Geranyl pyrophosphate synthase: Characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis)

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 271, No. 2, June, pp. 524-535,19X9

Geranyl Evidence

Pyrophosphate Synthase: Characterization of the Enzyme and That This Chain-Length Specific Prenyltransferase Is Associated with Monoterpene Biosynthesis in Sage (Salvia ol7icina/is)’ RODNEY

Keceived

December

CROTEAU’

12,1988,

AND

PALJL T. PURKETT

and in revised

form

February

Z&l989

Cell-free homogenates from sage (Sulvia oflcinalis) leaves convert dimethylallyl pyrophosphate and isopentcnyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [l-‘4C]dimethylallyl pyrophosphate and [l3H]isopentenyl pyrophosphate was verified as [‘4C,l-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradativc studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the Cl,, precursor for t,he production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases. CO19XYAcademicPress,Inc.

The fundamental chain elongation reaction in the biosynthesis of isoprenoid compounds is the prenyltransferase-catalyzed condensation between C1 of an allylic pyrophosphate and C, of isopentenyl pyrophosphate to afford the C, homolog of the allylic substrate (Fig. 1). A variety of prenyltransferases, each with differing specificities for the allylic substrate and

product, exist for the construction of the series of homologous prenyl pyrophosphate precursors of the various isoprenoid families. There has been considerable interest in these enzymes as they function at the branch points in the isoprenoid biosynthetic pathway and may thus have a regulatory function (1). The most thoroughly studied prenyltransferase is farnesyl pyrophosphate synthase. This enzyme supin isoprenoid plies the C15 intermediate biosynthesis, which may be further elongated or may serve as a precursor for the biosynthesis of sesquiterpenes or, after dimerization to squalene, as an intermediate in triterpene and sterol biosynthesis. This

1 This investigation was supported in part by National Science Foundation Grant DCB-8803504 and by Project 0268 from the Washington State University Agricultural Research Center, Pullman, WA 99164. ’ To whom correspondence should be addressed. 0003-9861/89 Copyright AU rights

$3.00

0 19X9 by Academic Press. Inc. of reproduction in any form reserved.

524

GERANYL

PYROPHOSPHATE

SYNTHASE

FROM

S&iu

o&in&s

525

TOPP ’ OPP +

R=ti R=C~H~

/3 t’ 4

2 R’= H Geranyl pyrophosphate

lsopentenyl pyrophosphate

Dlmethylallyl pyrophosphate

R’=CgHg

Geranyl pyrophosphate

Flc:. 1. The prenyltransferase

enzyme, and prenyltransferases in general, have been described in a comprehensive review by Poulter and Rilling (2j. It is now well established that geranyl pyrophosphate (Fig. l), the ubiquitous Cl0 intermediate of the general isoprenoid pathway (3), functions as the acyclic precursor of most monoterpenes (4), and it seems likely that a chain-length specific prenyltransferase should exist for the synthesis of this essential intermediate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate (5, 6) (Fig. 1). It is also known that the epidermal oil glands are the primary sites of synthesis and accumulation of monoterpenes in many herbaceous plants (7). This tissue, therefore, would be expected to contain the requisite enzyme for the synthesis of geranyl pyrophosphate. Conversely, such a prenyltransferase would be unlikely to be present in nonglandular tissue, since farnesyl pyrophosphate synthase catalyzes the condensation both of dimethylallyl pyrophosphate with isopentenyl pyrophosphate to form geranyl pyrophosphate and of the geranyl pyrophosphate with an additional isopentenyl pyrophosphate residue to form farnesyl pyrophosphate (2). The formation of geranyl pyrophosphate, as well as the &s-isomer, neryl pyrophosphate, and of mixed geometric isomers of farnesyl pyrophosphate, has been demonstrated in crude, cell-free extracts from several higher plant species (8-11). Yet, the existence of a chain-length specific geranyl pyrophosphate synthase has been adequately demonstrated in only two cases: in the bacterium Micrococcus where the enzyme apparently functions in generating the primer for solanesyl pyrophosphate biosynthesis en route to menaquin-

Farnesyl pyrophosphate

reaction.

one (12), and in cell cultures of Lithospermum erythrorhixon where the enzyme supplies the prenyl group for alkylation of p-hydroxybenzoate en route to shikonin (13). In this communication we demonstrate the existence of a distinct geranyl pyrophosphate synthase in cell-free preparations from leaves of the monoterpene-producing higher plant Sulvin oficinalis (common sage) and describe the general characteristics of the partially purified enzyme. Additionally, we provide strong suggestive evidence that this specific prenyltransferase, by its nearly exclusive location in the epidermal oil glands, is associated with monoterpene biosynthesis in this species. EXPERIMENTAL

PROCEDURES

Plant mul’erials, .subs;trates, and reagents. Sage plants (5’. ofiicinalis L.) were grown from seed under conditions previously described (14, 15). Immature, rapidly expanding leaves from 3- to 5-week-old plants (2-4 cm in length) were routinely used as the enzyme source. [4-“ClIsopentenyl pyrophosphate (53 Ci/mol) was purchased from New England Nuclear. 1RS-[1-3H]Isopentenyl pyrophosphate (22.5 Ci/mol) was obtained by pyrophosphorylation of [1-“Hlisopentenol according to previously published procedures (16). Thus, [l-3H]isopentenol (3-methyl-3-buten-l-01) was prepared by reduction of the corresponding aldehyde (1.12 mmol) with NaB”H, (100 mCi; 360 Ci/mol; New England Nuclear) in 95% EtOH, and was purified by column chromatography on silica [pentane:ether:benzene (15:4:1, v/v/v)] to afford the product free of ethanol (-17% radiochemical yield and >97%, radiochemical purity by radio-GLC”). The labeled product

‘Abbreviations used: GLC, gas-liquid raphy; TLC, thin-layer chromatography; morpholino)ethanesulfonic acid; Pipes,

chromatogMes, 2-(Npiperazine-

526

CROTEAU

AlUD PURKETT

was diluted with 0.5 mmol of authentic isopentenol to 22.5 Ci/mol and 1 ml of dried, distilled pyridine was added. The solvent volume (remaining from the column chromatography step) was reduced by distillation through a Vigreaus column until the head tertperature reached 8O”C, the labeled alcohol was tosyla&d in/. S&L and pyrophosphorylated, and the resulting pyrophosphate ester was purified essentially as described (16). An initial supply of [1-‘“CJdimethylallyl pyrophosphate (1 Ci/mol) was first oht,ained from New England Nuclear. When this material was no longer commercially available it was prepared by a strategy used previously to synt,hesize [‘%]geranyl pyrophosphate (17), which involves the Wadsworth and Emmans modification (18) of the Wittig reaction [condensation of acetone with diethyl methoxy[‘“C]carbonylmethyl phosphonatc generated by means of the Michael-Arbuzov reaction (1911.The resulting methyl ester was reduced with LiAlH, as before (17j, and the product (-12% isolated yield and >98% radiochemical purity by radio-GLC) was diluted with authentic dimethylallyl alcohol to 1.0 Ci/mol prior to pyrophosphorylation by modification of the Cramer and B6hm method (20, 21) and purification by ion-exchange chromatography on O-diethylaminocthyl-cellulose (22) as described in delail elsewhere (23,24). Dimethylallyl pyrophosphate was prepared from dimethylally1 alcohol as above, and was diluted with the “Clabeled product (to -0.03 Ci/mol) to assist in the purification by ion-exchange chromatography. The preparation of [I-“Hkeranyl pyrophosphate (87 Ci/mol) and [l-3H]farnesyl pyrophosphate (‘72 Ci/ mol) has been described previously (23, 25). The radiochemical purities of these substrates, and of the other pyrophosphate esters prepared for this investigation, wereverified Lo be the same or slightty higher than the corresponding starting materials by enzymatic hydrolysis and radio-GLC analysis of the liberated prenols using the assay procedure described below. Isopentenal and (*)-nerolidoi were gifts from R. Hopp, Haarmann and Reimer GmbH, Holzminden, West Germany. Isopentenol, dimethylallyl alcohol. dimethylvinyl carbinol, geraniol, nerol, (+)-linalool, farnesol (mixed geometric isomers), and geranylgeranlo! were purchased from Aldrich Chemical Co., CTC Organicu, or Research Organics, Inc. (?)-Linalylgeraniol was prepared by the mesylation and solvolysis (2Sj of geranylgeraniol. Biochemicals were obtained from Sigma Chemical Co.

niN’-bisf2-ethanesutfonic acid); Hepes, 4-(Z-hydroxyethyl).l-piperazineethanesulfonic acid; Tricine, br-tris(hydroxymethyljmethy1 glycine; Taps, tris(hydroxymethyl)methylaminopropanesuifonic acid.

Enxyrrw extra&on cwd prrrt~f.nl pw-Qicatirrr~ In preliminary experiments, prenyltransferase was extracted by homogenizing whole leaves under conditions generally employed previously for the preparation of prenyl cyclases (27). including the USC of phophatase inhibitors and thr adsorhents polyvinylpolypyrrulidone (28) and polystyrene resin (29). Although it was possible to demonstrate that the bulk of the prenyltransfcrase resided in the soluble fraction of these preparations, this method for obtaining geranyl pyrophosphate synthase was unsatisfactory for several reasons, including the coextraction of high levels of competing farnesyl pgrophosphale synthase and phosphatase activities, and the instability of the enzyme (40% loss of activity in 24 h at OOC, even in the presence of glycerol and dithiothreitol). For the selective extraction of enzymes from the leaf surface glands, a variatinn of a previous pubtished procedure was employed 130), which by several trials was optimized for the isolation of geranyl pyrophosphate synthasc frnm S oficinalis. Thus, 20 g of polystyrene resin beads and 4 g of polyvinylpolypyrrolidone were placed into a 10-0~ polycarbonate BeadBeater chamber (Biospec Products, Bartlesville, OKj and the chamber half filled with cold cxtraclion buffer consisting of 15 nlM Mes, pH 6.5, containing 1 mM dithiothreitol, 4 m;M sodium ascorbate, 100 mM N&l, and 20% glycerol (v/v), as well as 20 ~1%sodium phosphate and t.he proteinase inhibitors phenylmethylsulfonyl fluoride (29 PM) and hr-tosyl-L-phenylalanine chloromethgl ketone (14 FM). The leaves (20 g) were then added, and the container was filled with buffer and sealed with the rotor assembly which was affixed to the motor operated al a rheostat setting of 90 V. Tissue extraction was effected by running the Bead-Beater motor for 4 X 5-s bursts with 5-s intervals between bursts. The chamber was then placed in ice and the contents swirled manually for 15 min. The resulting extract was filtered through eight layers of cheesecloth to yield -250 ml of solution which was centrifuged at 27,000g for 15 min. the supernatant from which was filtered sequentially through 0.80-, 0.45-, and 0.22-Frn filters (Nalgene) to remove additional particulates. Although the suspension was quite dilute at this stage compared Lo a whole leaf homogenate, this preparation was somewhat more stable (leas than 20% loss of activity in 24 hj, and, notably, was enriched in geranyl pyrophosphate synthase relative to farnesyl pyrophosphate synthase and phosphatase. Attempts to concentrate the preparation by (NH&SO, precipitation indicated that all of the relevant activity precipitated al less than 25& saturation, giving a highly enriched preparation (>90% of the total prenyltransferase activity was geranyl pyrophosphate synthase following desalting), but one of low yield (~5% recovery of starting activity). Kevertheless, t.his procedure, which suggested that the en-

GERANYL

PYROPHOSPHATE

SYNTHASE

zyme was highly hydrophobic, led to the development of a more suitable partial purification. Thus, the filtered supernatant from above was routinely concentrated by ultrafiltration (Amicon YM-30) and the concentrate applied directly to a 20.ml column of phenylSepharose CL-4B (Pharmacia) equilibrated with the original extraction buffer. The column was washed with additional extraction buffer and eluted sequentially with 1 bed vol of the same buffer with 10 mM M&l, added and a reduced NaCl concentration (50 mM), and then with 2 bed vol of the same buffer without NaCl but with 50% ethylene glycol added. Finally, the geranyl pyrophosphate synthase was eluted with the 50% ethylene glycol buffer to which 0.5 M NaSCN (a chaotropic agent) had been added. This preparation was dialyzed against 15 mM Pipes, pH 6.75, containing 10 mM Mg&, 1 mM dithiothreitol, 20 ELM NaHzPOI, and 20% glycerol (v/v), concentrated by ultrafiltration (YM-30), and assayed directly, or applied to a 2.5 X 120-cm column of Sephadex G-150 equilibrated and developed (25 ml/h) with the same buffer containing 50 mM NaCI. Column fractions (3 ml) were assayed by the two methods described below. Ellz~n/e asscrys. In a typical assay for prenyltransferase, 1 ml of the preparation in the Pipes buffer system (pH 6.75, containing 10 mM MgCI, and the other additions) was incubated in the presence of 28 pM [l“Hlisopentenyl pyrophosphate and 56 fiM dimethylallyl pyrophosphate for 1 h at 31°C. For the “acid lability” assay (31, 32), which depends upon the solvolysis of enzymatically generated, labeled allylic pyrophosphates in the presence of radioactive isopentenyl pyrophosphate, 1 ml of pentane was added to the icechilled sample followed by 0.33 ml of 1 M HCI (to a final concentration of 0.25 M HCl), and the mixture was shaken for 30 min at 31°C. Following chilling in ice, addition of NaCl to saturation, vigorous mixing, and centrifugation to separate phases, an aliquot of the pentane layer was taken for determination of tritium content. Calibration of the assay with I-“H-labeled C,- to Cis-allylic pyrophosphates (5 nmol) indicated that >95% of the label originally present as py rophosphate ester was recovered as pentane soluble solvolysis products. Followingremoval of the remaining pentane layer and reextraction of the reaction mixture with an additional 1 ml of pentane, the combined extract was diluted with 1 mg each of the appropriate carrier standards (isopentenol, dimethylallyl alcohol, dimethylvinyl carbinol, geraniol, -nerol, linalool, farnesol (mixture of isomers), nerolidol (mixture of isomers), geranylgeraniol, and geranyllinalool), dried over anhydrous NaSO,, and concentrated under vacuum (Savant Speed Vat) in preparation for radio-GLC analysis. For the “enzymatic hydrolysis” assay (2, 33, 34), which involves the phosphatase-catalyzed hydrolysis of the labeled pyrophosphate esters and release of the corresponding prenols, 1 ml of a 0.2 M Tris solution,

FROM

Snlvicr

oficinn2is

527

pH 8.0, containing 2 units each of potato apyrase and calf intestine alkaline phosphatase (both from Sigma), was added to the ice-chilled incubation mixture and 1 ml of pentane was added as an overlay. The sample was incubated overnight at 31°C (adjustment of the pH to 8.0, coupled with the rapid hydrolysis of isopentenyl pyrophosphate, minimizes prenyl transferase activity during this postincubation treatment). Following enzymatic hydrolysis, the liberated prenols were extracted into diethyl ether (2 X 1 ml, with vigorous shaking and centrifugation to separate phases as before) and the ether extract treated with a few milligrams of NaBH, to convert any aldehydes present to the corresponding alcohols (the latter precaution was necessary because commercially available acid, or alkaline, phosphatase contains low but variable amounts of an apparent alcohol oxidase activity). The reaction mixture was passed through a short column of anhydrous NaZSO,, and 1 mg each of the appropriate carrier standards (isopentenol, dimethylallyl alcohol, nerol, geraniol, farnesols, and geranylgeraniol) was added prior to aliquot counting and concentration of the sample in preparation for radio-GLC analysis. Calibration of the assay with [l“Hlgeranyl pyrophosphate (5 nmol) gave about 85% yield of [1-“Hlgeraniol; the lack of quantitative recovery probably results, at least in part, from loss of label on oxidation to the aldehyde. For studies with inhibitors, a preincubation for 15 min at 30°C was employed and the reaction then initiated by addition of [:‘H]isopentenyl pyrophosphate and the allylic cosubstrate. Prepcuuticm (8 dwimfives. The diphenylurethane of geraniol was prepared (35) by treating 0.3 mmol of the product with 1.1 Equivalent of diphenylcarbamoyl chloride in 3 ml pyridine with stirring overnight at 60°C. The reaction products were partitioned between diethyl ether and 0.05 N aqueous HCI, and following removal of the organic solvent the diphenylurethane was crystallized from hexane after filtration of the insoluble diphenyl urea. Oxidation of geraniol to geranial was carried out by stirring 0.6 mmol of the alcohol with excess MnO, in hexane for several hours (36,37) followed by filtration of solids and separation of geranial from the &s-isomer (neral, formed in -8% yield) by argentation TLC on silica gel G-159, AgN03 (with hexane:ether, 1:l (v/v)). Geranial semicarbazone was prepared by treating 0.25 mmol of the aldehyde in 1 ml pyridine with an equivalent amount of semicarbazide hydrochloride in a few drops of water at room temperature overnight (38). Products were partitioned between diethyl ether and water, and on evaporation of the ether phase the semicarbazone was crystallized directly from methanol. Geranial (0.3 mmol) was oxidized to geranic acid with excess Tollen’s reagent (ammoniacal silver nitrate) (39). Following standard acid/base work up, the sodium salt was refluxed 1 h in excess 10% cu-p-dibromoaceto-

528

CROTEAU

AND

PURKETT

phenone in ethanol (38,40) to affordp-bromophenacyl geranoate which, following partitioning between ether and water, was crystallized from aqueous ethanol. Analytical methods. Thin-layer chromatography was done on l-mm layers of silica gel G (or silica gel G impregnated with 15% AgNOs) activated at 100°C for 3 h, or on buffered silica gel H prepared with 0.1 M (NH4)zHP04 (pH 6.8) and air-dried. Developing solvents are indicated elsewhere in the text. Radio-GLC was performed on a GOW-MAC 550P gas chromatograph (thermal conductivity detector) attached directly to a Nuclear Chicago 7357 gas proportional counter (27). Both thermal conductivity and radioactivity output channels were monitored with a SICA 7000A chromatogram processor, and the system was externally calibrated with [“Hltoluene. The chromatographic column used was 12 ft X 0.125 in. o.d. stainless steel coated with 15% SE-30 on Chromosorb WHP, and was programmed from 160°C (6 min hold) to 230°C at BO”C/min with He flow rate of 40 ml/min. Radioactivity in organic liquid and solid samples was determined in a solution consisting of 15 ml of 30% ethanol in toluene containing 0.4% (w/v) Omnifluor (New England Nuclear). The counting efhciency for 3H was 27% and that for ‘“C was 80%, and all samples were counted to a standard error of ~1%. RESULTS

AND

DISCUSSION

Demonstra,tion of geranyl pyrophosphate synthesis in a cell-free system. To examine

the synthesis of prenyl pyrophosphates in sage, homogenates were first prepared from intact, expanding leaves and centrifuged at 3000g to remove whole cells and debris. Following dialysis to the appropriate assay conditions (15 mM Pipes, pH 6.75, containing 10 mM MgC&, 1 mM dithiothreitol, 20 PM NaH2P04, and 20% glycerol), the preparation was incubated with 56 PM dimethylallyl pyrophosphate and 28 PM [l3H]isopentenyl pyrophosphate in the presence of 10 mM MgCl, and 1 mM MnC12. Subsequent treatment of the reaction products with alkaline phosphatase plus apyrase to hydrolyze the resulting prenyl pyrophosphates (partial hydrolysis had already occurred due to the presence of endogenous phosphohydrolases (33)) yielded a mixture of radiolabeled compounds which was analyzed by radio-GLC (Fig. 2A). In addition to [3H]isopentenol released from the substrate, the prenol fraction was composed of dimethylallyl alcohol (6.5%) via the action

Time

(min.)

FIG. 2. Radio-gas-liquid chromatograms of the prenols liberated by phosphatase treatment of the products generated by prenyltransferase activities when assayed with dimethylallyl pyrophosphate and[l-3H]isopentenyl pyrophosphate in the presence of MgCl,. (A) The radioactivity monitor response to the products generated in a homogenate of sage leaves; (B) the response to the TLC purified &-products generated by the partially purified prenyltransferase; (C) the response to the products generated by the geranyl pyrophosphate synthase following purification by hydrophobic interaction and gel permeation chromatography. The smooth lower tracing in (D) is the thermal conductivity detector response obtained from coinjected standards of isopentenol plus solvent (a), dimethylallyl alcohol (b), linalool (c), nerol (d), geraniol (e), cis- and tra?Ls-nerolidol (f), cis,trans-farnesol (g), tm7ls,trarLs-farnesol (h), cis- and trans-geranyllinalool (i), and geranylgeraniol (all frans) (j). The chromatographic column and operating procedures are described under Experimental Procedures.

of isopentenyl pyrophosphate isomerase (2,41)), geraniol (5.5%), trans,trans-farnesol (84%), and geranylgeraniol (-4%).

GERANYL

PYROPHOSPHATE

SYNTHASE

Trace levels (~0.5%) of nerol were noted, but no detectable amounts of labeled dimethylvinyl carbinol, linalool, nerolidol or cis,trans-farnesol could be found, nor were isomers of geranylgeraniol observed. Minor amounts of the cyclic sesquiterpenes humulene and caryophyllene and trace levels of cyclic monoterpenes were also noted, presumably arising by the action of endogenous cyclases (23,27,42,43) on the biosynthetic farnesyl and geranyl pyrophosphate, respectively, formed during the incubation. In the absence of added dimethylallyl pyrophosphate or divalent metal ion, both prenyltransferase and isopentenyl pyrophosphate isomerase activity were reduced about 20-fold. Centrifugation of the homogenate, first at 27,OOOg then at 15O,OOOg, revealed that essentially all of the observed prenyltransferase activity resided in the soluble enzyme fraction. Less than 6% of the total activity was associated with the 2’7,OOOg and 150,OOO.opellets when these membranous fractions were suspended and assayed under identical conditions. RadioGLC analysis of the labeled products derived from the membranous preparations (following enzymatic hydrolysis as before) indicated the presence of essentially only trclns,trun.s-farnesol as an elongation product; geraniol and geranylgeraniol were barely detected. Preparations designed to maximize the activity of particulate fractions, through the use of a soluble form of polyvinylpyrrolidone in the extraction buffer and immediate centrifugation of the homogenate without preliminary dialysis, served only to confirm the conclusion that the bulk of the prenyltransferase activity in sage leaf extracts (which forms Clo, C15, and CZOproducts) resided in the soluble enzyme fraction. The ability of the soluble enzyme preparations to synthesize geranyl pyrophosphate (as measured by the total geraniol released by endogenous pyrophosphates during the incubation and on subsequent phosphatase treatment) was rapidly lost (40% loss in 24 h at 0°C in assay buffer). The ability to synthesize geranylgeranyl pyrophosphate was similarly labile, whereas the loss of the farnesyl pyro-

FROM

Salviu

Fractton

oficinalis

529

Number

FIG. 3. Sephadex G-150 gel filtration of the soluble enzyme preparation from sage leaves. Absorbance at 280 nm (-), and farnesyl pyrophosphate (FPP) synthase (O---O) and geranyl pyrophosphate (GPP) sqnthase (e - l ) activities, when assayed with dimethylallyl pyrophosphate and [1-“Hlisopentenyl pyrophosphate as cosubstrates, are plotted. Chromatography and assay procedures are described under Experimental Procedures. V,, was at fraction 44.

phosphate synthase activity under these conditions was only 10-20s. To determine the number of prenyltransferases present, and to remove competing activities (pyrophosphate, isomerase), the preparation was concentrated by ultrafiltration and subjected to preliminary separation on Sephadex G-150. Fractions were examined using the acid-lability assay (based on the solvolysis of allylic pyrophosphates (2,31,32)), and the results showed a broad peak of prenyltransferase activity eluting at approximately 1.4 void vol (Fig. 3). Re-assay of fractions in t,he appropriate region, by enzymatic hydrolysis of the products (33, 34) and radio-GLC analysis (27) of the liberated prenols, indicated the major activity to be farnesyl pyrophosphate synthase as expected. Fractions centering on this activity peak produced l-2% geranyl pyrophosphate, probably resulting from the release of low levels of the Cl0 intermediate by the farnesyl pyrophosphate synthase. However, toward the backside of the broad activity peak, the proportion of geranyl pyrophosphate in the product mixtureincreased to nearly 60% (with -30% farnesyl pyrophosphate), suggesting the presence of a discrete geranyl pyrophosphate synthase activity (Fig. 3). Geranylgeranyl pyro-

530

CROTEAU AND PURKETT

phosphate synthase activity was spread throughout the broad peak of farnesyl pyrophosphate synthase activity, suggesting multiple species of this activity. Low levels of isopentenyl pyrophosphate isomerase activity essentially paralleled the farnesyl pyrophosphate synthase activity, whereas phosphatase activity (assayed by hydrolysis of [l-3H]geranyl pyrophosphate (33)) was widely distributed in the column fractions, with at least two activities broadly overlapping the region in which prenyltransferase(s) eluted. Product identification To confirm the identity of the putative geranyl pyrophosphate, column fractions enriched in the relevant synthase were combined (this preparation produced geranyl pyrophosphate in excess of 50% of the total prenyl pyrophosphate fraction and contained less than 10% of the isomerase and phosphatase activity of the original crude extract), and several large-scale incubations with such preparations (sorbitol was substituted for glycerol in the assay buffer) were carried out in the presence of 15 PM each of [l-‘4C]dimethy1allyl pyrophosphate (1 Ci/ mol) and [1-3H]isopentenyl pyrophosphate (22.5 Ci/mol) as cosubstrates. Following incubation, the reaction mixture was adjusted to pH 9.2 with NH40H and lyophilized. The resulting solid was suspended in a minimum quantity of 0.01 N NH,OH, then filtered and the filtrate subjected to TLC (silica gel H buffered with (NH&HPO,, pH 6.8, and developed with npropanol:NHs:HzO (6:3:1)). The gel from the region containing ClO-prenyl pyrophosphate (R, = 0.40-0.45, determined with [l3H]neryl and [ 1-3H ]g eranyl pyrophosphate as standards) was scraped directly into a vial containing 5 units each of apyrase and alkaline phosphatase in 7 ml of 0.2 M Tris buffer, pH 8.0, and the mixture incubated with gentle stirring overnight under a pentane overlay. The pentane soluble products were isolated (0.18 &i 3H; representing 86% of the label originally adhering to the gel), and an aliquot (2.7 X lo4 dpm) was analyzed by radio-GLC following the addition of geraniol, nerol, and (t-)-linalool as internal standards (Fig. 2B). This pentane extract was thus shown to consist essen-

tially of a single component coincident with authentic geraniol. The remaining product was diluted with 1 mmol of geraniol as carrier and one-third of the sample was converted to the diphenylurethane (35) [crystallized from hexane, mp = 8283”C, lit. mp = 82°C (44)] which exhibited a 3H:14C ratio of 23.5 + 0.5:1, consistent with the one-to-one condensation of dimethylallyl pyrophosphate with isopentenyl pyrophosphate. The remaining material was oxidized with Mn02 (36, 37) to a 937 mixture of trans.&s-aldehyde isomers, and one-half of the geranial isolated by preparative argentation TLC was converted to the semicarbazone (38) [crystallized from methanol, mp = 163”C, lit. mp = 161-163°C (45); “H:l*C = 12.4 t- 0.4:1]. The remaining geranial was oxidized to geranic acid with Tollen’s reagent (39), and thence converted to the p-bromophenacyl ester (40) [mp = 66-67°C from aqueous ethanol, lit. mp = 67°C (46)] which was essentially devoid of “H (i.e., 3H:‘“C - 0.4; the residual 3H likely resulting from the action of isopentenyl pyrophosphate isomerase (2, 41) during incubation, with the incorporation of minor levels of [l-3H]dimethylallyl pyrophosphate into the terminal C5 unit of geranyl pyrophosphate). On the basis of the chromatographic properties, and the preparation of crystalline derivatives exhibiting loss of half of the 3H on oxidation of geraniol to geranial and most of the remaining 3H on oxidation to geranic acid, it was concluded that the original biosynthetic product was [1-3H;5-‘4C]geranyl pyrophosphate generated by condensation of [l-‘4C]dimethylallyl pyrophosphate with [l-3H]isopentenyl pyrophosphate. Localixation of activity in epidermal oil glands. To examine the possibility that the

geranyl pyrophosphate synthase activity was specifically associated with the leaf epidermal glands, the presumptive site of monoterpene biosynthesis (7), a newly developed method (30) for the selective extraction of the contents of these structures was employed. This technique, which is based on gentle abrasion of the leaf surface, is capable of removing 70-100% of the surface oil glands while resulting in relatively little damage to the remaining leaf

GERANYL

PYROPHOSPHATE

SYNTHASE

epidermis and underlying mesophyll tissue, and the overall recovery (relative to whole-leaf extracts) of key enzymes associated with monoterpene and sesquiterpene biosynthesis (e.g., the geranyl and farnesyl pyrophosphate cyclases) is of comparable efficiency (70-100%) (30). The quantity of protein (47) extracted by this selective procedure, not surprisingly, is quite low (e.g., 0.5 mg protein/g leaf tissue). Soluble enzyme preparations from leaf surface extracts and from the leaves remaining after surface extraction were thus assayed for prenyltransferase activity, and the summation afforded a recovery in excess of 87% of the total prenyltransferase activities of a whole-leaf homogenate. The distribution of activities within the epidermis and residual leaf extracts was particularly revealing. Thus, the residual leaf extract contained the bulk of the prenyltransferase activity (1.07 X 10’ cpm on a total tissue basis), of which 97% of the product mix was farnesyl pyrophosphate, less than 2% was geranylgeranyl pyrophosphate, and only 1.5% (1.6 X lo4 cpm) was geranyl pyrophosphate, the latter perhaps arising only as an intermediate in the farnesyl pyrophosphate synthase reaction. The epidermal gland extract contained roughly 20% of the prenyltransferase activity of the residual leaf extract (i.e., 2.24 X lo” cpm on a total tissue basis), but with a product mixture consisting of 70% farnesyl pyrophosphate, 5% geranylgeranyl pyrophosphate, and over 22% (5.0 X lo4 cpm) geranyl pyrophosphate. Thus, not only were the epidermal gland extracts enriched in the presumptive geranyl pyrophosphate synthase (farnesyl to geranyl pyrophosphate ratios of less than 4:l compared to in excess of 65:l for the residual leaf extract), but they also produced more geranyl pyrophosphate on an absolute basis (5.0 X lo4 cpm) than did the residual leaf extracts (1.6 X lo4 cpm), despite the fact that the oil glands comprise but a small fraction (<5%) of the total leaf tissue. Although the interpretation of these results might appear to be complicated by the fact that geranyl pyrophosphate readily serves as an allylic substrate for farnesyl pyrophosphate synthase (2), it

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seems safe to assume that geranyl pyrophosphate produced from C5 precursors by farnesyl pyrophosphate synthase will be elongated and will not appreciably accumulate (2), whereas the geranyl pyrophosphate produced by geranyl pyrophosphate synthase will not serve as an effective substrate for the farnesyl pyrophosphate synthase since the solution concentration of this end product is quite low and is greatly exceeded by the concentration of the alternate allylic cosubstrate dimethylallyl pyrophosphate. Thus, the levels of geranyl pyrophosphate produced by the various extracts are likely a reliable indicator of gerany1 pyrophosphate synthase activity. Therefore, from the results it would follow that the geranyl pyrophosphate synthase resides primarily, if not exclusively, in the monoterpene-producing oil glands (i.e., about 70% of the overall activity associated with about 70% gland removal). Extension of the experiments described above indicated that roughly 10% of the total leaf isopentenyl pyrophosphate isomerase activity and the phosphatase activity (hydrolysis of [l-3H]geranyl pyrophosphate) was also located in the epidermal gland extracts. Partial purificution of geranyl pyrophnh.o.sphute synthnse. All subsequent experiments with the geranyl pyrophosphate synthase were carried out with the leaf surface extracts which were enriched in this enzyme. The soluble enzyme fractions from these preparations were routinely concentrated by ultrafiltration (Amicon YM-30) since (NH,),SO, precipitation resulted in significant loss (>95%) of geranyl pyrophosphate synthase activity, all of which precipitated at a concentration below 25% saturation. Attempts to separate the geranyl pyrophosphate synthase from farnesyl pyrophosphate synthase, which was the major competing activity in these preparations, by chromatography on Odiethylaminoethyl-cellulose or hydroxylapatite were partially successful; however, the resulting significant loss of geranyl pyrophosphate synthase activity (>90% ) made both procedures impractical. The fact that geranyl pyrophosphate synthase precipitated below 25%) saturation with

532

W&SO,

CROTEALJ

AND

suggested that the enzyme might be hydrophobic, and this ultimately lead to the exploitation of hydrophobic interaction chromatography as a practical method to make the needed separation. Thus, the concentrate from the surface gland extract was applied to a column of phenyl-Sepharose CL-4B which was eluted sequentially with buffers containing 20% glycerol, 50% ethylene glycol, and, finally, 50% ethylene glycol plus 0.5 N NaSCN as a chaotropic agent. The final step afforded the geranyl pyrophosphate synthase in reasonably good yield following dialysis to assay conditions (>50%) and substantially free of competing activities (i.e., following enzymatic hydrolysis of the elongation products, greater than 80% was shown to be [3H]geraniol by radio-GLC). This fraction was concentrated by ultrafiltration as before and applied to the Sephadex G-150 column as described in the preliminary studies. Activity was located by both the acid lability and enzymatic hydrolysis assays as before, and the appropriate fractions were pooled to afford a preparation representing about 20-fold purification from the initial extract and which produced, from C, precursors, geranyl pyrophosphate as the major elongation product (90%, with -2% farnesyl pyrophosphate and -8% geranylgeranyl pyrophosphate) (Fig. 2C). The c&isomer, neryl pyrophosphate, was not produced in detectable amounts by this system. Preparation of the geranyl pyrophosphate synthase by this method resulted in the removal of over 95% of the isopentenyl pyrophosphate isomerase activity of the crude extract, over 99% of the original farnesyl pyrophosphate synthetase activity, and over 87% of the original geranylgeranyl pyrophosphate synthase activity, when measured with C5 precursors, while affording the geranyl pyrophosphate synthase in 30-40% overall yields. When prenyltransferase activity was measured separately using dimethylallyl, geranyl, and farnesyl pyrophosphates as the allylic cosubstrates (each at 100 PM with [3H]isopentenyl pyrophosphate at 50 pM) the ratios of the corresponding geranyl to farnesyl to geranylgeranyl pyrophosphate

PURKETT

synthase activities in the preparation were shown to be 100:2:18 on a molar basis. Calibration of the above Sephadex G-150 column with a series of proteins of known molecular weight indicated that the geranyl pyrophosphate synthase eluted at a volume corresponding to a molecular weight of approximately 100,000, which is somewhat higher than the range observed (55,000-85,000) for a variety of other prenyltransferases of plant, microbial, and animal origin (1, 2, 13, 48-52). Sephacryl columns were unsuitable for estimation of molecular weight because of the tendency of the enzyme to bind to this matrix. Characterization of the enzyme. Preliminary studies with the partially purified geranyl pyrophosphate synthase established that, under standard assay conditions, product accumulation was linear up to at least 90 min at the 50 /Lg protein/ml level, and was also proportional to protein concentration up to at least 100 pg./ml for a l-h assay. All subsequent assays were carried out under these linear assay conditions (1 h at 30-50 pg protein/ml). The pH optimum for geranyl pyrophosphate synthesis was determined by dialysis of enzyme aliquots to a 15 mM concentration of a series of buffers of overlapping pH (Mes, Pipes, Hepes, Tricine, Taps) ranging from pH 5.0 to 9.0. The curve of enzyme response to pH was symmetrical with an optimum near 7.0 (in Pipes) and with half-maximum velocities at about pH 6.0 and pH 8.0. This optimum is in the range reported for a variety of other prenyltransferases of microbial, animal, and plant origin (1,2,10,11,31,50,52-56). Dialysis of the preparation to remove divalent cations reduced the prenyltransferase activity to a negligible level, indicating the absolute requirement for the metal ion as expected for enzymes of this type (1, 2, 49, 54, 56). Re-addition of Mg2+ restored maximum activity, and the concentration response curve was typically hyperbolic from which several plotting procedures gave a KVI value of 0.27 & 0.02 mM. The influence of several other cations was examined at concentrations up to 3 mM, but in no case did maximum activity approach that observed with Mg2+ at saturation (i.e.,

GERANYL

PYROPHOSPHATE

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(& > 20 mM). Such arginine-specific reMn2+ at 0.5 mM = 61% of maximum, Zn”+ agents have been shown to inhibit at least at 0.5 mM = 35%) Ni” at 1 mM = 39%) and some prenyltransferases responsible for Co’+ at 0.5 mM = 56%). Mg2+ is often prethe synthesis of longer chain prenols (51). ferred to Mn”+ by prenyltransferase (1,31, In general features, the geranyl pyro50), and other divalent metal ions are genphosphate synthase from sage resembles a erally less effective than either in promotvariety of other prenyltransferases from ing catalysis (10, 57). Earlier reports that the proportion of tramand cis-products diverse sources and of broad chain-length (i.e., neryl pyrophosphate) varied with the specificity. In selectively terminating elondivalent metal ion added to crude enzyme gation at the Cl0 stage, the geranyl pyropreparations from Citrus flavedo (10, 11) phosphate synthase is novel, and the present work constitutes the first detailed decould not be verified in preparations from sage leaf glands; neryl pyrophosphate was scription of such an enzyme from any not appreciably formed in these preparasource. The fact that geranyl pyrophostions under any conditions. phate has been shown to be the immediate precursor of most cyclic monoterpene The rate of formation of geranyl pyrophosphate as a function of isopentenyl py- types, and that the relevant synthase is lorophosphate concentration, at a saturatcated primarily in the epidermal glandular ing concentration of Mg2+ and dimethylalsite of monoterpene biosynthesis and acculyl pyrophosphate (50 PM), exhibited a mulation, strongly suggests that this entypical hyperbolic saturation curve. Com- zyme is responsible for producing the key acyclic precursor of the monoterpene famputer assisted Lineweaver-Burk, EadieHofstee, Hanes, and direct linear plotting ily of natural products. The possibility provided a rC,,,value for isopentenyl pyrothat geranyl pyrophosphate is supplied for phosphate of 7.3 + 1.1 pM. The response of this purpose as a by-product of farnesyl or the geranyl pyrophosphate synthase to in- geranylgeranyl pyrophosphate synthesis creasing concentrations of dimethylallyl can thus be eliminated. Similarly, alpyrophosphate, at a saturating level of iso- though neryl pyrophosphate has been propentenyl pyrophosphate (28 PM), was simposed as a possible precursor of cyclic ilarly evaluated and gave a K,,, value for monoterpenes (60), this &s-analog of the this allylic eosubstrate of 5.6 f 0.8 PM. V geranyl substrate is seemingly not prodetermined by both approaches was about duced by S. ojicinalis, thus arguing against 150 nmol/h. mg protein, with many prepa- this alternative route to cyclic products. rations approaching well over twice this Although the instability of geranyl pyrovalue. The K,,, values observed for the al- phosphate synthase and the presence of lylic and homoallylic cosubstrates are competing activities prevent accurate dewithin the range (l-10 FM) observed for termination of activity levels in oil gland many other prenyltransferases of differing extracts, a rough estimate based on such in chain-length specificity (1,2,10,31,49-57). vitro assay indicates that this prenyltransTo assessthe sensitivity of the enzyme ferase is present in oil glands at lo-fold to thiol-directed reagents, dithiothreitol higher activity levels than any monoterwas first removed by dialysis and the preppene cyclase from this tissue. aration then probed with a variety of such reagents over a broad concentration range ACKNOWLEDGMENTS (5,5’-dithiobis(nitrobenzoic acid), Iso - 4 We thank Mark Felton for technical assistance in PM; p-mercuribenzenesulfonic acid, I,,, Wichelns - 40 PM; N-ethylmaleimide, Is0 - 60 PM; the early phases of the research, Gregory for raising the plants, and Nancy Madsen for typing iodoacetamide, IS0> 500 PM). Sensitivity to the manuscript. thiol-directed reagents is characteristic of prenyltransferases (10, 11, 31, 58, 59). The REFERENCES arginine-directed reagent 2,3-butanedione, in borate buffer, was an ineffective in1. WEST, C. A., DUDLEY, M. W., AND DUEBER, M. T. hibitor of geranyl pyrophosphate synthase (1978) in Recent Advances in Phytochemistry

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