Interaction with the Small Subunit of Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Geranylgeranyl Diphosphate Synthase to Produce Geranyl Diphosphate (2002)

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C5 units, of the respective C10, C15, and C20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56–75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase. Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C5 units, of the respective C10, C15, and C20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56–75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase. geranyl diphosphate farnesyl diphosphate geranylgeranyl diphosphate isopentenyl diphosphate dimethylallyl diphosphate large subunit small subunit geranyl diphosphate synthase farnesyl diphosphate synthase geranylgeranyl diphosphate synthase dithiothreitol 3-(N-morpholino)-2-hydroxypropanesulfonic acid A subgroup of isoprenyl diphosphate synthases, referred to as the "short-chain prenyltransferases," consists of geranyl diphosphate (GPP1; C10) synthase, farnesyl diphosphate (FPP; C15) synthase, and geranylgeranyl diphosphate (GGPP; C20) synthase. These enzymes provide the acyclic branch point intermediates for the biosynthesis of numerous terpenoids, including monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes such as natural rubber. GGPP synthase and FPP synthase occur nearly ubiquitously in plants, animals, and bacteria (1Ogura K. Koyama T. Chem. Rev. 1998; 98: 1263-1276Crossref PubMed Scopus (291) Google Scholar). GPP synthase appears to be of much more limited distribution in nature, having been identified most frequently in essential oil (monoterpene)-producing plants (2Wise M.L. Croteau R. Cane D.E. Comprehensive Natural Products Chemistry: Isoprenoids Including Carotenoids and Steroids. 2. Elsevier Science Ltd., Oxford, UK1999: 97-153Google Scholar). Most isoprenyl diphosphate synthases, including the short-chain prenyltransferases, catalyze the divalent metal ion-dependent 1′-4 condensation of isopentenyl diphosphate (IPP) with an allylic prenyl diphosphate cosubstrate (3Poulter C.D. Rilling H.C. Porter J.W. Spurgeon S.L. Biosynthesis of Isoprenoid Compounds. 1. John Wiley & Sons, Inc., New York1981: 162-220Google Scholar), and they are distinguished by the specific chain length and double bond geometry at C2–C3 of the prenyl diphosphate product generated (Fig. 1). Thus, GPP synthase catalyzes a single condensation of IPP with dimethylallyl diphosphate (DMAPP) to form, specifically, GPP (C10). FPP synthase and GGPP synthase catalyze sequential condensations of IPP with an allylic primer (i.e. DMAPP, GPP, or FPP, as appropriate) to form the respective C15 and C20elongation products. Reaction parameters, such as substrate concentration (4Ohnuma S. Koyama T. Ogura K. J. Biochem. (Tokyo). 1992; 112: 743-749Crossref PubMed Scopus (33) Google Scholar, 5Ohnuma S. Hemmi H. Ohto C. Nakane H. Nishino T. J. Biochem. (Tokyo). 1997; 121: 696-704Crossref PubMed Scopus (13) Google Scholar) and metal ion cofactor (6Ohnuma S. Koyama T. Ogura K. Biochem. Biophys. Res. Commun. 1993; 192: 407-412Crossref PubMed Scopus (18) Google Scholar), are known to modify chain length specificity of some prenyltransferases. Evaluation of the crystal structure of homodimeric avian FPP synthase (7Tarshis L.C. Yan M. Poulter C.D. Sacchettini J.C. Biochemistry. 1994; 33: 10871-10877Crossref PubMed Scopus (372) Google Scholar), coupled with prenyltransferase sequence alignment data, has led to the directed mutagenesis of FPP synthase to alter the contour of the active site, thereby generating mutant enzymes that synthesize GGPP (8Tarshis L.C. Proteau P.J. Kellogg B.A. Sacchettini J.C. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15018-15023Crossref PubMed Scopus (315) Google Scholar) or GPP (9Stanley Fernandez S.M. Kellogg B.A. Poulter C.D. Biochemistry. 2000; 39: 15316-15321Crossref PubMed Scopus (60) Google Scholar,10Narita K. Ohnuma S. Nishino T. J. Biochem. (Tokyo). 1999; 126: 556-571Crossref Scopus (35) Google Scholar). A random chemical mutagenesis approach has also provided an altered FPP synthase capable of producing GGPP (11Ohnuma S. Nakazawa T. Hemmi H. Hallberg A.-M. Koyama T. Ogura K. Nishino T. J. Biol. Chem. 1996; 271: 10087-10095Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and has yielded homodimeric GGPP synthase mutants capable of producing FPP (5Ohnuma S. Hemmi H. Ohto C. Nakane H. Nishino T. J. Biochem. (Tokyo). 1997; 121: 696-704Crossref PubMed Scopus (13) Google Scholar) and polyprenols greater than C20 (12Ohnuma S. Hirooka K. Hemmi H. Ishida C. Ohto C. Nishino T. J. Biol. Chem. 1996; 271: 18831-18837Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This work eventually led to the direct mutagenesis of an archaeal GGPP synthase so as to produce FPP by the modified enzyme (13Ohnuma S. Hirooka K. Ohto C. Nishino T. J. Biol. Chem. 1997; 272: 5192-5198Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These experiments clearly indicate that amino acid substitutions to alter the contour of the active site may either restrict or allow sequential elongation of the polyprenyl chain. cDNAs encoding the large and small subunits of GPP synthase were isolated from a peppermint (Mentha piperita) oil gland library and were confirmed by functional coexpression of the heteromeric enzyme (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar). Thus, GPP synthase, in which both subunits are absolutely required for prenyltransferase activity, is unlike both FPP synthases and GGPP synthases that are functional homodimers (1Ogura K. Koyama T. Chem. Rev. 1998; 98: 1263-1276Crossref PubMed Scopus (291) Google Scholar). The GPP synthase large subunit (GPPS.lsu) shows a high level of deduced amino acid sequence identity (56–75%) with GGPP synthases of plant origin and a lower level of deduced amino acid identity (21–37%) with FPP synthases of plant origin. The large subunit sequence also contains two highly conserved aspartate-rich motifs found in other prenyltransferases (Fig. 2). Numerous lines of evidence have indicated these aspartate-rich clusters to be involved in substrate binding and product determination (15Marrero P.F. Poulter C.D. Edwards P.A. J. Biol. Chem. 1992; 267: 21873-21878Abstract Full Text PDF PubMed Google Scholar, 16Song L. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3044-3048Crossref PubMed Scopus (176) Google Scholar) by forming a salt bridge to the divalent metal ions that are coordinated to the diphosphate group of the reacting ester (8Tarshis L.C. Proteau P.J. Kellogg B.A. Sacchettini J.C. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15018-15023Crossref PubMed Scopus (315) Google Scholar). These aspartate-rich domains are noticeably absent in the GPP synthase small subunit (GPPS.ssu), which shares only 24–29% deduced amino acid sequence identity with plant GGPP synthases and shows no significant homology with FPP synthases. Because the small subunit lacks the aspartate-rich functional motifs and is alone inactive, it was hypothesized that the small subunit may bind to and modify the otherwise inactive large subunit to promote transfer catalysis while restricting chain length elongation specifically to the C10product. If the GPPS.ssu plays such a role, then it also seemed possible that the small subunit might be capable of influencing the chain length specificity of other types of prenyltransferases. To test this possibility, a His8-tagged version of the small subunit (GPPS.ssu.his) was coexpressed in Escherichia coliwith FPP synthase or GGPP synthase to permit affinity-based purification of any resulting chimeric species. Product analysis and kinetic evaluation of the resulting hybrid heterodimers, and comparison to the parent homodimeric transferases, demonstrated that the GPPS small subunit modifies the specificity of GGPP synthase by promoting the kinetically competent production of C10 chains. [4-14C]IPP (54 Ci/mol) was purchased from PerkinElmer Life Sciences. Unlabeled IPP, DMAPP, GPP, and FPP were purchased from Echelon Research Laboratories (Salt Lake City, UT). Authentic terpenol standards were from our own collection. Restriction enzymes were purchased from New England Biolabs. T4 ligase, PfuTurbo DNA polymerase, and CodonPlusE. coli competent cells were purchased from Stratagene. Synthesis of oligonucleotide primers was performed by Invitrogen. pET vectors were from Novagen. Alkaline phosphatase, apyrase, and protein molecular weight standards were purchased from Sigma. Standard molecular biology protocols were followed (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Protein concentrations were determined by UV absorbance using molecular weights and extinction coefficients calculated from the PeptideSort program (18Genetics Computer Group Program Manual for the Wisconsin Package, Version 10.0. Genetics Computer Group (GCG), Madison, WI1998Google Scholar). Proteins were analyzed by SDS-PAGE (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar), followed by staining with Coomassie Brilliant Blue R-250 (20Weber K. Pringle J.R. Osborn M. Methods Enzymol. 1972; 26: 3-27Crossref PubMed Scopus (1569) Google Scholar). Antibody preparation and immunoblotting protocols were described earlier (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar), as were radio-gas chromatography methods (21Tholl D. Croteau R. Gershenzon J. Arch. Biochem. Biophys. 2001; 386: 233-242Crossref PubMed Scopus (47) Google Scholar). A truncated version of the GPP synthase small subunit (designated GPPS.ssu), in which the plastidial transit peptide was deleted, was prepared from the original pSBET13.18 cDNA clone (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) for transfer into pET-37b using forward primer (5′-CC GCC GCC CAT ATG CAG CCG-3′) to create an NdeI site and code for a starting methionine (in place of amino acid Ser-48 of the original protein) and reverse primer (5′-G GAT CCG AAT AAG CTT CTA AGC CG-3′) to generate a HindIII restriction site downstream of the stop codon. The resulting amplicon was digested with NdeI and HindIII, gel-purified, and directionally ligated into theNdeI/HindIII-digested pET-37b vector to yield pETGPPS.ssu. Mutation of the stop codon, to permit translation through the carboxyl-terminal His8-tag provided on the pET-37b vector, was accomplished by PCR amplification using pETGPPS.ssu as template and forward primer (5′-GGA GAT ATA CAT ATG CAG CCG-3′) and reverse primer (5′-CC CGC AAG CTT CCC AGC CGC G-3′), thereby resulting in a lysine substitution for the stop codon six residues from the His8-tag. The resulting amplicon (designated GPPS.ssu.his) was digested and gel-purified as before and directionally ligated into pET-37b that had been digested with NdeI andHindIII. A similarly truncated version of the GPP synthase large subunit was prepared from the original pMp23.10 cDNA clone (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) using forward primer (5′-GCG CCT TCG ACT TCG CAT ATG TTC GAT TTC GAC GG-3′) to create an NdeI site and code for a starting methionine (in place of amino acid Ala-83 of the original protein) and reverse primer (5′-CAC TAT AGG GCG AAT TGG GAT CCG GGC CCC CCC TCG AG-3′) to convert the downstream KpnI site (GGTACC) in the original pBluescript SK− version (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) into a BamHI site (GGATCC). The amplified sequence (designated GPPS.lsu) was digested withNdeI and BamHI, and the gel-purified fragment was ligated into pET-32a that had been similarly digested to yield pETGPPS.lsu. A truncated version of the Taxus canadensis GGPP synthase, from which the amino-terminal plastid targeting peptide was also deleted, was prepared from the original full-length cDNA clone (22Hefner J. Ketchum R.E.B. Croteau R. Arch. Biochem. Biophys. 1998; 360: 62-74Crossref PubMed Scopus (136) Google Scholar) using forward primer (5′-CC CGA AGA CAT ATG TTT GAT TTC AAC G-3′), to install an NdeI site and thus mutate Glu-98 to Met-1, and reverse primer (5′-CTA GCC CGG TCG ACC TCA GTT TTG CCT GAA TGC-3′), to create a SalI restriction site downstream of the stop codon. The amplified sequence (designated GGPPS) was digested withNdeI and SalI, gel-purified, and ligated into the pET-32c vector that had been identically digested to yield pETGGPPS. A truncated version of the Abies grandis GGPP synthase was similarly prepared. An FPP synthase cDNA clone was prepared from a full-length sequence located in a peppermint (M. piperita) oil gland expressed sequence tag library (23Lange B.M. Wildung R.M. Stauber J.E. Sanchez C. Pouchnik D. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 97: 2934-2939Crossref Scopus (276) Google Scholar) using primer (5′-GGG TGA TTA CAT ATG GCG AAT C-3′) to create an NdeI restriction site at the starting methionine and reverse primer (5′-GTA TTT CAA AGC TCG AGT TTA TTT CTG C-3′) to create an XhoI site downstream of the stop codon. The resulting amplicon was digested with NdeI andXhoI, gel-purified, and ligated into similarly digested pET-32b to yield pETFPPS. Cotransformations with two plasmids (e.g. both GPP synthase subunits and pETGPPS.ssu.his with pETFPPS or pETGGPPS) were performed in a single transformation event. Positive transformants were screened for multiple resistance with kanamycin (pETGPPS.ssu and pETGPPS.ssu.his), carbenicillin (pETGPPS.lsu, pETGGPPS, and pETFPPS), and chloramphenicol (conferred by the pACYC-based plasmid of the BL21-Codon Plus host cells that also contains extra copies of the argU, ileY, andleuW tRNA genes). Transformants were initially grown in 5 ml of Luria-Bertani medium and then transferred to 1 liter of the same medium and grown at 20 °C until A600 reached 0.6. The temperature of the culture was then lowered to 15 °C prior to induction with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside, and incubation was continued for an additional 16 h. Following induction and incubation, the transformed cells were pelleted by centrifugation (20 min at 2500 × g) and resuspended in either 20 ml of buffer A (see buffer description below) or in 10 ml of His-tagged lysis buffer (50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, 1 mm dithiothreitol (DTT), 1 mm benzamidine, and 10 mm histidine). The suspended cells were disrupted by brief sonication (VirSonic, 100% power, two 30-s bursts at 4 °C), and the homogenate was centrifuged at 12,000 × g (30 min) to pellet debris and then at 195,000 × g (1.5 h) to provide the soluble enzyme fraction that was filtered through a cellulose acetate membrane (Nalgene, 0.2 μm). Crude recombinant GPP synthase was purified by fast protein liquid chromatography (Amersham Biosciences) using an HR 10/10 column (Amersham Biosciences) containing Poros PI anion-exchange medium (PerSeptive Biosystems) that was previously equilibrated with buffer A (25 mm Mopso (pH 7.2), 10% (v/v) glycerol, 1 mm DTT and 1 mm benzamidine) and eluted with a 30-ml linear gradient (0–50%) with buffer B (buffer A containing 2m NaCl). Recombinant GPP synthase eluted at 200–300 mm NaCl, and the pooled fractions were adjusted to 2m NaCl and applied to an HR 10/10 column containing phenyl-Sepharose (Amersham Biosciences) previously equilibrated with buffer B and eluted with a 30-ml linear gradient (0–100%) to buffer A. GPP synthase eluted in fractions containing less than 500 mm NaCl, and the combined material was desalted on Econo-Pac10DG Columns (Bio-Rad) and applied to an HR 5/5 column containing a strong anion-exchange matrix (Source 15, Amersham Biosciences) that was previously equilibrated with buffer A. A 30-ml linear gradient (0–50% buffer B) eluted recombinant GPP synthase at 250–270 mm NaCl to provide the protein at >90% purity as determined by SDS-PAGE. Recombinant GPPS.ssu, GGPP synthase, and FPP synthase were similarly purified. For metal ion-affinity chromatography of the His8-tag recombinant proteins, the initial extracts (10 ml; ∼150 mg of protein) were combined with 0.5 ml of nickel-nitrilotriacetic acid matrix (Qiagen), mixed on a rotary shaker for 1 h at 4 °C, and then poured into a 10-ml Poly-Prep column (Bio-Rad) to gravity drain. The nickel-nitrilotriacetic acid matrix was then washed with 16 column volumes of 50 mm NaH2PO4 buffer (pH 8.0) containing 300 mm NaCl and 20 mmhistidine, followed by elution with 2 ml of the same buffer containing 250 mm histidine. The protein fraction eluted with 250 mm histidine was desalted (Bio-Rad Econo-Pac10DG) into buffer A, loaded onto a HR 5/5 column containing Source 15 strong anion-exchange matrix that was equilibrated with buffer A, and then eluted with a 10-ml linear gradient (0–50%) of buffer B. For size exclusion chromatography, 200 μg of purified enzyme was loaded onto a calibrated XK 16/70 column of Superdex 200 (Amersham Biosciences) and eluted (at 0.3 ml/min while monitoring at A280) with 25 mm Mopso buffer (pH 6.7) containing 10% v/v glycerol, 1% ethylene glycol, 100 mm NaCl, and 2 mm DTT. Sedimentation equilibrium experiments were performed at 4 °C on a Beckman Optima XL-A analytical ultracentrifuge equipped with electronic speed control and photoelectronic scanning. GPP synthase (purity >90%) in 25 mm NaH2PO4 (pH 7.5) containing 150 mm NaCl and 5 mmMgCl2 was loaded into a 12-mm double-sector cell containing 3-mm columns, and molecular weight was determined from the average of scans at three different velocities and three radii using two enzyme concentrations, by calculation in SEDNTERP (24Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar) using sigma (ς >95% confidence level, determined using NONLIN (25Johnson M.L. Correia J.J. Yphantis D.A. Halvorsen H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar)) and a partial specific volume (0.7427 ml/g determined from amino acid sequence) calculated in SEDNTERP (24Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). Assays used to determine steady-state kinetic constants and to evaluate product chain length distribution were based on the acid-lability procedure (26Holloway P.W. Popjack G. Biochem. J. 1967; PubMed Scopus Google Scholar, H. U. Chem. Google Scholar) in which the product allylic are to of the and allylic that are from the IPP by For kinetic of enzyme was combined with 100 mm Mopso (pH 6.7) containing the of and the allylic cosubstrate GPP, or in a volume of 100 The were for 2 and the linear was then with 100 of a (v/v) The was then with 1 ml of for 10 min to allow of and then centrifuged to the from the allylic an of which was by liquid were using and the for substrate are the of three the for are the of two used for product analysis were to described with the that of protein was at substrate concentrations, and the was to 10 min followed by the of 1 ml of 100 mm buffer (pH containing 10 each of and to the diphosphate The was with and to for h at °C, which the were mixed and centrifuged to The procedure was with of and the combined was with and 20 for radio-gas Unlike other short-chain prenyltransferases that are homodimers (1Ogura K. Koyama T. Chem. Rev. 1998; 98: 1263-1276Crossref PubMed Scopus (291) Google Scholar), GPP synthase is of two a large subunit (GPPS.lsu) that (but expressed alone is inactive in and a similarly inactive, subunit that any known prenyltransferase appears to and chain length specificity in the subunit (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar). To GPPS.ssu with other prenyltransferases and GGPPS) to chain length distribution of the prenyl diphosphate it was to and purification methods for the in coli of functional GPPS had been demonstrated previously (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar), yet the of soluble recombinant protein was to the to form GPPS M. Gershenzon J. Croteau R. 2000; PubMed Scopus Google Scholar, G. M. R. M. C. 1992; PubMed Scopus Google Scholar) and M. S. C. R. B. J. 1992; Google Scholar, K. T. T. M. 2000; PubMed Scopus Google Scholar) of plant origin have been to Thus, these are to be as an amino-terminal plastidial transit peptide for and to the form K. S.M. Rev. Biol. Google Scholar, Rev. Biol. 1998; PubMed Scopus Google Scholar). GPPS.ssu and to be as such based on the comparison of the deduced amino-terminal (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) to of other transit G. J. J. Biochem. PubMed Scopus Google Scholar). with other plastidial including and synthases Croteau R. Biochemistry. 1998; PubMed Scopus Google Scholar, Wildung M.R. D. R.M. Croteau R. Arch. Biochem. Biophys. 2000; PubMed Scopus Google Scholar) and (22Hefner J. Ketchum R.E.B. Croteau R. Arch. Biochem. Biophys. 1998; 360: 62-74Crossref PubMed Scopus (136) Google Scholar), have demonstrated of of more purified truncated to the both the GPPS small and large subunits were truncated for from the pET The (Fig. yielded of a size with the molecular weights of the subunits previously determined by SDS-PAGE (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar). Coexpression in coli of the truncated (pETGPPS.ssu and demonstrated the previously and a yield of recombinant enzyme both subunits were expressed of the recombinant GPPS coexpressed pETGPPS.ssu and by the procedure described yielded greater than 5 mg of protein liter of as by SDS-PAGE the subunit of GPPS (14Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) the truncated subunit of the enzyme in these at a of (Fig. and analysis of the confirmed that the truncated GPPS geranyl diphosphate (Fig. of purified prenyltransferases. The SDS-PAGE of purified prenyltransferases and the and using directed the GPP synthase large and small for description of the prenyltransferases. The molecular standards are and radio-gas analysis of the by of prenyl diphosphate of the prenyltransferases. A is the to standards of dimethylallyl and and The are the to the generated from by GPPS with by