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THEJOURNAL OF BIOLOGICALCHEMISTRY Vol. 257. No. 5, Issue of March 10. pp. Y335-2342,zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1982 PrintedzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAinzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA US.A.zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Biosynthesis of (&)-a-Pineneand (-)-p-Pinene from Geranyl Pyrophosphate by a Soluble Enzyme Systemfrom Sage (Saluia officinalis)* (Received for publication, August 17, 1981) Herve Gambliel and Rodney CroteauS From the Institute of Biological Chemistry, and Biochemistry/Biophysics Program, Washington State University, Pullman, Washington 99164 The volatile oil of immature sage (Salvia officinalis) distributed monoterpenes in the plant kingdom and are the leaves was shown to contain (f)-a-pinene and (--)+- major constitutentsof the various turpentines (1). Both opti- pinene as major constituents, and solubleenzyme prep- cal antipodes of a-pinene are natural products, and theymay arations from this tissue catalyzed the divalent cation-co-occur with either isomer predominant (1-3). By contrast, dependent conversion of the acyclic Clo precursor ger- P-pinene almost always occurs as the optically pure (--)-[lS any1 pyrophosphate to a-pinene and P-pinene and to 5S] isomer (I), the (+)-[1R:5R] antipode apparently being of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA lesser amountsof other monoterpeneolefins. The iden- rather limited distribution (2). Ruzicka et al. (4)proposed an tities of the principalbiosynthetic products as a-pinene ionic scheme fort.he formation of the pinenes (Fig. 2, Scheme and P-pinenewere confirmed by chromatographic I) involving cyclization of the neryl cation to a monocyclic Downloaded from analysis andby the synthesisof crystalline derivatives. a-terpinyl intermediatewhich undergoes internal Markowni- The biosynthetic pinenes derived from [l-3H]geranyl koff addition to a piny1 cation that is subsequently stabilized pyrophosphatewere stereospecifically converted to of vivo camphor, and exchangeof the a-hydrogens of this ke- by two variants proton loss. On the basis of recent in tone established that all the tritium of the original time course studies with Pinuspinaster(5), the acyclic trienes pinane nucleus was located on the methylene bridge cis-ocimene and myrcene were suggested as precursors of a- carbon (C-7), thus demonstratingspecific conversion of and /I-pinene, respectively (Fig.2, Scheme ZI),reviving eariier www.jbc.org the acyclic precursor. Stereospecific conversion of the radical-type cyclization schemes for the construction of the biosynthetic pinenes to fenchone oxime, followed by pinenes (4, 6). Finally, the favorableisomerization of the resolution of thecorresponding diastereomeric (-)- exocyclic to the endocyclic isomer (7, 8) raises the possibility methyloxymethyl ethers, confirmed the stereochemis- of a single cyclization of an acyclic precursor to P-pinene, by guest, on December 7, 2011 try of the original products as (k)-[7-3H]a-pinene and followed by enzymic (or nonenzymic) conversion to 0-pinene (-)-[7-3H]P-pinene. The possibility of isomerization of (Fig. 2, Scheme IZI).The labeling pattern of a-pinene derived P-pinene to a-pinene was eliminated, as was the in- from exogenous [2-’4C]mevalonate in Pinus attenuata (9) is volvement of other free intermediates, thus indicating consistent with any of the above biogenetic schemes and no direct cyclization of geranyl pyrophosphateto themix- experimental evidence to distinguish among the alternatives ture of pinene isomers. NeryI pyrophosphate andIina- is available. While the origin of the pinenes is thus uncertain, loyl pyrophosphate could also function as acyclic pre- the results of chemotaxonomic studies (10) and in vivo tracer cursors of the pinenes and of the other monoterpene studies (11) suggest that the various isomers probably arise olefins, but only geranylpyrophosphate afforded a via distinct routes involving variants both in the stereochem- product distribution comparable thatto formed in vivo. istry of ring formation and in the generation of the double Preliminary examination of the cell-free systemzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA sug- bond in either the endocyclic or exocyclic position. The fact gested the presence of more than one pinene cyclase activity. that positionally isomeric and enantiomeric pinenesco-occur, with the combination (+)-a-pinene and (-)-P-pinene being particularly common (12, 13), furtherimplies that such mul- tiple routes are operativein the same organism. &-Pinene’ and/I-pinene (Fig. 1) are among the mostwidely Crude cell-free extracts from Citrus Zimonum (14), Pinus radiata (15), and common sage (Salvia officinalis) (16, 17) * This investigationwas supported in part by grants from the convert the acyclic C10 precursors GPP and NPP to products National Science Foundation (PCM 78-19417) and Haarmann and chromatographically coincident with a-pinene and ,&pinene. Reimer GmbH. Thisis Scientific Paper6014, Project 0268, College of Agriculture Research Center, WashingtonState University, Pullman, However, the identitiesof the biosynthetic productswere not Washington99164. The costs of publication of this article were confirmed, and, although both tritium atoms from [1-”H2,U- defrayed in part by the payment of page charges. This article must I4C]GPP were shownto be retained in the pinenes inthe latter therefore be hereby marked “advertisement” in accordance with 18 study (17), the labeling pattern and the stereochemistryof the U.S.C. Section 1734 solely to indicate this fact. products were not verified. Furthermore, the possible isom- $ To whom correspondence should be addressed. ’ The abbreviations and trivial names used are: a-pinene, 2,6,6- -__ trimethylbicyclo[3.1.l]hept-2-ene;GPP, pyrophosphate ester of ger- myrcene, 3-methylene-7-methyl-1,6-octadiene;ocimene, 3,7-di- aniol (3,7-dimethylocta-2(trans),6-dienol);NPP, pyrophosphate ester methyl-l,3(cis-or trans-),6-octatriene;a-terpineol, l-methyl-4-(2-hy- of nerol (3,7-dimethylocta-2(cis),6-dienol);LPP, pyrophosphate ester droxyisopropy1)-1-cyclohexene;borneol, 1,7,7-trimethylbicyclo[2.2.1] of linalool (3,7-dimethylocta-l,6-dien-3-ol);P-pinene, 6,6-dimethyl-2- heptan-2-oUendo);1,8-cineole, 1,3,3-trimethyl-2-oxabicyclo[2.2.2]0~- methylenebicyclo[3.1.l]heptane; camphene, 2,2-dimethyl-3-methyl- tane; MES, 2-[N-morpholino]ethanesulfonic acid; Tricine, N-[2-hy- enebicyclo[2.2.l]heptane; terpinolene, l-methyl-4-isopropropyl~ene- droxy-1,l-bis(hydroxymethyl)ethyl]glycine;GLC, gas-liquid chroma- 1-cyclohexene; limonene, I-methyl-4-isopropenyl-1-cyclohexene; tography; TLC, thinlayer chromatography. 2335 2336 Biosynthesis of Isomeric Pinenes from Geranyl Pyrophosphate zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAverifiedzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA as before (16) by analysis of the products of alkaline phos- phatase-apyrase hydrolysis and acid hydrolysis. (+)-a-Pinene, (-)-a-pinene, and (-)+pinene were obtained from Aldrich Chemical Co., cis- and trans-pinanol were from PCR Re- search Chemicals Inc., (+)-limonene was from ICN Pharmaceuticals, terpinolene was from Haarmann and Reimer GmbH, Holzminden, b West Germany, and the ocimenes were from International Flavors and Fragrances. All other monoterpenes were purchased from Pfaltz a b C and Bauer, Inc. and were purified by distillation or chromatography before use. For isotopic dilution experiments, the monoterpenes were zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAsuspended in water with the aid of Tween 20 (40 pg/pmol of terpene) and sonication (needle probe of a Biosonik 111 at full power). Insoluble polyvinylpyrrolidone (Polyclar AT, GAF Corp.) and Amberlite XAD- 4 polystyrene resin (Rohm and HaasCorp.) were purified by standard procedures for use as adsorbents (23,24). All other biochemicals and reagents were obtained from Sigma or Aldrich. Enzyme Preparation-Sage leaves (2-3 g) were homogenized in a Ten-Broeck homogenizer containing a slurry of an equivalent leaf weight of insoluble polyvinylpyrrolidone in cold 75 mM MES-5 mM sodium phosphate buffer, pH 6.5, containing 20% glycerol, 5 mM f dithioerythritol, 10 mM Na2S205,10 mM sodium ascorbate, 15 mM MgC12. The homogenate was then slurried with an equal tissue weight P of hydrated XAD-4 polystyrene resin and passed through four layers d e of cheesecloth. Centrifugation at 27,000 X g for 20 min (pellet dis- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAcarded), then at 105,000 X g for 1 h, provided the soluble supernatant that, after dialysis to assay conditions, was used as theenzyme source. Downloaded from Enzyme Assays-A typical reaction mixture contained 0.2 to 0.5 mg of protein in a total volume of 1 ml of 10 mM MES-5 mM sodium phosphate buffer or 10 mM Tricine-5 mM sodium phosphate buffer, pH 6.5, containing 0.5 mM dithioerythritol, 15 mMMgC12 (or 1 mM MnC12) in a Teflon-sealed screw cap vial. In some experiments, 1 mM sodium ascorbate and 1 mM Na2S205 were included in the assay medium, and the buffer contained 10% glycerol. The reaction was started by the addition of up to10 p~ (10 nmol) of either of the three www.jbc.org acyclic precursors ([1-3H]GPP, [l-”H]NPP, or (*)[1-3H]LPP), and 1 h I ml of pentane was carefully