Monoterpene Synthases from Common Sage (Salvia officinalis )

Common sage (Salvia officinalis ) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep -menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli . The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes. Common sage (Salvia officinalis ) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep -menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli . The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes. The cyclization of the universal precursor geranyl diphosphate to form monocyclic and bicyclic monoterpenes is catalyzed by a group of enzymes termed monoterpene synthases (or cyclases). The biochemical transformation of geranyl diphosphate to cyclic products has been investigated using enzymes from a variety of plants, including both angiosperms (1Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (465) Google Scholar) and gymnosperms (2Lewinsohn E. Gijzen M. Croteau R. Arch. Biochem. Biophys. 1992; 293: 167-173Crossref PubMed Scopus (59) Google Scholar, 3Savage T.J. Hatch M.W. Croteau R. J. Biol. Chem. 1994; 269: 4012-4020Abstract Full Text PDF PubMed Google Scholar, 4Savage T.J. Ichii H. Hume S.D. Little D.B. Croteau R. Arch. Biochem. Biophys. 1995; 320: 257-265Crossref PubMed Scopus (35) Google Scholar), and a mechanistic paradigm for these transformations (Scheme FS1) is well established (1Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (465) Google Scholar, 5Wise M.L. Croteau R. Cane D.E. Comprehensive Natural Products Chemistry: Isoprenoids. 2. Elsevier Science, Oxford, UK1998Google Scholar). Thus, geranyl diphosphate is initially ionized and isomerized to form either (3R )- or (3S )-linalyl diphosphate, depending on the particular enzyme. This step permits rotation about the C2–C3 single bond of the bound allylic isomer to the cisoid conformer which, upon subsequent ionization, promotes electrophilic attack by C1 on the C6–C7 double bond, resulting in the formation of the α-terpinyl cation as a central intermediate. Further transformations of this reactive intermediate may be effected by additional intramolecular electrophilic additions, hydride shifts, or other rearrangements before termination of the sequence by deprotonation of the final cation or capture by an external nucleophile, such as a hydroxyl ion or the diphosphate group. Although the fate of the substrate has been well characterized in numerous monoterpene cyclization reactions, the molecular mechanisms by which the enzymes effect these transformations is still poorly understood. Culinary sage (Salvia officinalis ) produces a number of monoterpenes, including (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, (+)- and (−)-camphene, (+)-sabinene, (+)- and (−)-limonene, myrcene, 1,8-cineole, and (+)-bornyl diphosphate (SchemeFS1) (1Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (465) Google Scholar). Because sage produces this broad range of acyclic, monocyclic, and bicyclic monoterpenes, including several olefin isomers, a cyclic ether and a diphosphate ester, this plant has provided an ideal system for the study of a variety of synthases, all of which utilize the same substrate but produce different products by variations on a single reaction mechanism (1Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (465) Google Scholar, 5Wise M.L. Croteau R. Cane D.E. Comprehensive Natural Products Chemistry: Isoprenoids. 2. Elsevier Science, Oxford, UK1998Google Scholar). These include (+)-bornyl diphosphate synthase (the enzyme producing the precursor of (+)-camphor) (6Croteau R. Karp F. Arch. Biochem. Biophys. 1979; 198: 512-522Crossref PubMed Scopus (83) Google Scholar, 7Croteau R. Karp F. Arch. Biochem. Biophys. 1979; 198: 523-532Crossref PubMed Scopus (51) Google Scholar), 1,8-cineole synthase (8Croteau R. Alonso W.R. Koepp A.E. Johnson M.A. Arch. Biochem. Biophys. 1994; 309: 184-192Crossref PubMed Scopus (89) Google Scholar), (+)-sabinene synthase (the enzyme producing the precursor of (−)-3-isothujone) (9Croteau R. Hopp R. Mori K. Recent Developments in Flavor and Fragrance Chemistry. VCH, Weinheim, Germany1992: 263-273Google Scholar, 10Croteau R. Am. Chem. Soc. Symp. Ser. 1992; 490: 8-20Google Scholar), and several pinene synthases (11Gambliel H. Croteau R. J. Biol. Chem. 1982; 257: 2335-2342Abstract Full Text PDF PubMed Google Scholar, 12Gambliel H. Croteau R. J. Biol. Chem. 1984; 259: 740-748Abstract Full Text PDF PubMed Google Scholar, 13Wagschal K.C. Pyun H.-J. Coates R.M. Croteau R. Arch. Biochem. Biophys. 1994; 308: 477-487Crossref PubMed Scopus (28) Google Scholar, 14Pyun H.-J. Wagschal K.C. Jung D.-I. Coates R.M. Croteau R. Arch. Biochem. Biophys. 1994; 308: 488-496Crossref PubMed Scopus (13) Google Scholar). As is typical of monoterpene cyclases (5Wise M.L. Croteau R. Cane D.E. Comprehensive Natural Products Chemistry: Isoprenoids. 2. Elsevier Science, Oxford, UK1998Google Scholar, 15Wagschal K. Savage T.J. Croteau R. Tetrahedron. 1991; 47: 5933-5944Crossref Scopus (55) Google Scholar), many of these enzymes from sage appear to generate multiple products from geranyl diphosphate. Investigations with the partially purified native enzymes have suggested that a single enzyme, termed (+)-pinene synthase (cyclase I), is responsible for the synthesis of both (+)-α-pinene and (+)-camphene, with lesser amounts of (+)-limonene and myrcene, whereas a second enzyme, (−)-pinene synthase (cyclase II), has been shown to produce (−)-α-pinene, (−)-β-pinene, and (−)-camphene, with minor amounts of (−)-limonene, terpinolene, and myrcene (11Gambliel H. Croteau R. J. Biol. Chem. 1982; 257: 2335-2342Abstract Full Text PDF PubMed Google Scholar, 12Gambliel H. Croteau R. J. Biol. Chem. 1984; 259: 740-748Abstract Full Text PDF PubMed Google Scholar). More recently, a third synthase from sage, termed cyclase III, has been described which produces a mixture of (+)-α-pinene and (+)-β-pinene, along with minor amounts of myrcene (13Wagschal K.C. Pyun H.-J. Coates R.M. Croteau R. Arch. Biochem. Biophys. 1994; 308: 477-487Crossref PubMed Scopus (28) Google Scholar, 14Pyun H.-J. Wagschal K.C. Jung D.-I. Coates R.M. Croteau R. Arch. Biochem. Biophys. 1994; 308: 488-496Crossref PubMed Scopus (13) Google Scholar). Evidence that these reactions are catalyzed by individual multifunctional enzymes is provided by co-purification and differential inhibition studies (12Gambliel H. Croteau R. J. Biol. Chem. 1984; 259: 740-748Abstract Full Text PDF PubMed Google Scholar), as well as by isotopically sensitive branching experiments (13Wagschal K.C. Pyun H.-J. Coates R.M. Croteau R. Arch. Biochem. Biophys. 1994; 308: 477-487Crossref PubMed Scopus (28) Google Scholar, 15Wagschal K. Savage T.J. Croteau R. Tetrahedron. 1991; 47: 5933-5944Crossref Scopus (55) Google Scholar, 16Croteau R.B. Wheeler C.J. Cane D.E. Ebert R. Ha H.J. Biochemistry. 1987; 26: 5383-5389Crossref PubMed Scopus (60) Google Scholar). Despite considerable effort, the (+)-pinene synthase has never been chromatographically separated from the aforementioned (+)-bornyl diphosphate synthase (17McGeady P. Croteau R. Arch. Biochem. Biophys. 1995; 317: 149-155Crossref PubMed Scopus (16) Google Scholar), nor has the (−)-pinene synthase been fully resolved from 1,8-cineole synthase, although stereochemical considerations indicate that the latter two are probably distinct enzyme species (8Croteau R. Alonso W.R. Koepp A.E. Johnson M.A. Arch. Biochem. Biophys. 1994; 309: 184-192Crossref PubMed Scopus (89) Google Scholar, 18Croteau R. Satterwhite D.M. Wheeler C.J. Felton N.M. J. Biol. Chem. 1989; 264: 2075-2080Abstract Full Text PDF PubMed Google Scholar). In this report, we describe the homology-based cloning, and subsequent sequencing and heterologous expression, of three monoterpene synthase cDNA genes from sage, the recombinant enzymes from which produce three different major types of cyclic monoterpene products, (+)-sabinene (a bicyclic olefin), 1,8-cineole (a bicyclic ether), and (+)-bornyl diphosphate (a bicyclic diphosphate ester), respectively (Scheme FS1). Comparison of the sizes, subunit architectures, and product distributions of these multiple-product enzymes clarifies the assignment of specific catalytic functions to defined monoterpene synthases, and the deduced sequences provide information on the relatedness of these enzymes within the species and to other terpenoid synthases of plant origin. Additionally, comparison of the primary structures of these mechanistically different monoterpene synthases from the same plant, the first examples of this type thus far available, allows preliminary assessment of active site interactions and provides the foundation for more detailed study of structure-function relationships. Sage plants (S. officinalis L.) were grown from seed as described previously (19Croteau R. Karp F. Arch. Biochem. Biophys. 1976; 176: 734-746Crossref PubMed Scopus (79) Google Scholar). [1-3H]Geranyl diphosphate (250 Ci/mol) was prepared by an established method (8Croteau R. Alonso W.R. Koepp A.E. Johnson M.A. Arch. Biochem. Biophys. 1994; 309: 184-192Crossref PubMed Scopus (89) Google Scholar). Terpenoid standards were from our own collection. Unless otherwise stated, all reagents were obtained from Sigma or Aldrich Chemical Co. DNA sequences were assembled and analyzed using GCG software (20Genetics Computer Group Wisconsin Package version 9.0. Genetics Computer Group (GCG), Madison, WI1996Google Scholar). Approximately 15 g of emerging sage leaves (shoot tips) from 3-week-old plants were ground to a fine powder in liquid nitrogen and extracted into buffer composed of 200 mm Tris-HCl (pH 8.5), 300 mm LiCl, 5 mm thiourea, 1 mm aurintricarboxylic acid, 10 mm dithiothreitol, and 10 mm EDTA, and containing 1% (w/v) polyvinylpyrrolidone (M r∼40,000). Total RNA thus extracted was prepared by precipitation with isopropyl alcohol, followed by CsCl density gradient centrifugation, as described previously (21Lewinsohn E. Steele C.L. Croteau R. Plant Mol. Biol. Rep. 1994; 12: 20-25Crossref Scopus (62) Google Scholar). Poly(A)+ mRNA was isolated by chromatography on oligo(dT)-cellulose (Qiagen) and 6.3 μg of the resulting mRNA was used to construct a λZAPII cDNA library according to the manufacturer's instructions (Stratagene). A general strategy for the homology-based PCR cloning of terpenoid synthases of higher plant origin has been suggested (22Steele C.L. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (62) Google Scholar) based upon a comparison of the cDNA sequences of a monoterpene synthase (23Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar), a sesquiterpene synthase (24Facchini P.J. Chappell J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11088-11092Crossref PubMed Scopus (212) Google Scholar), and a diterpene synthase (25Mau C.J.D. West C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8497-8501Crossref PubMed Scopus (112) Google Scholar) of angiosperm origin. Thus, three regions of deduced amino acid sequence (corresponding to residues 180–188, 197–203, and 380–387 of limonene synthase from spearmint (23Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar)) were employed to design primers corresponding to 1F, 5′-A(G/A)(G/A)A(C/T)GA(G/A)(G/A)AIGGI(G/A)A(G/A)TA(C/T)AA(G/A)GA-3′; 2F, 5′-ATG(T/C)TICA(G/A)(C/T)TITA(T/C)GA(G/A)GC-3′; and 3R 5′-CTI(G/T)(C/T)I(G/A)AIGGICT(G/A)AT(G/A)TAC(G/T)T(C/T)-3′. Using purified sage leaf cDNA library phage as template (5 μl at 1.5 × 109 plaque-forming units/ml), PCR was performed under a wide range of amplification conditions (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar, 27Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. Academic Press, San Diego, CA1990: 3-12Google Scholar) in a total volume of 50 μl containing 10 mm Tris-HCl (pH 9.0), 50 mm KCl, 2.5 mm MgCl2, 200 μm of each dNTP, 0.5 μm of each primer, and 2.5 units of Taq polymerase (Life Technologies, Inc.). Analysis of the PCR reaction products by agarose gel electrophoresis (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar) indicated that only the combination of primers 2F and 3R amplified a discrete product of approximately 600 bp, which was ligated into pT7Blue (Novagen), and transformed into Escherichia coli NovaBlue cells. Plasmid DNA was prepared from 32 individual transformants; seven of these had inserts of the predicted size (∼600 bp). These inserts were partially sequenced (DyeDeoxy Terminator Cycle Sequencing; Applied Biosystems) to reveal two distinct “terpenoid synthase-like” sequences. The relative ability of these two potential probes to hybridize with expressed genes was evaluated by DNA-RNA hybridization. Two samples of sage leaf mRNA isolated as above (3 μg each) were electrophoresed on 1% (w/v) agarose under denaturing conditions and blotted onto separate polyvinylidene difluoride membranes using standard techniques (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar). Each membrane was evaluated with 32P-labeled probe, generated from one or the other of the 600-bp fragments using random hexamer priming (28Tabor S. Struhl K. Scharf S.J. Gelfand D.H. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1991: 3.5.9-3.5.10Google Scholar), by standard hybridization and washing protocols (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar). Autoradiography of the membrane revealed that both probes hybridized to a 2-kilobase transcript, although one probe generated a significantly stronger signal (∼10-fold) than the other. This probe was subsequently employed to screen the cDNA library in an attempt to isolate full-length cDNA sequences encoding the corresponding presumptive terpene synthase. UV cross-linked nitrocellulose lifts containing 3–5 × 104 primary plaques (plated onE. coli XL1-Blue-MRF′), after pre-hybridization (in 1.25 × SSPE, 0.5 × Denhart's reagent, 9% formamide, 0.002% SDS, and 10 μg/ml denatured E. coli DNA, for 2 h at 42 °C), were hybridized in the same medium with approximately 8 μCi of the 32P-labeled probe for 48 h. Filters were washed, first at room temperature (in 2 × SSC with 0.1% SDS) then at 55 °C (in 1 × SSC with 0.1% SDS), and subsequently exposed to x-ray film at −70 °C (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar). Plaques yielding positive signals were purified through two additional rounds of hybridization. A total of 77 purified λZAP clones so isolated were excised in vivo to generate BluescriptII SK(−) phagemids and transformed into E. coli SOLR cells according to the Stratagene protocol. The size of each cDNA insert was determined by PCR using T3 and T7 promoter primers, and transformed clones containing an insert >1.6 kilobases were either expressed to assay for monoterpene synthase activity or sequenced at the 5′ terminus using the T3 promoter primer. Bluescript plasmids expressing monoterpene synthase activity in cell-free extracts of transformed E. coli (see below) were fully sequenced on both DNA strands by primer walking or by the method of nested deletions using exonuclease III and mung bean nuclease (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar). To improve the efficiency of functional expression and facilitate subsequent enzyme purification, each of the apparent full-length pBluescript clones that expressed monoterpene synthase activity was subcloned, in-frame, into pGEX vectors (Pharmacia) using a convenientBam HI (SBS and SSS) or Eco RI (SCS) restriction site at the 5′-end, and the Xho I restriction site at the 3′ terminus. Fidelity in subcloning was confirmed by complete sequencing, and these plasmid constructs were expressed in E. coli XL1-Blue-MFR′ cells. The Bluescript plasmids expressed in E. coli strain XL1-Blue were grown in 5 ml of LB medium (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 2.69-2.76Google Scholar), supplemented with 100 μg of ampicillin/ml, to anA 600 = 0.5 at 37 °C with constant shaking, then induced with 1 to 3 mmisopropyl-1-thio-β-d-galactopyranoside. The cells were allowed an additional 4 h growth at 37 °C before harvesting by centrifugation (2000 × g , 10 min) and lysis by sonication (Braun-Sonic 2000 with microprobe at maximum power for 15 s), on ice, in 50 mm Mopso buffer containing 10% glycerol, 10 mm MgCl2, and 5 mmdithiothreitol (either pH 6.5 or 7.1, as appropriate for the assays described below). The sonicates were cleared by centrifugation (18,000 × g , 10 min) and the resulting supernatant was used as the enzyme source. The pGEX constructs in E. coli XL1-Blue-MFR′ cells were similarly grown at 37 °C toA 600 = 1.0 to 1.5, then induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside and incubated overnight at 20 °C with constant shaking. The cells were then harvested and lysed, and the soluble supernatant prepared as before. Purification of the resulting fusion proteins was attempted using the glutathione-Sepharose affinity column according to the manufacturer's instructions (Pharmacia). Of the three expressed monoterpene synthases (see below), only one (SBS) bound to the matrix but, even in this case, affinity-based purification proved to be unreliable. Therefore, partial purification of the heterologously expressed synthases was achieved by ion-exchange chromatography onO -diethylaminoethyl-cellulose (Whatman DE-52) using a 0–400 mm NaCl gradient. The partially purified preparations were desalted by repeated ultrafiltration and dilution using an Amicon Centriprep 30 concentrator (30 kDa cutoff) and the appropriate assay buffer. The pGEX-expressed fusion proteins were also subjected to gel permeation chromatography (Pharmacia FPLC system) using a Pharmacia XY 16 × 70 column packed with Superdex S-200 and equilibrated with the appropriate 50 mm Mopso buffer system. The column was developed at a flow rate of 0.3 ml/min and calibrated using the Sigma MW-GF-200 molecular weight marker kit. K avvalues of the recombinant enzymes were compared with the calibration standards to establish molecular weights (29Cooper T.G. The Tools of Biochemistry. John Wiley & Sons, New York1977: 169-193Google Scholar), which were then corrected for the engineered fusion and transit peptide to estimate the molecular weight of the corresponding native form. Monoterpene synthase activities were assayed by methods previously described (6Croteau R. Karp F. Arch. Biochem. Biophys. 1979; 198: 512-522Crossref PubMed Scopus (83) Google Scholar, 8Croteau R. Alonso W.R. Koepp A.E. Johnson M.A. Arch. Biochem. Biophys. 1994; 309: 184-192Crossref PubMed Scopus (89) Google Scholar, 12Gambliel H. Croteau R. J. Biol. Chem. 1984; 259: 740-748Abstract Full Text PDF PubMed Google Scholar, 30Croteau R. Cane D.E. Methods Enzymol. 1985; 110: 383-405Crossref Google Scholar). Briefly, an aliquot of the bacterial cell lysate, appropriate column fractions, or partially purified and desalted enzyme preparation, in 0.5 or 1.0 ml of 50 mm Mopso buffer (pH 6.1 for SBS, and 7.1 for SCS and SSS) containing 10 mm MgCl2, 5 mmdithiothreitol, and 10% (v/v) glycerol, was transferred to a 7-ml glass Teflon sealed, screw-capped tube, and the mixture was overlaid with 1 ml of pentane to trap volatile products. The reaction was initiated by the addition of 4.5 μm[1-3H]geranyl diphosphate (1.3 μCi), with incubation at 31 °C with gentle shaking for 0.5 to 3.0 h. The pentane layer and an additional pentane extract (2 × 1 ml) were passed over a short column of silica gel surmounted by anhydrous MgSO4(in a Pasteur pipette) to afford the monoterpene olefin fraction. Subsequent extraction of the remaining aqueous phase with diethyl either (2 × 1 ml), and passage of this extract through the same column, yielded the oxygenated monoterpene fraction. The residual aqueous phase was then treated with excess potato apyrase and wheat germ acid phosphatase to hydrolyze monoterpenol diphosphate esters (6Croteau R. Karp F. Arch. Biochem. Biophys. 1979; 198: 512-522Crossref PubMed Scopus (83) Google Scholar,30Croteau R. Cane D.E. Methods Enzymol. 1985; 110: 383-405Crossref Google Scholar). The liberated alcohols were then extracted into diethyl ether (2 × 1 ml) and the combined extract dried over anhydrous MgSO4. Radioactivity in the various fractions was determined by liquid scintillation counting of aliquots (Packard 460 CD with external standard quench correction) and the remaining material was concentrated for radio-GC and GC-MS analysis. Kinetic analyses were carried out with the partially purified, recombinant pGEX fusion proteins by determination of initial reaction rates at a minimum of 10 substrate concentrations ranging from 0.45 to 45 μm [1-3H]geranyl diphosphate, at saturating levels of the divalent metal ion cofactor. The results were analyzed by non-linear regression of the Michaelis-Menten equation using the curve-fitting capabilities of Sigma-Plot (Jandel Corp.). Radio-GC was performed on a Gow-Mac 550P gas chromatograph with thermal conductivity detector directly coupled to a Nuclear-Chicago 8731 gas proportional counter (31Satterwhite D.M. Croteau R. J. Chromatogr. 1988; 454: 61-73Crossref Scopus (28) Google Scholar). An AT-1000 packed column (Alltech) was used with helium as carrier at 30 ml/min and with temperature programming from 70 to 200 °C (at 5 °C/min) for analysis of monoterpene olefins, and from 100 to 200 °C (at 5 °C/min) for analysis of oxygenated monoterpenes. Authentic standards (10–20 μg/component) were included with each injection in order to correlate the retention times determined by mass and radioactivity detectors. GC-MS was performed on a Hewlett-Packard 6890 GC-quadrupole mass selective detector system interfaced with a Hewlett-Packard Chemstation for data analysis. An Alltech AT-1000 fused silica capillary column (30 m × 0.25 mm inner diameter) was employed. Inleting was done by cool, on-column injection at 40 °C, with oven programming from 40 °C (50 °C/min) to 50 °C (5 min hold) then to 160 °C (10 °C/min), under a constant flow of 0.7 ml of helium/min. Full spectra were recorded for major reaction products which were identified by comparison of retention times to authentic standards and by comparison of spectra to those of the NSB75K library using the G1033A NIST probability based matching algorithm. Chiral phase separations were performed on a Hewlett-Packard 5890 GC by split injection (80:1) on a 30-m cyclodex-B capillary column (J & W Scientific) using H2 as carrier at 0.6 ml/min and temperature programming from 70 to 200 °C at 10 °C/min with flame ionization detection. Compound identification was based on retention time identity with the authentic standard. Because S .officinalis (culinary or common sage) produces such a broad structural variety of monoterpenes, this species has been utilized extensively for studies on the enzymology, stereochemistry, and mechanism of monoterpene cyclization reactions (1Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (465) Google Scholar, 5Wise M.L. Croteau R. Cane D.E. Comprehensive Natural Products Chemistry: Isoprenoids. 2. Elsevier Science, Oxford, UK1998Google Scholar). Structural analyses of the responsible monoterpene synthases, and more detailed study of the cyclization mechanisms, require the isolation of cDNA species encoding these target enzymes. Protein purification from sage, as the basis for cDNA isolation, has been of limited success (17McGeady P. Croteau R. Arch. Biochem. Biophys. 1995; 317: 149-155Crossref PubMed Scopus (16) Google Scholar) because of the number of synthases present and their similarity in physical properties (32Alonso W.R. Croteau R. Methods Plant Biochem. 1993; 9: 239-260Google Scholar), and thus far has not permitted a reverse genetic approach to cloning of any of the monoterpene synthases from this species. As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was recently proposed (22Steele C.L. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (62) Google Scholar) that was developed by comparison of the deduced amino acid sequences of cDNAs encoding a monoterpene synthase (23Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar), a sesquiterpene synthase (24Facchini P.J. Chappell J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11088-11092Crossref PubMed Scopus (212) Google Scholar), and a diterpene synthase (25Mau C.J.D. West C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8497-8501Crossref PubMed Scopus (112) Google Scholar) of phylogenetically distant angiosperm species. Three conserved regions of sequence were identified that appeared to be useful for the design of degenerate PCR primers. Two of these primers ultimately amplified a 600-bp fragment using cDNA from a sage leaf library as template. Cloning and sequencing showed the amplified products to correspond to two distinct sequence groups, both of which showed similarity to sequences of cloned terpene synthases, but only one of which hybridized strongly to a 2-kilobase target upon Northern blot analysis of sage leaf mRNA. This more efficient probe was utilized to screen the sage leaf cDNA library, from which 77 positive phagemids were purified. Size selection of the purified and in vivo excised clones yielded a subset of 44 with insert size greater than 1.6 kilobases, and these were expressed in E. coli XL1-Blue cells and the resulting extracts were assayed for functional monoterpene synthase activity by monitoring the conversion of [1-3H]geranyl diphosphate to monoterpene olefins, oxygenated monoterpenes, and monoterpenyl diphosphate esters. Nine functionally active clones were identified by this means, four types of which s