ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

184, 77-86 (1977)

Demonstration of a Cyclic Pyrophosphate Intermediate in the Enzymatic Conversion of Neryl Pyrophosphate to BorneoIl RODNEY Department

ofAgFicu~tuFal

CROTEAU2

AND

FRANK

KARP

Chemistry and the Program in Biochemistry and Biophysics, University, Pullman, Washington 99164 Received

April

Washington

State

18, 1977

A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borne01 and the oxidation of (+)-borne01 to (+)-camphor. Attempts to purify the borne01 synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH,) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borne01 is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.

Borneo13 and camphor are bicyclic monoterpenes found in the volatile oil of a large number of plant species, where they may function in an allelopathic role (11. Both compounds possess penetrating odors and find wide use in flavoring agents and medicinals. Ruzicka and associates (2) suggested a hypothetical scheme for the formation of the camphane (bornane) family of bicyclic monoterpenes in which nerol, or more likely neryl pyrophosphate

(3, 4), is cyclized to an a-terpinyl cation which participates in an internal alkylation of the olefin to yield the camphane nucleus (i.e., a 2-bornyl cation). The labeling pattern of borne01 and camphor derived from exogenous [2J4C]mevalonic acid and [2J4C]geraniol in intact tissue is consistent with this hypothesis (5, 6); yet, the pathway for the formation of the camphane monoterpenes remains uncertain, and virtually nothing is known about the enzymes involved in the biosynthesis of these compounds. The volatile oil of sage (Saluia of,&alis) contains an appreciable amount of camphor (15% by weight) and a lesser amount of borne01 (0.5%) (7). Recently, we described a soluble enzyme preparation from this tissue that converts neryl pyrophosphate to borne01 and, in the presence of NAD, dehydrogenates this alcohol to camphor (8). cY-Terpineol is not an intermediate in the biosynthesis of borneol, and the diastereomeric isoborneol (which theoretically could function as a precursor of camphor) is not synthesized by the prep-

’ This is Scientific Paper No. 4791, Project 0268, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164. This investigation was supported in part by a National Science Foundation grant (PCM76-23632) and by a Cottrell research grant from the Research Corporation. * Author to whom all correspondence should be addressed. 3 Abbreviations used: nerol, 3,7-dimethylocta2(cis),6-dienol; geraniol, 3,7-dimethylocta-2(trans), 6-dienol; borneol, 1,7,7-trimethylbicyclo[2.2.11-2heptanol(endo); isoborneol, 1,7,7-trimethylbicyclo[2.2.1]-2-heptanol(exo); camphor, 1,7,7-trimethylbicyclo[2.2.11-2-heptanone; a-terpineol, p-menth-len-8-01. 77 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

78

CROTEAU

aration. In this communication we describe further studies with this enzyme system and report our finding that borne01 is synthesized in two discrete enzymatic steps: the cyclization of neryl pyrophosphate to bornyl pyrophosphate followed by the hydrolysis of this intermediate to borneol. EXPERIMENTAL

PROCEDURES

Plant materials. Sage (Salvia officinalis L.) plants were grown from seed in peat moss in a growth chamber (14-h day under 15,000-1x light intensity with a day/night temperature cycle of 30/ 19°C). Unfurled leaves (l-2 cm in length) from the shoot apex were collected from la-day-old plants and washed with 0.5 mM EDTA solution followed by distilled water. Substrates and reagents. [l-3HlNerol (300 Cilmol) was synthesized and purified as described previously (7). (+)-[G-3H]Borneol (24.3 Ci/mol) was prepared by exposure of (+)-borne01 [obtained by mixed-hydride reduction of (+)-camphor (911 to 6 Ci of 3H2 according to Wilzbach’s method (23). Rigorous purification by preparative thin-layer chromatography gave chemically and radiochemically pure material (verified by radio gas-liquid chromatographic analysis of several derivatives). [l-3H]Nerol and (+)-[G-3H1borneol were phosphorylated and the pyrophosphates were separated from the monophosphates by standard techniques (10, ll), described in detail elsewhere (7). Thin-layer chromatography, phosphate determination, and radiochromatographic analyses of the enzymatic hydrolysis products were employed to confirm identifications and verify purities of the phosphorylated substrates (71, which were lyophilized and dissolved in 0.01 M (NH&CO, (pH 9.0) for use in the assays. (+)-Borneol was obtained from Nutritional Biochemicals Corp., and (+)- and (-)-camphor were from Aldrich Chemical Co. Other reagents and biochemicals were purchased from Aldrich Chemical Co. or Sigma Chemical Co. Enzyme preparation. The 105,OOOg supernatant used as the enzyme source was prepared from a homogenate of young sage leaves (10 to 15 g) as previously described (8). This soluble preparation was then concentrated, and its pH and ionic strength adjusted, by dialysis against cold 0.01 M sodium phosphate buffer (pH 7.5) containing 5 mM MgCl,, 1 mM ascorbic acid, and 0.5 mM dithioerythritol and saturated with Carbowax 6000 (Union Carbide). The concentrate was then centrifuged at 27,000g to remove any denatured protein. This soluble concentrate was used as such to prepare [3H]borneol and [3H]camphor for product identification (see below), or it was applied to a 110 x 2-cm column of Sephadex G-150 that was equilibrated

AND

KARP

and developed (12 ml/h, 3.5-ml fractions) with the buffer described above (but without the Carbowaxl. Preparation of biosynthetic borne01 and camphor. To prepare [3Hlborneol for product identification, an aliquot of the 105,OOOgconcentrate was incubated for 3 h at 30°C in the presence of 20 mM MgCl, and 50 ELM [l-3H]neryl pyrophosphate. For the preparation of [3Hlcamphor, the same incubation procedure was employed except that 1.5 mM NAD was added to the medium. The biosynthetic products were isolated as previously described (8). Enzyme assays. To assay the formation of borne01 from [l-3H]neryl pyrophosphate, 0.2 ml of the appropriate column fraction was incubated for 1 h at 30°C in a sealed centrifuge tube in the presence of 10 mM MgCl, and 50 PM [l-3H1neryl pyrophosphate. At the end of incubation, the sample was extracted with diethyl ether (21x 1 ml) and 10 mg of (*)-borne01 was added as an internal standard. The extract was concentrated at 0°C under a stream of N, and subjected to thin-layer chromatographic analysis [silica gel G with hexane:ethyl acetate (4:1, v/v) as developing solvent]. The borne01 region was located and the silica gel from this region was transferred to a counting vial for determination of 3H content by liquid scintillation spectrometry. If the radioactive products were to be analyzed further, they were eluted from the gel with diethyl ether and an aliquot was taken for determination of 3H content. To assay the conversion of neryl pyrophosphate to bornyl pyrophosphate, the ether-extracted reaction mixture from above was first boiled and then cooled and adjusted to pH 5.0 with 0.1 M sodium acetate; it was then incubated again, either for 1 h with an excess of the bornyl pyrophosphate pyrophosphohydrolase (described later in the text) or for 4 h with 2 units of acid phosphatase and 5 units of apyrase (both type 1 from Sigma). At the end of this hydrolysis period, the PHIborneo released was extracted and assayed by the thin-layer chromatographic procedure indicated above. To monitor the hydrolysis of synthetic (+I-[G3H]bornyl pyrophosphate [or (+)-[G-3H1bornyl phosphate] to borneol, 0.1 ml of the appropriate column fraction was incubated in a sealed centrifuge tube containing 0.4 ml of 0.1 M sodium acetate buffer (pH 5.01, 10 mM MgCl,, and 20 @M substrate. At the end of a 30-min incubation period at 3O”C, 1.5 ml of hexane was added and the tube was shaken vigorously. After centrifugation, the aqueous phase was frozen in a dry-ice bath and the hexane phase was poured off into a scintillation vial for the determination of 3H by liquid scintillation spectrometry. The accuracy and reproducibility of this assay procedure were confirmed by calibration against standard solutions of [G-3Hlborneol and by comparison to the more tedious thin-layer chromatographic assay. To examine the conversion of bornyl pyrophosphate to bornyl phosphate, the hexane-extracted

ENZYMATIC

CONVERSION

OF NERYL

reaction mixture from above was boiled briefly and centrifuged to remove denatured protein. An aliquot of the supernatant was then streaked on a silica gel G plate, and the chromatogram was developed with isopropanol:ammonia (sp grav, 0.88):water (4:2:1, vl v/v). Authentic standards were run on every plate (R, of the monophosphate = 0.68-0.75; R, of the pyrophosphate = 0.08-0.12). Either the chromatograms were scanned with a Berthold thin-layer scanner or the silica gel from the appropriate region was transferred to a scintillation vial for determination of 3H content by liquid scintillation spectrometry. The recovery of radioactive products for each assay procedure was determined with the appropriate labeled standard, and corrections were made. Boiled controls were included in each experiment and in all cases nonenzymatic activity was negligible. of camphor. Exchange of the a-hydrogen 13H]Camphor was diluted with 50 mg of unlabeled (*)-camphor to known specific activity and dissolved in 50% aqueous dioxane. Two drops of 40% NaOH were added and the mixture was heated on a steam bath for 15 min and then allowed to stand overnight. Under these conditions, the C-3 ezo-ahydrogen is selectively exchanged (12). The reaction mixture was then acidified and extracted with pentane, and the camphor was quantitatively reisolated by thin-layer chromatography [silica gel G, with hexane:ethyl acetate (4:1, v/v) as developing solvent] for determination of specific activity. To exchange both cY-hydrogens of the ketone, the above procedure was repeated, but the reaction mixture was refluxed for 45 min. Controls run with water instead of 40% NaOH resulted in negligible decrease in specific activity. Resolution of (+camphor. 13H]Camphor was diluted with 100 mg of (r)-camphor and refluxed (with a water trap) for 14 h in 25 ml of anhydrous benzene containing 60 mg of n(-)-2,3-butanediol (K & K Laboratories) and 1 mg of toluene-p-sulfonic acid to form a mixture of diastereomeric ketals (84% yield) (13). The reaction products were washed with water, dried over Na*SO,, and concentrated under vacuum, and the concentrate was subjected to thin-layer chromatography (silica gel G with benzene as developing solvent). The mixture of diastereomeric ketals (R, = 0.48) was eluted from the gel with diethyl ether and resolved by radio gas-liquid chromatography under conditions described later in the text. Chemical conuersions. Acetylation was done with a 2:l mixture of acetic anhydride and pyridine at room temperature overnight. Benzoylation was performed by refluxing benzoyl chloride in pyridine containing 20% 4-dimethylaminopyridine for 3 h. Trimethylsilyl ethers were prepared by heating with Tri-Sil (Pierce Chemicals) for 15 min at 80°C.

PYROPHOSPHATE

TO BORNEOL

79

The above derivatives were isolated by thin-layer chromatography [silica gel G, with hexane:diethyl ether (9:1, v/v) as developing solvent]. Osmylation of oletins was carried out in diethyl ether:pyridine (lO:l, v/v) or water, and the osmic esters were decomposed with Na,SO,. Two-phase chromic acid oxidation of borne01 (50 mg in ether) was done by published procedures (14). The preparation of bornyl phenylurethane and camphor oxime and the purification and recrystallization of these derivatives have been described (8). 13H]Camphor (30 mg) was oxidized to camphoric acid by refluxing for several hours with 300 mg of CrO, in 5 ml of glacial acetic acid. The reaction mixture was diluted with water, and the camphoric acid was extracted with ethyl acetate and purified by thin-layer chromatography [silica gel G with hexane:diethyl ether:acetic acid (25:25:1, v/v/v) as developing solvent] (R, = 0.29). Chromatography. Thin-layer chromatography was done on l.O-mm layers of silica gel G activated at 110°C for 4 h. Developing solvents are indicated elsewhere. After development, the chromatograms were sprayed with a 0.2% ethanolic solution of 2,7dichlorofluorescein and viewed under ultraviolet light to locate the appropriate component. Paper chromatography of bornyl pyrophosphate (R, = 0.53-0.60) was done by the ascending technique (Whatman No. 3MM) using t-butanol:formic acidwater (20:5:8, v/v/v) as developing solvent. Purification of the water-soluble biosynthetic products (i.e., bornyl pyrophosphate) by diethylaminoethyl cellulose column chromatography was carried out essentially like the purification of phosphorylated substrates (7). Experimental details are given under the appropriate figure legends. Gas-liquid chromatography was performed on a Varian chromatograph, with the effluent connected to a Nuclear Chicago Model 7357 radioactivity monitor. The chromatographic columns and conditions are indicated under the appropriate figures. Determination of rudioactiuity. Radioactivity in organic liquid samples and in thin-layer chromatographic fractions was assayed with a Packard liquid scintillation spectrometer in a counting solution consisting of 0.55% (w/v) Permablend III (Packard) dissolved in 30% ethanol in toluene (15 ml). The counting efficiency for 3H was 37%. Aqueous samples (0.1 ml) and thin-layer fractions containing phosphorylated products were assayed as above in 15 ml of Aquasol (New England Nuclear Corp.). The counting efficiency for 3H was 35%. All assays were conducted with a standard deviation of less than 3%. RESULTS

AND

DISCUSSION

Conversion of Neryl Pyrophosphate neol and Camphor

to Bor-

A soluble enzyme system obtained from

80

CROTEAU

homogenates of young sage leaves converts [l-3Hlneryl pyrophosphate to borne01 and, in the presence of NAD, oxidizes the borne01 to camphor (6). According to the hypothesis of Ruzicka and associates (2), borne01 and camphor derived from [l3Hlneryl pyrophosphate should be labeled specifically at C-3. To test this possibility, biosynthetic [3H]camphor (sp act, 2685 dpmlmg) was treated with NaOH in aqueous dioxane to selectively exchange the exo-a-hydrogen (12). Reisolation of the 13Hlcamphor after such treatment indicated that approximately 50% of the 3H had, in fact, been lost (sp act, 1270 dpml mg). Under more vigorous conditions, both the exo- and endo-a-hydrogens could be exchanged, and the specific activity was reduced to 194 dpm/mg. Identical treatment of [3H]camphor obtained by chromic acid oxidation of biosynthetic [3Hlborneol gave similar results: a decrease in specific activity from 2444 to 1178 dpm/mg, with a further decrease to 143 dpm/mg under more vigorous conditions. In addition, oxidation of [3H]camphor to camphoric acid resulted in the complete (>99%) loss of 3H. Thus, the bulk of the 3H was located at C-3, demonstrating that borne01 and camphor were derived directly from [l3Hlneryl pyrophosphate in a manner consistent with the earlier hypothesis (2) and with previous in uiuo studies (5, 6). Enantiomer Composition thetic Products

of the Biosyn-

Camphor isolated from steam-distilled sage oil by thin-layer chromatography (99+% as determined by combined gasliquid chromatography/mass spectrometry) was shown to be the (+)-isomer ([aIF +44; C = 10, ethanol), in agreement with earlier reports (15, 16). An indication to the contrary (6) could not be verified. Only a small amount of borne01 is available from sage oil (0.5%); however, a sample of this material yielded a positive deflection when examined by optical rotatory dispersion (600-400 nm), consistent with the role of (+)-borne01 as a precursor of (+)-camphor. To examine the enantiomer composition of the [3-3Hlcamphor derived from [ l-3H]neryl pyrophosphate by the cell-free

AND

KARP

preparation, the labeled ketone was diluted with racemic camphor and converted to a mixture of diastereomeric ketals by with n( -)-2,3-butanediol condensation (13). The ketal fraction was isolated by thin-layer chromatography and subjected to radio gas-liquid chromatography (Fig. la). Only the ketal derived from (+)-camphor was labeled. Attempts to resolve the biosynthetic [3-3Hlborneol by chromatographic separation of the diastereomeric (+)- a-methoxy-a-trifluoromethylphenylacetate or (-)-methoxyacetate esters (17, 18) were insufficient to allow the unambiguous assignment of enantiomers. Therefore, the [3-3Hlborneol was oxidized to [3-

W’



2 ‘16 18 Time (min)



20



FIG. 1. (a) Radio gas-liquid chromatogram of the diastereomeric ketals obtained by condensation of n(-)-2,3-butanediol with [3-3Hlcamphor derived from [l-3H]neryl pyrophosphate by the enzyme preparation from sage. (b) Similar chromatogram of the ketals obtained from [3-3H]camphor which was acquired by CrO, oxidation of [3-3Hlborneol. The borneol was derived from [l-3Hlneryl pyrophosphate by the enzyme preparation from sage. The top two tracings show the radioactivity recorded by the monitor attached to the gas chromatograph. The smooth bottom tracing is the flame ionization detector response obtained from a coinjected mixture of the diastereomeric ketals derived from (+)- and (-)-camphor. The order of elution of the ketals was determined by separate injection of each authentic diastereomer. Gas-liquid chromatography was performed on a 9.5-ft x 0.125in.-o.d. stainless-steel column packed with 15% cyanoethylsucrose on 60/ 80-mesh Gas Chrome R. The column was programmed from 100 to 125°C at 5”/min with an argon flow rate of 160 cmVmin.

ENZYMATIC

CONVERSION

OF NERYL

3Hlcamphor with Cr03 and the ketone was chromatographically resolved as above. Only the ketal derived from (+)-camphor was labeled (Fig. lb), indicating that the borneol, from which it was obtained, was also the (+)-isomer. Thus, both borne01 and camphor of the appropriate stereochemistry were obtained directly from [l3H]neryl pyrophosphate by the cell-free preparation. Demonstration of an Intermediate neol Biosynthesis

in Bor-

To examine the enzymes involved in camphor biosynthesis in greater detail and, in particular, to determine if any free intermediates were involved in the cyclization of neryl pyrophosphate to borneol, we attempted to purify the borne01 synthetase activity by chromatography on Sephadex G-150. Although preliminary studies indicated that maximum borne01 formation was observed in crude preparations in 0.01 M sodium phosphate buffer (pH 7.5) in the presence of MgCl, (10 mM), assay of column fractions under these conditions indicated that no individual fraction possessed the ability to convert [l3Hlneryl pyrophosphate to borneol. However, recombination of column fractions followed by concentration (ultrafiltration, Amicon PM 10 membrane) restored activthat borne01 formation ity, suggesting from neryl pyrophosphate required two or more separable enzyme components or subunits. More detailed recombination studies subsequently revealed that the combination of protein eluting at approximately 1.25 void volumes (designated F,) with protein eluting at about 1.75 void volumes (designated F,) restored activity to the full extent observed in the unfractionated preparation (Table I). Furthermore, incubation of F, and F, separated by a dialysis membrane still allowed borneol formation from [1-3H]neryl pyrophosphate (Table I), suggesting the involvement of a free dialyzable intermediate and the requirement for two distinct enzyme components. Subsequent experiments revealed that the deproteinized (ultrafiltration) water-soluble products produced by F, from neryl pyrophosphate could be con-

PYROPHOSPHATE

81

TO BORNEOL TABLE

I

CONVERSION OF NERYL PYROPHOSPHATE TO BORNEOL BY SOLUBLE ENZYMES FROM Salvia officinalis”

Enzyme preparation

105,OOOgsupernatant F, F* F, + F, F, + F, (boiled) F, (boiled) + F, F, + F, (in dialysis bag) F, (water-soluble products) F, (water-soluble products) phosphatase + apyrase

Borneo1 formed (dpm x 10-1)

+ F, + acid

10.54 0.09 0.02 10.44 0.08 0.03 4.06 10.65 8.78

(1In each case, 5% of the appropriate fraction (105,OOOg supernatant, F, and F, as described in Fig. 5) was incubated in a total volume of 0.2 ml of 0.01 M sodium phosphate buffer (pH 7.5) containing 10 mM MgCl,, 1 mM ascorbic acid, 0.5 mM dithioerythritol, and 50 PM ll-3Hlneryl pyrophosphate for 1 h at 30°C. 13H1Borneol was assayedby the thinlayer chromatographic procedure described under Experimental Procedures. In the dialysis experiment, equilibrium was not achieved in 1 h, and the efficiency of borne01 formation was correspondingly reduced. The F, water-soluble products were prepared by preincubation as above, followed by heating, ultrafiltration, and thorough extraction with diethyl ether.

verted to borne01 by F, (Table I). The ether-soluble metabolites from F, were inactive with FP, and neither the water-soluble products nor the ether-soluble products produced by F, from neryl pyrophosphate could be converted to borne01 by F, (results not shown). Thus, the data strongly suggested that F, was capable of transforming neryl pyrophosphate to a water-soluble, dialyzable intermediate(s) that could be converted to borne01 by Fz. Identification of the Intermediate neol Biosynthesis

in Bor-

The water-soluble nature of the unknown intermediate suggested the possibility that it was phosphorylated. To examine this possibility, the water-soluble products obtained from the incubation of F, with 11-3Hlneryl pyrophosphate were treated with a mixture of apyrase and alkaline phosphatase. Recovery of the de-

82

CROTEAU

proteinized water-soluble products after ether extraction revealed that they no longer gave rise to borne01 when incubated with F,. Thus, phosphate ester hydrolysis had destroyed the intermediate. Radio gas-liquid chromatographic analysis of the ether-soluble products, obtained by apyrase:alkaline phosphatase hydrolysis, revealed the presence of two labeled components (Fig. 2). The major product was coincident with nerol, the expected hydrolysis product of the substrate, while the minor product was coincident with borneol. Treatment of the hydrolysis products with 0~0, to convert nerol to the corresponding water-soluble pentaol, followed by pentane extraction, allowed the thinlayer chromatographic isolation of essentially pure [3H]borneol (Fig. 3a). Oxidation of this material with CrO,, followed by radio thin-layer chromatography and radio gas-liquid chromatography, yielded a single radioactive component coincident with camphor (Figs. 3b and 3d). A portion

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KARP

of the 13Hlcamphor was reduced with LiAlH,. Isolation of the products followed by thin-layer chromatography gave rise to a single radioactive component coincident with the diastereomeric isoborneol (Fig. 3c), as expected by stereoselective reduction of the ketone (9). Another portion of the 13Hlcamphor obtained on oxidation was treated with hydroxylamine hydrochloride. Radio thin-layer chromatography and radio gas-liquid chromatography indicated the presence of a single radioactive component coincident with camphor oxime, and the oxime was crystallized to constant specific activity (5410 2 280 dpml mg, through six crystallizations). The identity of the minor hydrolysis product as 13Hlborneol was further confirmed by crystallization of bornyl phenylurethane to constant specific activity (3840 2 190

I

I

4 Time

a

(min.)

FIG. 2. Radio gas-liquid chromatogram of the ether-soluble products obtained by alkaline phosphatase:apyrase hydrolysis of the water-soluble material derived from [1-3H]neryl pyrophosphate by incubation with the F, enzyme preparation. The top tracing shows the radioactivity. The smooth bottom tracing is the Dame ionization detector response obtained from coinjected standards of linalool (a), a-terpineol and isobomeol (b), borne01 (cl, nerol (d), and geraniol (e). Gas-liquid chromatography was performed on a 10-R x 0.125-in.-o.d. stainlesssteel column packed with 12% Carbowax 4000 on 80/100-mesh Gas Chrome Q held at 175°C with an argon flow rate of 150 cm3/min.

I -

TirL

(min

FIG. 3. (a) Radio thin-layer chromatogram of the OsO,-treated products isolated from the ether-soluble material described in Fig. 2. (b) Radio thinlayer chromatogram of the products obtained by oxidizing the bomeol, described in (a) above, with CrG,. (cl Radio thin-layer chromatogram of the products obtained by reduction of the camphor, described in (b) above, with LiAlH,. Thin-layer chromatography was performed on silica gel G with hexane:ethyl acetate (4:1, v/v) as the developing solvent. The bar graph represents 3H contained in &mm sections of gel. The standards indicated are borne01 (B), isoborneol (I), and camphor (0. Or is the origin. (d) Radio gas-liquid chromatogram of the camphor fraction isolated from the thin-layer chromatogram shown in (b). The top tracing shows radioactivity. The smooth bottom tracing represents the flame ionization detector response obtained from coinjected authentic camphor. Gas-liquid chromatography was performed on the Carbowax column described in Fig. 2 held at 135°C with an argon flow rate of 150 cmVmin.

ENZYMATIC

CONVERSION

OF NERYL

dpm/mg) and by the preparation of several other derivatives including the acetate, the benzoate, and the trimethylsilyl ether, as described previously (8). In each case, a single radioactive component was formed, and it was coincident with the appropriate synthetic borne01 derivative on both radio thin-layer and radio gas-liquid chromatographic analyses. These results clearly implicated a phosphorylated borne01 intermediate in the conversion of [l-3Hlneryl pyrophosphate to (+)-[3-3Hlborneol. To examine this possibility in greater detail, the deproteinized water-soluble products obtained by large-scale incubation of F, with [1-3Hlneryl pyrophosphate were prepared. Treatment of aliquots of the soluble products with 0.1 M CH3C02H, 0.1

M N&OH

(30

IIIin

at

3o”c),

Or oSo4

(1

h at 3O”C), followed by lyophilization and assay with F,, gave the same quantity of borne01 as the untreated products. Thus, the intermediate was stable to acid and alkali under these conditions and contained no unsaturation, consistent with a phosphorylated borne01 derivative. Therefore, to remove residual neryl pyrophosphate and other acid-labile materials before further purification of the intermediate, the water-soluble products were heated at 30°C for 30 min with 0.1 M CH,CO,H and then extracted with ether. The water-soluble products were next treated with 0~0, to convert any remaining olefinic products to the corresponding diols. This preparation was then lyophilized and separated into neutral, monophosphorylated, pyrophosphorylated, and more polar materials by column chromatography on diethylaminoethyl cellulose (Fig. 4). The elution pattern of the major product was identical to that of synthetic [G-3Hlbornyl pyrophosphate. Each of the four main components obtained by diethylaminoethyl cellulose chromatography was then lyophilized and dissolved in water, and an aliquot (lo5 cpm) was incubated with F2. Only the pyrophosphorylated material possessing the same elution profile as synthetic bornyl pyrophosphate gave rise to borne01 on incubation with FO, and, more important, this product was quantitatively converted to borne01 by F,.

PYROPHOSPHATE

TO BORNEOL

83

FrOct~on Number

cellulose column FIG. 4. Diethylaminoethyl chromatography of the acid- and OsO,-treated water-soluble products obtained by incubating the F, enzyme preparation from sage with [l-3HIneryl pyrophosphate (lower tracing). The column (1.5 x 16 cm) was developed with a linear gradient of 150 ml each of 10 and 55 mM (NH&CO, (-----I. Fractions of 4.5 ml were colIected, and a O.l-ml aliquot of each was assayed for 3H content (0-O). V, was at fraction number 5. The upper tracing is the elution pattern of a mixture of synthetic [G-3H]bornyl phosphate (BP) and [G-3H]bornyl pyrophosphate (BPP) run on the same column under identical conditions.

the pyrophosphorylated Furthermore, fraction was also converted to borne01 bv incubation with a mixture of alkaline phosphatase and apyrase, or a mixture of acid phosphatase and apyrase, but not by any other hydrolytic enzyme tested (e.g., glucuronidase, (Y- and p-glucosidases, esterase). Hydrogenolysis of the pyrophosphorylated material with LiAlH, also gave rise to borneol, and the identity of this product, as well as the borne01 formed by enzymatic hydrolysis, was verified by formation of derivatives as described above. In addition, direct analysis of the radioactive pyrophosphorylated products by both paper chromatography and thin-layer chromatography on silica gel G gave rise to a single radioactive component coincident with synthetic bornyl pyrophosphate. On heating at 100°C for 30 min in 1 N HCl to hydrolyze the pyrophosphate bond (19), the product was largely converted to a less polar material coincident with synthetic bornyl phosphate on thin-layer chromatography. Thus, the intermediate obtained by incubation of F, with neryl pyrophosphate was shown to be bornyl pyrophosphate. Subsequent assay of G-150 column fractions for the ability to cyclize neryl pyro”

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CROTEAU

phosphate to bornyl pyrophosphate (utilizing a hydrolysis step with a mixture of acid phosphatase and apyrase) revealed the precise location of F,, now designated bornyl pyrophosphate synthetase (Fig. 5). To determine whether bornyl phosphate was also formed by the synthetase, the products derived from neryl pyrophosphate that possessed an elution pattern similar to bornyl phosphate (Fig. 4) were examined further. Hydrolysis of this material with alkaline phosphatase or acid phosphatase yielded very little ether-soluble product, and no borne01 was detected. Thus, bornyl phosphate was not produced by the synthetase. (The material with the elution behavior of monophosphates on diethylaminoethyl cellulose column chromatography could have been degradation products derived from acid hydrolysis or 0~0, treatment.) The bornyl pyrophosphate synthetase was also devoid of borneol kinase activity when incubated with [G-3Hlborneol, ATP, MnCl,, and MgC12, thus eliminating this route as an alternative means of bornyl pyrophosphate (or bornyl phosphate) formation. The above evidence demonstrating the formation of bornyl pyrophosphate strongly suggested that the F, enzyme component was functioning in the hydrolysis of bornyl pyrophosphate to borneol. This suggestion was confirmed by incubation of F, with synthetic ( +)-[G-3Hlbornyl pyrophosphate, and preliminary studies indicated that the rate of hydrolysis of bornyl pyrophosphate to borne01 by F, was about lo-

Fraction

AND KARP

fold greater in 0.1 M acetate (pH 5.0) buffer than under previous assay conditions (0.01 M phosphate, pH 7.5). Subsequent assay of G-150 column fractions for the ability to hydrolyze (+)-[G-3Hlbornyl pyrophosphate to borne01 allowed the precise location of F2, now termed bornyl pyrophosphate phosphatase (Fig. 5). Repetition of this assay with the F, (synthetase)-derived water-soluble products as substrate, rather than synthetic bornyl pyrophosphate, showed an identical elution pattern. Thinlayer chromatographic analysis of the water-soluble products remaining after incubation of the phosphatase with either synthetic [G-3Hlbornyl pyrophosphate or biosynthetic [3-3Hlbornyl pyrophosphate revealed the presence of low, but detectable, levels of labeled bornyl phosphate. Subsequent assay of the phosphatase with (+)[G-3Hlbornyl phosphate showed that this substrate was hydrolyzed at a rate comparable to that observed with (+)-[G3Hlbornyl pyrophosphate. Thus, a two-step hydrolysis seemed probable. Although the phosphatase was capable of hydrolyzing bornyl phosphate, incubation of this preparation with the products of the synthetase that behaved like monophosphates on chromatography (Fig. 4) did not yield borneol. Thus, the fact that the synthetase did not produce bornyl phosphate was confirmed. CONCLUSION

Although we have not yet examined the bornyl pyrophosphate synthetase or the

Number

FIG. 5. Sephadex G-150 gel filtration of the 105,OOOg supernatant preparation from sage leaves..Absorbance at 280 nm (-), bornyl pyrophosphate synthetase (FJ activity (O-----O), and bomyl pyrophosphate phosphatase (F,) activity (O-----O) are plotted. Chromatography and assay procedures are described under Experimental Procedures. V, was at fraction number 6.

ENZYMATIC

CONVERSION

OF NERYL

phosphatase activities in detail, it is quite clear from the evidence presented that borne01 is synthesized by the initial cyclization of neryl nyrophosphate to bornyl pyrophosphate followed by hydrolysis of this intermediate to the alcohol (which may then be oxidized to camphor) (Fig. 6). The intermediate role of bornyl pyrophosphate in the biosynthesis of borne01 has also been demonstrated in soluble preparations from rosemary (Rosmarinus off;cinalis) leaves, by means similar to that described above. The oil of rosemary is reported to contain (-)-borne01 (15). Thus, the proposed pathway for borne01 formation is probably widespread in the plant kingdom. Cyclic pyrophosphates such as copalyl pyrophosphate (201, pre-squalene pyrophosphate (211, and pre-lycopersene pyrophosphate (22) are involved in the formation of diterpenes, triterpenes, and tetraterpenes, respectively; however, this is the first report of a cyclic pyrophosphorylated intermediate in the biosynthesis of monoterpenes. Furthermore, unlike the other cyclic pyrophosphates cited, bornyl pyrophosphate is unique in that the pyrophosphate moiety of the acyclic precursor appears to have migrated during cyclization and to have participated in the formation of the 1,4 bridge. A possible mechanism for the conversion of neryl pyrophosphate to (+)-bornyl pyrophosphate is shown in Fig. 7. The enzyme may be involved in stabilizing the transient cation.

FIG. 6. Proposed pathway (+)-borne01 and (+)-camphor phate.

for the biosynthesis of from neryl pyrophos-

FIG. 7. A possible mechanism for the conversion of neryl pyrophosphate to (+)-bornyl pyrophosphate.

PYROPHOSPHATE

TO BORNEOL

a5

We are now in the process of purifying and characterizing these enzymes involved in borne01 biosynthesis and are attempting to determine if cyclic pyrophosphate intermediates are also involved in the biosynthesis of other cyclic monoterpene alcohols such as thujyl alcohol and fenchyl alcohol. ACKNOWLEDGMENTS We thank Dr. J. I. Legg for assistance in obtaining the optical rotatory dispersion spectrum and for helpful discussions. D1: P. E. Kolattukudy REFERENCES 1. MULLER, C. H., AND CHOU, C. H. (1972) in Phytochemical Ecology (Harborne, J. B., ed.), pp. 201-215, Academic Press, New York. 2. RUZICKA, L., ESCHENMOSER, A., AND HEUSSER, H. (1953) Experientia 9, 357-367. 3. LOOMIS, W. D. (1967) in Terpenoids in Plants (Pridham, J. B., ed.), pp. 59-82, Academic Press, New York. P., BEYTIA, E., CORI, O., AND 4. VALENZUELA, YUDELEVICH, A. (1966) Arch. Biochem. Biophys. 113, 536-539. D. (1970) 5. BANTHORPE, D. V., AND BAXENDALE, J. Chem. Sot. (C), 2694-2696. 6. BATTERSBY, A. R., LAING, D. G., AND RAMAGE, R. (1972) J. Chem. Sot. (Perkin I), 2743-2748. 7. CROTEAU, R., AND KARP, F. (1976) Arch. Biothem. Biophys. 176, 734-746. 8. CROTEAU, R., AND KARP, F. (1976) Biochem. Biophys. Res. Commun. 72, 440-447. 9. ELIEL, E. L., AND NASIPURI, D. (1965) J. Org. Chem. 30, 3809-3814. 10. CORNFORTH, R. H., AND POPJAK, G. (1969) in Methods in Enzymology (Clayton, R. B., ed.), Vol. 15, pp. 359-390, Academic Press, New York. 11. DUGAN, R. E., RASSON, E., AND PORTER, J. W. (1968) Anal. Biochem. 22, 249-259. 12. THOMAS, A. F., SCHNEIDER, R. A., AND MEINWALD, J. (1967) J. Amer. Chem. Sot. 89, 6870. 13. CASANOVA, J., AND COREY, E. J. (1961) Chem. Ind., 1664-1665. 14. BROWN, H. C., AND GARG, C. P. (1961)J. Amer. Chem. Sot. 83, 2952-2953. 15. GUENTHER, E. (1949) The Essential Oils, Vol. 3, pp. 695-724, Van Nostrand, New York. 16. PARRY, J. W. (1969) Spices, Vol. 2, p. 207, Chemical Publishing Co., New York. 17. DALE, J. A., DULL, D. L., AND MOSHER, H. W. (1969) J. Org. Chem. 34, 2543-2549. 18. INGERSOLL, A. W. (1944) in Organic Reactions (Adams, R., Bachman, W. E., Fieser, L. F.,

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Johnson, J. R., and Snyder, H. R., eds.), Vol. 2, pp. 398-400, Wiley, New York. 19. TIDD, B. K. (1971) J. Chem. Sot. (B), 1168-1176. 20. SHECHTER, I., AND WEST, C. A. (1969) J. Biol. Chem. 244, 3200-3209. 21. EPSTEIN, W. W., AND RILLING, H. C. (1970) J.

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KARP

Biol. Chem. 245, 4597-4605. 22. BARNES, F. J., QURESHI, A. A., SEMMLER, E. J., AND PORTER, J. W. (1973) J. Biol. Chem. 248, 2768-2773. 23. WILZBACH, K. E. (1957) J. Amer. Chem. Sot. 79. 1013.

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 184, 77-86 (1977) Demonstration of a Cyclic Pyrophosphate Intermediate in the Enzymatic Conversion of Ne...
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