular trichome secretory cell clusters essentially as previously described (6, 15, 16), except that sonication of the isolated secretory cell clusters was carried out in a buffer of 25 mM potassium phosphate, pH 6.0, containing 10% (v/v) glycerol, 10 mM Na2S206, 1 mM EDTA, and 1 mM dithiothreitol, to which was added 1% (w/v) polyvinylpyrrolidone (M, 10,000). The sonicated extract was filtered through 20-pm nylon mesh and the filtrate centrifuged at 27,000g (15 min, pellet discarded) and then again at 195,000g (90 min). The 195,000g supematant was subjected to anion-

exchange chromatography on DEAE-cellulose (20 g DE-52, Whatman). The column was equilibrated with 15 mM sodium phosphate buffer, pH 6.0, containing 1 mM sodium ascorbate, 0.5 mM dithiothreitol, and 0.2 mM EDTA. After loading, the column was washed with 100 ml equilibration buffer containing 100 mM NaCl, and the limonene synthase activity was eluted with 100 ml of the same buffer containing 250 mM NaCl. Enzyme assay. Limonene synthase preparations were desalted or dialyzed to assay conditions. Assays were carried out in a buffer of 10 mM Mopso or Hepes, pH 7.0, containing 5 mM sodium phosphate, 10%

* Abbreviations used: Mes, 2-(N-morpholino)ethanesulfonic acid; Mopso, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate, Tes, N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid.

geranyl

pyrophosphate

neryl pyrophosphate

3S-linalyl pyrophosphate

a-terpinyl pyrophosphate

3R-linalyl pyrophosphate

sulfonium analog of linalyl cation

FIG. 1. Substrates and analogs employed with (4S)-limonene synthase. OPP denotes the pyrophosphate moiety, and the asterisk indicates the position of the ‘H label. The sulfonium analog of the linalyl cation was designed to mimic this proposed reaction intermediate.

CHARACTERIZATION

AND

MECHANISM

(v/v) glycerol, 1 mM sodium ascorbate, and 0.5 mM dithiothreitol. A typical reaction mixture (1 ml) contained 5.0 pg protein and 1 mM MnCls in a Teflon-sealed, screw-capped glass test tube. The reaction was initiated by addition of 7 FM [l-3H]geranyl pyrophosphate. Pentane (1 ml) was carefully layered on top of the assay mixture to trap volatile products. After incubation for 45 min at 30°C with gentle agitation, the reaction was stopped by vigorous mixing and the product isolated chromatographically as previously described (6). Radioactivity was determined by liquid scintillation counting in 10 ml of cocktail containing 0.4% (w/ v) Omnifluor (Du Pont) dissolved in 30% (v/v) ethanol in toluene, using a Packard Tricarb 460 CD liquid scintillation spectrometer (3H efficiency, 42%). For product identification by GLC or radio-GLC, aliquots of the olefin fraction and the oxygenated monoterpenoid fraction (17) were diluted with the appropriate internal standards or authentic carriers, respectively, and concentrated under a stream of Nz. Protein concentrations were estimated by the dye-binding technique of Bradford (18) using the Bio-Rad protein assay kit with bovine serum albumin as standard. Both enzyme activity measurements and protein determinations were reproducible to within 10%. For inhibitor studies, the assay buffer was formulated without sodium ascorbate or dithiothreitol. Enzyme samples were preincubated with inhibitor for 5 min at 30°C. After addition of substrate (7 pM [lsH]geranyl pyrophosphate) and divalent metal cofactor (1 mM MnCl,), incubation was carried out for an additional 40 min and the assay processed as described above. For substrate protection studies, enzyme was preincubated with protectant for 5 min at 3O”C, then with inhibitor for 15 min and, finally, incubation was carried out for an additional 30 min at 3O’C. For analog protection studies, the procedures were the same, except that the samples were dialyzed to remove excess reactants prior to addition of the substrate at the start of the assay. Appropriate controls (with and without protectants) were included in each set of experiments. Analytical procedures. Essential oil was extracted from peppermint buds and young leaves by soaking the tissues in diethyl ether for 1 h at 4°C. The extract was decolorized with activated charcoal, washed with water, dried over MgSO,, and concentrated under Nz in preparation for capillary GLC. GLC was performed on a Hewlett-Packard 5890A gas chromatograph with a 3392A integrator. A chiral phase, fused silica capillary column (0.25 mm id. X 30 m) coated with a 0.25-pm film of peralkylated j3-cyclodextrin (Cyclodex B, J & W Scientific) was used: Hz carrier (1.5 ml/min); split injection (220°C); temperature programming [6O”C (15 min hold) to 220°C (5”C/min)]; flame ionization detector (250°C). GLC analyses of the products of limonene synthase incubation with [l-3H]geranyl pyrophosphate and with [3-3H]a-terpinyl pyrophosphate were carried out under similar conditions. Radio-GLC was performed on a Gow-Mac 550P gas chromatograph as previously described (6). SDS-PAGE was carried out with the system of Laemmli (19) in 7.5% polyacrylamide vertical slab gels (16 cm X 18 cm X 1 mm, Hoefer SE600 vertical gel apparatus). Samples were dialyzed against water, lyophilized, resuspended in SDS sample buffer (19) and denatured in a steam bath for 2 min before loading. Gels were run at a constant current of 20 mA for about 3 h. After silver staining (Bio-Rad Laboratories kit), gels were scanned at 633 nm with an LKB 2202 ultroscan laser densitometer. Preparative isoelectric focusing was carried out in free solution with a Rotofor cell (Bio-Rad Laboratories) at a constant power of 12 W for 4 h at -5°C. A limonene synthase preparation that had been partially purified as described above was precipitated with 50% ammonium sulfate and dialyzed against a buffer of 1 mM sodium phosphate, pH 7.0, containing 10% (v/v) glycerol and 0.5 mM dithiothreitol before loading. The Rotofor cell was filled with a solution of 20% (w/v) sucrose, 0.5 mM dithiothreitol, and 1.5% (w/v), ampholytes (pH 4-6, Bio-F&d). Twenty fractions of 2 ml were collected and the pH of each was measured. For determination of limonene synthase activity, each fraction was adjusted to 1.5 M NaCl, dialyzed against assay buffer, and assayed by standard

OF (4S)-LIMONENE

51

SYNTHASE

procedures. A single peak of activity was observed. The overall recovery of limonene synthase activity from this procedure was 7%.

RESULTS

AND

DISCUSSION

Enzyme purity, stability, and assay. (4S)-Limonene synthase from peppermint (M. X piperita) and spearmint (M. spicata) has previously been purified to apparent homogeneity by three chromatographic steps starting from a crude extract of glandular trichome secretory cells (6). To quickly obtain large amounts of partially purified enzyme for characterization, secretory cell extracts from peppermint were subjected to a one-step fractionation via anion-exchange chromatography on DEAE-cellulose. This procedure afforded a good recovery of limonene synthase (80-90% of the activity in the crude extract) with virtually no detectable competing phosphohydrolase activity. As judged by SDS-PAGE, limonene synthase made up 30-40% of the total protein in this partially purified preparation (data not shown). Limonene synthase activity was relatively unstable in crude secretory cell extracts. However, after DEAE-cellulose chromatography, the enzyme could be stored in assay buffer for 1 week at 4°C without significant loss of activity. Addition of bovine serum albumin, protease inhibitors (e.g., phenylmethylsulfonyl fluoride), or higher concentrations of thiol reagents, reducing agents, or polyol (up to 30% glycerol) did not markedly improve stability. Preparations that were lyophilized from buffers of low polyol content (e.g., 1% sorbitol) and kept at -2O”C, retained at least 50% activity for 6 months. Considerable losses of limonene synthase activity resulted (presumably by adsorption) from attempts to concentrate enzyme solutions by ultrafiltration using Amicon-stirred cells or centrifugal concentrators. The basic parameters of the enzyme assay were determined with the partially purified preparation using, as a guide, previously published assay conditions for monoterpene cyclases (7, 17). Activity was optimal with Mn2+ (l-2 mM MnC1.J as the required divalent metal ion, and geranyl pyrophosphate at 7 PM provided a saturating concentration of substrate. GLC and radio-GLC analyses showed that (4S)-limonene was the principal product of the enzyme (>94%). In a series of trial assays, the rate of conversion of geranyl pyrophosphate to (4S)-limonene increased linearly with protein concentration to 15 pg/ ml for a 45min assay. At the 5 pg/ml protein level, product formation was proportional to incubation times up to 45 min, with less than 5% substrate conversion. All subsequent assays were conducted within these operational limits. Boiled controls showed insignificant levels of (4S)limonene synthase activity. Optimum requirement.

pH,

isoelectric

point,

and divalent

metal

ion

The effect of pH on (4S)-limonene synthase activity was examined with a series of Mes, Pipes, Mopso, Hepes, and Tes buffers of overlapping pH. A bell-

52

RAJAONARIVONY,

GERSHENZON,

shaped curve was obtained with a peak of activity at pH 6.7 and half-maximum velocities at pH 6.1 and pH 7.2. Monoterpene, sesquiterpene, and diterpene cyclases generally exhibit pH optima between 6.0 and 7.5 and halfmaximum velocities within a half pH unit of the optimum (1, 20-33). The pH dependence of cyclase activity may be a consequence of the properties of the pyrophosphorylated substrate, as well as the characteristics of the enzyme (1). For example, the degree of substrate ionization, which varies with pH, may influence the formation of complexes with the divalent metal ion (34). In addition, at pH values below 6.0, geranyl pyrophosphate and other allylic pyrophosphates are subject to appreciable, nonenzymatic solvolysis (35). The pIvalue of (4S)-limonene synthase was determined to be 4.35 + 0.05 by isoelectric focusing in free solution. Isoelectric points between 4 and 5 appear to be typical for terpene cyclases (24, 29, 32, 33). (4S)-Limonene synthase had an absolute requirement for a divalent metal ion cofactor, either Mg2+ or Mn2+. When divalent cations were removed by dialysis or complexation with EDTA, negligible enzyme activity was observed. A plot of enzyme activity as a function of metal ion concentration showed an increasing rate of catalysis up to 10 mM for MgC12 and up to 2 mM for MnC12, with inhibition observed at higher concentrations, particularly in the case of MnC12 (Fig. 2). At the concentration optima, Mn2+ afforded somewhat higher rates of activity than did Mg2+ and was therefore used in the standard assay at a concentration of 1 mM. (Higher concentrations of Mn2+ were not employed because it was difficult to avoid the precipitation of Mn2+ salts in the assay buffer.) No synergism was detected between these metal ions. While several other cyclases exhibit different proportions of products with Mg2+ as compared to Mn2+ (29, 32), limonene synthase showed no alterations in product distribution in response to metal ion cofactor. A variety of other divalent metal ions tested at 0.5 mM (Ca”‘, Cd2+,Co2+,Cu2+,Fe2+, Ni2+, Zn2+; all as chlorides, except Fe’+ as sulfate) did not support limonene synthase catalysis at greater than 5% of the rate obtained with 0.5 mM Mn2+. All other terpene cyclases investigated to date require either Mg2+ or Mn2+ for catalysis. The role of the metal ion is seemingly to neutralize the negative charge of the pyrophosphate moiety and thus assist in the initial ionization of the substrate (1, 34, 35). As a group, monoterpene cyclases show no clear preference for either Mg2+ or Mn’+. Some show highest activity with Mg2+ (with Mn2+ being less effective) (27,29,31,32), whereas others show highest activity with Mn2+ (26, 28, 36, 37). By contrast, sesquiterpene and diterpene cyclases generally show marked preferences for Mg2+, with Mn2+ giving only lo50% of the activity achieved with Mg2+ (20-25, 33, 38). For nearly all cyclases, concentrations of Mn2+ greater than l-2 mM are inhibitory to activity. The suppression

AND

CROTEAU

01 0

I

I

I

I

I

10

20

30

40

50

Concentration (mlvl) FIG. 2. Effect of divalent metal ion concentration, Mg2+ (0) and Mn’+ (O), on (4S)-limonene synthase activity. Assays were carried out under standard conditions as described under Experimental Procedures. The K, value for Mn2+ (0.15 f 0.02 mM) was determined from an expanded scale plot.

of cyclase activity by high concentrations of Mg2+ (>20 mM), as observed in this study for limonene synthase, has not been previously reported for other cyclases. Multiple product formation. When incubated with geranyl pyrophosphate, partially purified (4S)-limonene synthase preparations produced in addition to limonene (94% of total product) several other monoterpene olefins [myrcene (1.5%), cY-pinene (2.0%), and P-pinene (2.4%)], as revealed by radio-GLC analysis (Fig. 3). A nearly identical mixture of products was generated by the apparently homogeneous enzyme confirming that these other olefins were formed by the action of (4S)-limonene synthase in vitro. The enzyme may conceivably produce these minor products in uivo, as well, since the oil of young peppermint leaves and buds (the same tissue used for enzyme isolation) was found to contain small amounts (~1%) of myrcene, cy-pinene, and ,&pinene by GLC analysis, in agreement with previous reports (39,40). Other monoterpene and sesquiterpene cyclases also synthesize multiple products (29-33, 37). The biosynthesis of multiple products by (4S)-limonene synthase can be rationalized by assuming that the minor products observed arise from presumptive carbocationic intermediates (l-3) formed in the course of the conversion of geranyl pyrophosphate to limonene (Scheme I) (1). Myrcene can be formed by deprotonation of either the geranyl (1) or the linalyl (2) cation. Deprotonation of the cY-terpinyl cation (3) leads to limonene, the principal product of the enzyme. However, internal addition of the a-terpinyl cation to generate the pinyl cation (4) could yield, by alternative deprotonations, either a-pinene or /3-pinene. Therefore, all the minor olefinic products of

CHARACTERIZATION

AND

MECHANISM

OF (4S)-LIMONENE

53

SYNTHASE

phate would be expected to perturb the product distribution from that usually observed with [ l-3H]geranyl pyrophosphate. A primary deuterium isotope effect on the deprotonation leading to limonene should increase partitioning of the a-terpinyl cation toward (Y-and @-pinene. In fact, both the rate of production and the relative proportion of the pinenes were detectably enhanced with the deuterated substrate (Table I), in spite of a significant overall rate reduction relative to the control, suggesting that both pinenes were indeed minor coproducts of the limonene cyclization. 0

1

I

I

,

10

20

30

40

Substrate kinetics linalyl intermediate.

Time (minutes) FIG. 3. Radio-GLC separation of the monoterpene olefins generated by (4S)-limonene synthase from [l-3H]geranyl pyrophosphate. Product isolation and conditions for GLC analysis are described under Experimental Procedures. The smooth lower tracing is the thermal conductivity detector response to authentic standards of a-pinene (l), ,9-pinene (2), myrcene (3), and limonene (4). The upper tracing is the radioactivity recorded by the monitor attached to the chromatograph. Output signal was electronically integrated and for three independent analyses gave peak integration values with an SE of less than 10% of the mean.

(4S)-limonene synthase can be generated from intermediates of the proposed reaction sequence. To confirm whether the olefins limonene, a-pinene, and P-pinene are products of the same enzyme, rather than arising through the combined action of several closely related enzymes that fortuitously copurify, an attempt was made to exploit the phenomenon of isotopically sensitive branching (41) using methods previously developed for examining other monoterpene cyclizations (42,43). A preparation of limonene synthase was incubated with [8,92H6,1-3H]geranyl pyrophosphate and the product distribution compared with that resulting from a parallel incubation with [ l-3H]geranyl pyrophosphate as a control. If (4S)-limonene, cr-pinene, and fl-pinene all arise from the same monoterpene cyclase via the a-terpinyl cation (3), administration of [8,9-2H6,1-3H]gerany1 pyrophos-

TABLE

Isotopically

Sensitive Branching

in Monoterpene

and enantiomer

preference for the

A plot of the rate of formation of limonene as a function of geranyl pyrophosphate concentration exhibited typical hyperbolic saturation, from which several computer-assisted kinetic evaluation methods provided an apparent K,,, value of 1.8 t- 0.3 PM. Most other monoterpene, sesquiterpene, and diterpene cyclases investigated have K, values in the range 0.5-3.0 PM for their respective prenyl pyrophosphate substrates (20, 21, 23-25, 27, 29, 30, 32, 38). Geranyl pyrophosphate is assumed to be the natural substrate for cyclization since, in preparations free of competing phosphohydrolase activity, all cyclases investigated efficiently convert this ubiquitous isoprenoid intermediate to cyclic monoterpene products without the formation of free intermediates (1). However, other prenyl pyrophosphates, including neryl pyrophosphate and linalyl pyrophosphate (Fig. l), can also serve as precursors for monoterpene cyclases. To examine the geometric specificity and enantioselectivity of the limonene cyclase, the efficiency of conversion of the alternate substrates [l3H]neryl pyrophosphate, (3S)-(lZ)-[1-3H]linalyl pyrophosphate, and (3R)-(lZ)-[1-3H]linalyl pyrophosphate was investigated. Each of these substrates exhibited typical saturation kinetics with the enzyme, and the K,,, values obtained were very similar to that calculated for gerany1 pyrophosphate (Table II). However, the velocity of the reaction with neryl pyrophosphate was much lower than that with geranyl pyrophosphate, and the velocities

I

Olefin Biosynthesis

Rate of product formation

Catalyzed

by (4S)-Limonene

(nmol . h-’ . mg protein-‘)

[Product

Synthase

distribution

(%)l”

Substrate

Limonene

ol-Pinene

/3-Pinene

Myrcene

Total olefins

[l-3H]Geranyl pyrophosphate [8,9-2H,J-3H]Geranyl pyrophosphate

575 [94.2] 375 [92.5]

10.9 [1.8] 12.1 [3.0]

12.2 [2.0] 13.0 [3.2]

12.4 [2.0] 5.3 [1.3]

610 [loo] 405 [loo]

’ Monoterpene production was determined by assay with each substrate (at saturation; 7 pM) using the same preparation of (4S)-limonene synthase. Standard conditions were used as described under Experimental Procedures. The distribution of olefins was determined by electronic integration of radio-GLC traces (see Experimental Procedures and Fig. 3). Data reported are the averages of three independent assays with SE 40%.

54

RAJAONARIVONY, TABLE

II

GERSHENZON,

AND

CROTEAU

nene. Kinetic analysis gave an apparent K,,, of 0.38 PM and V of 180 nmol per hour per milligram protein (compared to 750 nmol per hour per milligram protein for a parallel incubation with geranyl pyrophosphate). The inSubstrate K,n (PM) VI2 efficient transformation of a-terpinyl pyrophosphate is consistent with earlier studies that have also discounted Geranyl pyrophoephate 1.8 100 the role of a-terpinyl pyrophosphate in monoterpene bioNeryl pyrophosphate 1.0 23 synthesis (26, 31). The fact that the cyclase was able to (3R)-Linalyl pyrophosphate 1.9 153 (3S)-Linalyl pyrophosphate 2.3 260 utilize, at a modest rate, the tertiary pyrophosphate ester (presumably by ionization to the corresponding cation ’ Kinetic constants were determined for each substrate using standard and deprotonation) is somewhat surprising as is the relconditions as described under Experimental Procedures. Results are atively low Km value observed with this “substrate.” Resexpressed as the averages of at least four determinations. *The rate of reaction with geranyl pyrophosphate was 750 olution of both the limonene product and the residual nmol * h-l. mg protein-‘. substrate (after hydrolysis to a-terpineol) via capillary GLC on a chiral phase column revealed both to be racemic. Thus, the cyclase did not discriminate between antipodes with both linalyl pyrophosphate antipodes were higher. of this tertiary pyrophosphate ester. More detailed studies Similar patterns of substrate utilization have been ob- on the anomalous binding and transformation of a-terserved with other monoterpene cyclases, and the ineffi- pinyl pyrophosphate by monoterpene cyclases will be cient utilization of neryl pyrophosphate is not unusual presented elsewhere. (9, 27-30). The rapid rate of conversion of both linalyl Inhibitors. Limonene synthase activity was inhibited pyrophosphate antipodes is consistent with the inter- by sodium phosphate (Ki = 30 mM) and sodium pyromediacy of this tertiary allylic isomer in the proposed phosphate (Ki = 1 mM), two substances widely known to reaction sequence (Scheme I) and indicates that the cybe inhibitors of terpene cyclases and prenyltransferases clization component of the coupled isomerization-cycli(12,20,23, 26, 27, 29,31,32,36,49). At lower concentrazation sequence proceeds at a faster rate than does the tions (5-20 mM), sodium phosphate stimulated limonene isomerization step (44, 45). cyclase activity two- to threefold over controls without Based on the well-founded assumption that the interphosphate (perhaps by stabilizing the protein), and so mediate, enzyme-bound precursor to cyclization possesses was routinely added to assay buffers at 5 mM. Although an anti-en&-conformation (1, 46), the (3S)-linalyl antithe precise mode of inhibition by sodium phosphate is pode would be the stereochemically predicted intermediate unknown, sodium pyrophosphate is believed to act as a in the formation of (4S)-limonene (as in Scheme I). Thus, simple substrate analog and to bind competitively to the it was not surprising that (3S)-linalyl pyrophosphate exactive site of the cyclase. The pyrophosphate moiety of hibited a significantly higher reaction velocity than did geranyl pyrophosphate is the principal determinant of the (3R)-antipode. Nevertheless, the velocity of the stesubstrate binding for the monoterpene cyclases (1,50). reochemically “incorrect” (3R)-antipode was substantial Another strong inhibitor of limonene synthase activity (Table II). This lack of strict enantiospecificity is not was a sulfonium ion analog of the linalyl cation thought uncommon among monoterpene cyclases (9, 11, 47, 48). to be generated during the enzymatic formation of liIn fact, there is no need for the enzyme to discriminate between these enantiomers in Go. Stereochemistry is monene (Fig. 1). The magnitude of this inhibition (Ki determined at the isomerization step (1,8,44) and, thus, -1-2 PM) was similar to that shown for other monotera given cyclase encounters only one specific linalyl py- pene cyclases (3, 12, 51), providing further evidence for rophosphate antipode as a reaction intermediate. The the intermediacy of a carbocationic linalyl species in the aberrant cyclization of (3R)-linalyl pyrophosphate is ex- biosynthesis of limonene, while confirming the general pected to lead to (4R)-limonene, based on analogy to other electrophilic nature of the reaction (3, 12,52). Inhibition caused by the linalyl sulfonium analog could be synergized cyclases (9). The intermediacy of the cu-terpinyl cation (3) in the with sodium pyrophosphate. For example, concentrations cyclization reaction suggested the possibility that a-ter- of both the linalyl analog and sodium pyrophosphate that each limited limonene synthase activity to approximately pinyl pyrophosphate might mimic this ionic intermediate and potentially serve as a precursor of limonene. Incu- 20% of that measured in an uninhibited control when tested separately afforded complete inhibition when tested bation of limonene synthase with (4&S’)-[3-3H]a-terpinyl pyrophosphate followed by radio-GLC analysis of the re- together. Limonene synthase thus appears to bind more sulting olefins showed the principal product of the reaction tightly to the combination of the linalyl analog and sodium to be limonene (95% of total), along with a small amount pyrophosphate than to either partner alone, lending supanion remains (5%) of terpinolene, the isopropylidene isomer of limo- port to the notion that the pyrophosphate Substrate Specificity and Enantioselectivity of (4S)-Limonene Synthase”

CHARACTERIZATION

AND

MECHANISM

paired with the carbocationic intermediates formed during the course of the reaction (1, 53,54). The effect of a number of amino acid-directed reagents on limonene synthase activity was also investigated. The methionine-directed reagent benzyl bromide (Iho = 0.15 mM at pH 6.0) and the histidine-directed reagent diethyl pyrocarbonate (& = 0.15 mM at pH 7.0) were both inhibitors of catalysis by limonene synthase. Both of these substances have been previously reported as inhibitors of several other monoterpene and sesquiterpene cyclases (23, 32, 33, 55). Several amino acid-directed reagents shown to be active against other terpene cyclases (28, 51) had no effect on limonene synthase over a broad concentration range, including phenylmethylsulfonyl fluoride (serinedirected) and the arginine-specific reagents phenylglyoxal and 2,3-butanedione (tested in tricine-borate buffer at pH 8.0 in the dark). Sensitivity to thiol-directed reagents is a near universal characteristic of terpene cyclases and prenyltransferases (1, 23, 26-33, 49, 55, 56). Limonene synthase does not deviate from this pattern in being inhibited by both phydroxymercuribenzoate (& = 0.35 pM) and N-ethylmaleimide (&,, = 1.8 mM) when tested in buffer without dithiothreitol or sodium ascorbate. Inhibition by p-hydroxymercuribenzoate could be completely reversed by subsequent treatment with 5 mM cysteine for 30 min at

OF (4S)-LIMONENE

55

SYNTHASE TABLE

III

Protection of (4S)-Limonene Synthase against Inactivation by the Thiol-Directed Reagent, p-Hyciroxymercuribenzoate (pHMB) Inhibitor pHMB pHMB pHMB pHMB

and protectants”

rateb

5.9 6.3 2.8 71.2

+ GPP + MnClz t GPP + MnC&

pHMB pHMB t PP; pHMB t PPi t MnCl, pHMB + linalyl sulfonium pHMB t linalyl sulfonium + PP, t MnClx

Relative

1.1

analog analog

2.5 0.6 0.6

51.7

’ Abbreviations used: pHMB, p-hydroxymercuribenzoate; GPP, gerany1 pyrophosphate; PP; , sodium pyrophosphate. Concentrations of protectants were: GPP, 7 pM; MnClx, 1 mu; PPi , 1 mM; linalyl sulfonium of pHMB was 3 pM in the first set of analog, 5 FM. The concentration experiments (top) and 1 FM in the second set (bottom). Procedures for preincubation with protectants and inhibitor, and details of the assay, are described under Experimental Procedures. In the second set of experiments, substrate analogs and excess inhibitor were removed by dialysis prior to the assay. b Rates are given relative to uninhibited controls (with or without the addition of the appropriate protectant) set at 100.

30°C. To assess the presence of an essential thiol group(s) at the active site, combinations of substrate, substrate analogs, and metal ion cofactor were tested as possible protectants against inactivation withp-hydroxymercuribenzoate. The addition of geranyl pyrophosphate (7 PM) and MnCl, (1 mM) prior to treatment with p-hydroxymercuribenzoate (1 PM) almost completely prevented inhibition (Table III), indicating the presence of at least one essential cysteine residue at or near the active site of limonene synthase. Neither geranyl pyrophosphate nor MnCl, alone had any protective effect, suggesting that prior to catalysis, the substrate binds to the active site in the form of a complex with the metal ion cofactor (1,34). Further protection studies were undertaken with the sulfonium ion analog of the presumptive linalyl reaction intermediate, shown above to be a good reversible inhibitor of limonene synthase. Addition of the sulfonium ion analog in conjunction with MnC12 and sodium pyrophosphate provided significant protection against inactivation by p-hydroxymercuribenzoate, protection that was not obtainable without the presence of all three of these substances together (Table III). This result strengthens the case for the participation of a pyrophosphate anion-paired linalyl carbocation as an intermediate in the enzymatic cyclization of geranyl pyrophosphate to limonene, and it provides further evidence for the presence of an essential thiol residue at the active site of limonene synthase.

Other monoterpene cyclases may also possess an essential thiol group at their active sites. Preliminary studies with 1,8-cineole synthase from Salvia oficinalis showed that inhibition by the thiol-directed reagent methyl methanethiosulfonate was abolished by prior incubation with either geranyl pyrophosphate or inorganic pyrophosphate plus MnClz (57). Protection against thiol-directed reagents with a combination of prenyl pyrophosphate substrates and divalent metal ion has also been described for prenyltransferases (58), enzymes that bear several mechanistic similarities to terpene cyclases (4,5). Concl~.~ions. The basic properties of (4S)-limonene synthase from peppermint (molecular weight (6), subunit architecture (6), pH optimum, pl, hydrophobicity (6), Km for geranyl pyrophosphate, catalytic constant (6), and requirement for divalent metal ion) are quite similar to those of most other monoterpene cyclases (26-32, 37). In addition, limonene synthase is also sensitive to many of the same inhibitors effective against other monoterpene cyclases, including sodium phosphate, sodium pyrophosphate, and reagents directed toward cysteine, methionine, and histidine residues. The research described in this paper provides the first direct evidence for the presence of an essential cysteine residue at the active site of a monoterpene cyclase. A unique feature of limonene synthase is that this enzyme is the only well-characterized mono-

56

RAJAONARIVONY,

GERSHENZON,

terpene cyclase to synthesize predominantly (>90%) one product. Other enzymes of this class that have been studied in detail have been found to produce complex mixtures of monoterpenes (29,31,32,37,38). Experiments with a variety of inhibitors and substrate analogs have established that the mechanism of (4S)limonene synthase catalysis (Scheme I) is similar to that proposed for other monoterpene cyclases. The results of studies assessing protection against p-hydroxymercuribenzoate inactivation show that the substrate, geranyl pyrophosphate, likely binds to the enzyme as a complex with the divalent metal ion cofactor. The metal ion is thought to neutralize the negative charge of the pyrophosphate moiety and thus assist in the ionization of the substrate (34,59). After ionization, the resulting geranyl cation is isomerized to linalyl pyrophosphate, an enzymebound intermediate capable of cyclization. The rapid rate of utilization of linalyl pyrophosphate by (4S)-limonene synthase, the potent inhibitory effect of the linalyl sulfonium analog, and the high degree of protection afforded by the linalyl analog (in concert with divalent metal ion and sodium pyrophosphate) against inactivation by p-hydroxymercuribenzoate are all consistent with the proposed intermediacy of linalyl pyrophosphate in the reaction sequence. Finally, the inhibition displayed by the linalyl sulfonium ion analog, and its synergism with sodium pyrophosphate, provide evidence for the electrophilic nature of the isomerization-cyclization mechanism and for the existence of carbocation:pyrophosphate anion pairs as reaction intermediates. Further mechanistic studies of (4S)Jimonene synthase, including detailed investigations of the structure of the active site, are currently under way. ACKNOWLEDGMENTS We thank our colleagues Al Koepp, D. Michael Satterwhite, and Kurt Wagschal for generously providing isotopically labeled substrates, Greg Wichelns for raising the plants, and Joyce Tamura-Brown for typing the manuscript.

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CHARACTERIZATION 39. Lawrence,

AND

MECHANISM

B. M. (1980) Perfum. Flau. 5,49-55.

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OF (4S)-LIMONENE 50. Wheeler,

57

SYNTHASE

C. J., and Croteau, R. (198’7) J. Biol. Chem. 262,

8213-

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M.

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58. de la Fuente, M., Perez, L. M., Hashagen, U., Chayet, L., Rojas, C., Portilla, G., and Cori, 0. (1981) Phytochemistry 20, 1551-1557. 59. Brems,D.

8352.

N., andRilling,

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