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• W2047532982 abstract "ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase andent-kaurene synthase. A cell-free system of the fungusPhaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA inEscherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10−6m, whereas it did not inhibit the ent-kaurene synthase activity even at 10−4m. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions. ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase andent-kaurene synthase. A cell-free system of the fungusPhaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA inEscherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10−6m, whereas it did not inhibit the ent-kaurene synthase activity even at 10−4m. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions. Gibberellins (GAs) 1The abbreviations used are: GA, gibberellin; Amo-1618, 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenylpiperidine-1-carboxylate; AS, abietadiene synthase; CDP, copalyl diphosphate; CPS, copalyl diphosphate synthase (formerly ent-kaurene synthase A); FCPS, fungal-type CPS; GC-MS, gas chromatograph-mass spectrometry; GGDP, geranylgeranyl diphosphate; GST, glutathione S-transferase; KS,ent-kaurene synthase (formerly ent-kaurene synthase B); IPTG, isopropyl-1-thio-β-d-galactopyranoside; PCR, polymerase chain reaction; TLC, thin layer chromatography.1The abbreviations used are: GA, gibberellin; Amo-1618, 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenylpiperidine-1-carboxylate; AS, abietadiene synthase; CDP, copalyl diphosphate; CPS, copalyl diphosphate synthase (formerly ent-kaurene synthase A); FCPS, fungal-type CPS; GC-MS, gas chromatograph-mass spectrometry; GGDP, geranylgeranyl diphosphate; GST, glutathione S-transferase; KS,ent-kaurene synthase (formerly ent-kaurene synthase B); IPTG, isopropyl-1-thio-β-d-galactopyranoside; PCR, polymerase chain reaction; TLC, thin layer chromatography. are one of an important group of phytohormones regulating many aspects of plant growth and development. Some fungal species produce GAs as secondary metabolites (1Rademacher W. J. Plant Growth Regul. 1994; 15: 303-314Crossref Scopus (66) Google Scholar). Gibberella fujikuroi is a rice pathogenic fungus producing high amount of GAs. The fermentation and biosynthesis of GA in G. fujikuroi were well characterized, since some GAs are produced industrially using the fungus (2Brückner B. Blechschmidt D. Crit. Rev. Biotechnol. 1991; 11: 163-192Crossref Scopus (60) Google Scholar, 3Bearder J.R. Crozier A. The Biochemistry and Physiology of Gibberellins. I. Praeger, New York1983: 251-387Google Scholar).GA is unequivocally synthesized from ent-kaurene in both fungi and plants (4Cross, B. E., Galt, R. H. B., and Hanson, J. R. (1964) J. Chem. Soc. 295–300.Google Scholar, 5Graebe J.E. Hedden P. Gaskin P. MacMillan J. Planta. 1974; 120: 307-309Crossref PubMed Scopus (27) Google Scholar). ent-Kaurene is a tetracyclic diterpene hydrocarbon formed from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP). The pathway of ent-kaurene biosynthesis was first confirmed using cell-free systems from G. fujikuroi (6Shechter I. West C.A. J. Biol. Chem. 1969; 244: 3200-3209Abstract Full Text PDF PubMed Google Scholar). The two-step cyclization was thought to involve two different enzymes: copalyl diphosphate synthase (CPS, formerlyent-kaurene synthase A) and ent-kaurene synthase (KS, formerly ent-kaurene synthase B). These enzymes were partially purified from Fusarium moniliforme, an anamorph ofG. fujikuroi. (7Fall R.R. West C.A. J. Biol. Chem. 1971; 246: 6913-6928Abstract Full Text PDF PubMed Google Scholar). It was suggested that CPS and KS in the fungus might be tightly associated, because the two activities could not be separated. On the other hand, the two activities were successfully separated from a plant enzyme preparation from Marah macrocarpus by chromatographic methods (8Duncan J.D. West C.A. Plant Physiol. 1981; 68: 1128-1134Crossref PubMed Google Scholar). The two enzymes are possibly localized in plastids (9Aach H. Böse G. Graebe J.E. Planta. 1995; 197: 333-342Crossref Scopus (43) Google Scholar). Quite recently, genes encoding both enzymes have been cloned from plants: CPS from Arabidopsis(10Sun T.-p. Kamiya Y. Plant Cell. 1994; 6: 1509-1518Crossref PubMed Scopus (420) Google Scholar), maize (11Bensen R.J. Johal G.S. Crane V.C. Tossberg J.T. Schnable P.S. Meeley R.B. Briggs S.P. Plant Cell. 1995; 7: 75-84Crossref PubMed Scopus (356) Google Scholar) and pea (12Ait-Ali T. Swain S.M. Reid J.B. Sun T.-p. Kamiya Y. Plant J. 1997; 11: 443-454Crossref PubMed Scopus (98) Google Scholar), and KS from pumpkin (13Yamaguchi S. Saito T. Abe H. Yamane H. Murofushi N. Kamiya Y. Plant J. 1996; 10: 203-213Crossref PubMed Scopus (102) Google Scholar). Several other GA biosynthetic enzymes have been cloned from plants as well (14Hedden P. Kamiya Y. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 431-460Crossref PubMed Scopus (502) Google Scholar). In contrast, no GA biosynthetic enzymes have been cloned from fungi.GA biosynthesis in another GA-producing fungus,Phaeosphaeria sp. L487 has been characterized recently (15Kawaide H. Sassa T. Kamiya Y. Phytochemistry. 1995; 39: 305-310Crossref Scopus (22) Google Scholar). This fungus produces significant amounts of GA1 through a pathway similar to that in higher plants: namely, 3β-hydroxylation of GA9 and GA20 occurs to form GA4 and GA1, respectively (15Kawaide H. Sassa T. Kamiya Y. Phytochemistry. 1995; 39: 305-310Crossref Scopus (22) Google Scholar, 16Kawaide H. Sassa T. Biosci. Biotechnol. Biochem. 1993; 57: 1403-1405Crossref Scopus (36) Google Scholar). This contrasts to G. fujikuroi, in which 3β-hydroxylation occurs early in the pathway of GA12-aldehyde and GA9 is not converted to GA4. Although ent-kaurene is an intermediate of GA biosynthesis in both fungi and plants (17Coolbaugh R.C. Crozier A. The Biochemistry and Physiology of Gibberellins. I. Praeger, New York1983: 53-98Google Scholar), we were unable to compare the characteristics of the plant CPS and KS with fungal CPS and KS at molecular levels. Little is known about enzymes and genes involved in ent-kaurene biosynthesis ofPhaeosphaeria sp. L487. In addition, none of the diterpene cyclases from fungi have been isolated yet. Therefore, we focused on fungal-type CPS (FCPS) of Phaeosphaeria sp. L487. In this paper we report cDNA cloning and characterization of an enzyme involved in ent-kaurene biosynthesis in the fungusPhaeosphaeria sp. L487.DISCUSSIONWe report the isolation and characterization of a cDNA encoding the fungal ent-kaurene synthase, FCPS/KS, fromPhaeosphaeria sp. L487. The genes for the fungal sesquiterpene cyclases, trichodiene synthase from Fusarium sporotrichoides (28Horn T.M. Beremand P.D. Gene ( Amst .). 1989; 79: 131-138Crossref PubMed Scopus (174) Google Scholar) and aristolochene synthase fromPenicillium roqueforti, have been characterized (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). However, although many fungal species produce interesting bioactive diterpene compounds, such as the GAs and fusicoccins (36Turner W. Aldridge D. Fungal Metabolites II. Academic Press, London1983: 272-302Google Scholar), none of the genes encoding diterpene cyclases have been isolated from fungi. Thus, this is the first report of the cloning of a diterpene cyclase of fungal origin. We initially focused on the cDNA cloning of fungal CPS, since the information about the sequence of CPS was obtained from various plants. The predicted amino acid sequence of FCPS/KS isolated from Phaeosphaeria sp. L487 has homology with the plant CPS and KS. The phylogenetic tree (Fig. 5) indicates that FCPS/KS has a significant evolutional relationship with plant CPS. However, whereas both CPS and KS have a transit peptide in their amino-terminal regions, this is absent in FCPS/KS. FCPS/KS contains neither membrane translocation sequences or membrane spanning regions. Therefore, FCPS/KS is likely to be localized in the cytoplasm, as are the fungal sesquiterpene cyclases (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). This is also consistent with the presence of enzyme activity in the soluble fraction of the cell-free preparation from Phaeosphaeria sp. L487. In plants, all GGDP-derived carbon skeletons are synthesized within plastids and sesquiterpene hydrocarbons within the cytoplasm or endoplasmic reticulum (37Kleinig H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 39-59Crossref Google Scholar).We revealed that FCPS/KS is a bifunctional cyclase having both activities of CPS and KS in plants. FCPS/KS catalyzes the two-step cyclization of GGDP to ent-kaurene via CDP with a single polypeptide. Amo-1618 inhibits CPS activity in FCPS/KS at low concentrations. In contrast, it does not inhibit the KS activity, even at high concentrations. Similar inhibition characteristics are observed in enzyme preparations of G. fujikuroi and Marah macrocarpus (35Hedden P. Phinney B.O. MacMillan J. Sponsel V.M. Phytochemistry. 1977; 16: 1913-1917Crossref Scopus (23) Google Scholar). It has been suggested that, in plants, CPS and KS may interact with each other to synthesize ent-kaurene from GGDP (6Shechter I. West C.A. J. Biol. Chem. 1969; 244: 3200-3209Abstract Full Text PDF PubMed Google Scholar). This suggests that the catalytic sites for the two cyclizations are separated. Furthermore, the similar modes of action of Amo-1618 on ent-kaurene biosynthesis in fungi and plants indicate that FCPS/KS of Phaeosphaeria sp. L487 and CPS-KS complex of plants have similar structure. At present, the two active sites responsible for CPS and KS activities within the FCPS/KS cannot be determined by sequence comparison. In contrast, AS is also a bifunctional enzyme which catalyzes the cyclization of GGDP to abietadiene via labdadienyl diphosphate (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The amino-terminal region of AS, containing the DXDDTA motif, resembles CPS, whereas the carboxyl-terminal region, containing the DDXXD motif, resembles KS (Fig. 2). The two active sites within the AS suggested a fusion of elements of CPS-type enzymes and KS-type enzymes (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, there are some examples where biosynthetic genes are separate in one organism but linked in another (38Yourno J. Kohno T. Roth J.R. Nature. 1970; 228: 820-824Crossref PubMed Scopus (69) Google Scholar, 39Kirschner K. Bisswanger H. Annu. Rev. Biochem. 1976; 45: 143-166Crossref PubMed Scopus (152) Google Scholar, 40Samson S.M. Dotzlaf J.E. Slisz M.L. Becker G.W. Van Frank R.M. Veal L.E. Yeh W.-K. Millar J.R. Queener S.W. Ingolia T.D. Bio/Technology. 1987; 5: 1207-1214Crossref Scopus (139) Google Scholar). FCPS/KS is an attractive model for understanding of structure-activity relationship of the enzymes involved in ent-kaurene biosynthesis between fungi and plants. To understand the catalytic sites of FCPS/KS, more detail studies will be required. Comparison of FCPS/KS with diterpene cyclases from G. fujikuroi and other fungi will be necessary for understanding the evolution of GA biosynthesis in fungi. Gibberellins (GAs) 1The abbreviations used are: GA, gibberellin; Amo-1618, 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenylpiperidine-1-carboxylate; AS, abietadiene synthase; CDP, copalyl diphosphate; CPS, copalyl diphosphate synthase (formerly ent-kaurene synthase A); FCPS, fungal-type CPS; GC-MS, gas chromatograph-mass spectrometry; GGDP, geranylgeranyl diphosphate; GST, glutathione S-transferase; KS,ent-kaurene synthase (formerly ent-kaurene synthase B); IPTG, isopropyl-1-thio-β-d-galactopyranoside; PCR, polymerase chain reaction; TLC, thin layer chromatography.1The abbreviations used are: GA, gibberellin; Amo-1618, 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenylpiperidine-1-carboxylate; AS, abietadiene synthase; CDP, copalyl diphosphate; CPS, copalyl diphosphate synthase (formerly ent-kaurene synthase A); FCPS, fungal-type CPS; GC-MS, gas chromatograph-mass spectrometry; GGDP, geranylgeranyl diphosphate; GST, glutathione S-transferase; KS,ent-kaurene synthase (formerly ent-kaurene synthase B); IPTG, isopropyl-1-thio-β-d-galactopyranoside; PCR, polymerase chain reaction; TLC, thin layer chromatography. are one of an important group of phytohormones regulating many aspects of plant growth and development. Some fungal species produce GAs as secondary metabolites (1Rademacher W. J. Plant Growth Regul. 1994; 15: 303-314Crossref Scopus (66) Google Scholar). Gibberella fujikuroi is a rice pathogenic fungus producing high amount of GAs. The fermentation and biosynthesis of GA in G. fujikuroi were well characterized, since some GAs are produced industrially using the fungus (2Brückner B. Blechschmidt D. Crit. Rev. Biotechnol. 1991; 11: 163-192Crossref Scopus (60) Google Scholar, 3Bearder J.R. Crozier A. The Biochemistry and Physiology of Gibberellins. I. Praeger, New York1983: 251-387Google Scholar). GA is unequivocally synthesized from ent-kaurene in both fungi and plants (4Cross, B. E., Galt, R. H. B., and Hanson, J. R. (1964) J. Chem. Soc. 295–300.Google Scholar, 5Graebe J.E. Hedden P. Gaskin P. MacMillan J. Planta. 1974; 120: 307-309Crossref PubMed Scopus (27) Google Scholar). ent-Kaurene is a tetracyclic diterpene hydrocarbon formed from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP). The pathway of ent-kaurene biosynthesis was first confirmed using cell-free systems from G. fujikuroi (6Shechter I. West C.A. J. Biol. Chem. 1969; 244: 3200-3209Abstract Full Text PDF PubMed Google Scholar). The two-step cyclization was thought to involve two different enzymes: copalyl diphosphate synthase (CPS, formerlyent-kaurene synthase A) and ent-kaurene synthase (KS, formerly ent-kaurene synthase B). These enzymes were partially purified from Fusarium moniliforme, an anamorph ofG. fujikuroi. (7Fall R.R. West C.A. J. Biol. Chem. 1971; 246: 6913-6928Abstract Full Text PDF PubMed Google Scholar). It was suggested that CPS and KS in the fungus might be tightly associated, because the two activities could not be separated. On the other hand, the two activities were successfully separated from a plant enzyme preparation from Marah macrocarpus by chromatographic methods (8Duncan J.D. West C.A. Plant Physiol. 1981; 68: 1128-1134Crossref PubMed Google Scholar). The two enzymes are possibly localized in plastids (9Aach H. Böse G. Graebe J.E. Planta. 1995; 197: 333-342Crossref Scopus (43) Google Scholar). Quite recently, genes encoding both enzymes have been cloned from plants: CPS from Arabidopsis(10Sun T.-p. Kamiya Y. Plant Cell. 1994; 6: 1509-1518Crossref PubMed Scopus (420) Google Scholar), maize (11Bensen R.J. Johal G.S. Crane V.C. Tossberg J.T. Schnable P.S. Meeley R.B. Briggs S.P. Plant Cell. 1995; 7: 75-84Crossref PubMed Scopus (356) Google Scholar) and pea (12Ait-Ali T. Swain S.M. Reid J.B. Sun T.-p. Kamiya Y. Plant J. 1997; 11: 443-454Crossref PubMed Scopus (98) Google Scholar), and KS from pumpkin (13Yamaguchi S. Saito T. Abe H. Yamane H. Murofushi N. Kamiya Y. Plant J. 1996; 10: 203-213Crossref PubMed Scopus (102) Google Scholar). Several other GA biosynthetic enzymes have been cloned from plants as well (14Hedden P. Kamiya Y. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 431-460Crossref PubMed Scopus (502) Google Scholar). In contrast, no GA biosynthetic enzymes have been cloned from fungi. GA biosynthesis in another GA-producing fungus,Phaeosphaeria sp. L487 has been characterized recently (15Kawaide H. Sassa T. Kamiya Y. Phytochemistry. 1995; 39: 305-310Crossref Scopus (22) Google Scholar). This fungus produces significant amounts of GA1 through a pathway similar to that in higher plants: namely, 3β-hydroxylation of GA9 and GA20 occurs to form GA4 and GA1, respectively (15Kawaide H. Sassa T. Kamiya Y. Phytochemistry. 1995; 39: 305-310Crossref Scopus (22) Google Scholar, 16Kawaide H. Sassa T. Biosci. Biotechnol. Biochem. 1993; 57: 1403-1405Crossref Scopus (36) Google Scholar). This contrasts to G. fujikuroi, in which 3β-hydroxylation occurs early in the pathway of GA12-aldehyde and GA9 is not converted to GA4. Although ent-kaurene is an intermediate of GA biosynthesis in both fungi and plants (17Coolbaugh R.C. Crozier A. The Biochemistry and Physiology of Gibberellins. I. Praeger, New York1983: 53-98Google Scholar), we were unable to compare the characteristics of the plant CPS and KS with fungal CPS and KS at molecular levels. Little is known about enzymes and genes involved in ent-kaurene biosynthesis ofPhaeosphaeria sp. L487. In addition, none of the diterpene cyclases from fungi have been isolated yet. Therefore, we focused on fungal-type CPS (FCPS) of Phaeosphaeria sp. L487. In this paper we report cDNA cloning and characterization of an enzyme involved in ent-kaurene biosynthesis in the fungusPhaeosphaeria sp. L487. DISCUSSIONWe report the isolation and characterization of a cDNA encoding the fungal ent-kaurene synthase, FCPS/KS, fromPhaeosphaeria sp. L487. The genes for the fungal sesquiterpene cyclases, trichodiene synthase from Fusarium sporotrichoides (28Horn T.M. Beremand P.D. Gene ( Amst .). 1989; 79: 131-138Crossref PubMed Scopus (174) Google Scholar) and aristolochene synthase fromPenicillium roqueforti, have been characterized (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). However, although many fungal species produce interesting bioactive diterpene compounds, such as the GAs and fusicoccins (36Turner W. Aldridge D. Fungal Metabolites II. Academic Press, London1983: 272-302Google Scholar), none of the genes encoding diterpene cyclases have been isolated from fungi. Thus, this is the first report of the cloning of a diterpene cyclase of fungal origin. We initially focused on the cDNA cloning of fungal CPS, since the information about the sequence of CPS was obtained from various plants. The predicted amino acid sequence of FCPS/KS isolated from Phaeosphaeria sp. L487 has homology with the plant CPS and KS. The phylogenetic tree (Fig. 5) indicates that FCPS/KS has a significant evolutional relationship with plant CPS. However, whereas both CPS and KS have a transit peptide in their amino-terminal regions, this is absent in FCPS/KS. FCPS/KS contains neither membrane translocation sequences or membrane spanning regions. Therefore, FCPS/KS is likely to be localized in the cytoplasm, as are the fungal sesquiterpene cyclases (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). This is also consistent with the presence of enzyme activity in the soluble fraction of the cell-free preparation from Phaeosphaeria sp. L487. In plants, all GGDP-derived carbon skeletons are synthesized within plastids and sesquiterpene hydrocarbons within the cytoplasm or endoplasmic reticulum (37Kleinig H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 39-59Crossref Google Scholar).We revealed that FCPS/KS is a bifunctional cyclase having both activities of CPS and KS in plants. FCPS/KS catalyzes the two-step cyclization of GGDP to ent-kaurene via CDP with a single polypeptide. Amo-1618 inhibits CPS activity in FCPS/KS at low concentrations. In contrast, it does not inhibit the KS activity, even at high concentrations. Similar inhibition characteristics are observed in enzyme preparations of G. fujikuroi and Marah macrocarpus (35Hedden P. Phinney B.O. MacMillan J. Sponsel V.M. Phytochemistry. 1977; 16: 1913-1917Crossref Scopus (23) Google Scholar). It has been suggested that, in plants, CPS and KS may interact with each other to synthesize ent-kaurene from GGDP (6Shechter I. West C.A. J. Biol. Chem. 1969; 244: 3200-3209Abstract Full Text PDF PubMed Google Scholar). This suggests that the catalytic sites for the two cyclizations are separated. Furthermore, the similar modes of action of Amo-1618 on ent-kaurene biosynthesis in fungi and plants indicate that FCPS/KS of Phaeosphaeria sp. L487 and CPS-KS complex of plants have similar structure. At present, the two active sites responsible for CPS and KS activities within the FCPS/KS cannot be determined by sequence comparison. In contrast, AS is also a bifunctional enzyme which catalyzes the cyclization of GGDP to abietadiene via labdadienyl diphosphate (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The amino-terminal region of AS, containing the DXDDTA motif, resembles CPS, whereas the carboxyl-terminal region, containing the DDXXD motif, resembles KS (Fig. 2). The two active sites within the AS suggested a fusion of elements of CPS-type enzymes and KS-type enzymes (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, there are some examples where biosynthetic genes are separate in one organism but linked in another (38Yourno J. Kohno T. Roth J.R. Nature. 1970; 228: 820-824Crossref PubMed Scopus (69) Google Scholar, 39Kirschner K. Bisswanger H. Annu. Rev. Biochem. 1976; 45: 143-166Crossref PubMed Scopus (152) Google Scholar, 40Samson S.M. Dotzlaf J.E. Slisz M.L. Becker G.W. Van Frank R.M. Veal L.E. Yeh W.-K. Millar J.R. Queener S.W. Ingolia T.D. Bio/Technology. 1987; 5: 1207-1214Crossref Scopus (139) Google Scholar). FCPS/KS is an attractive model for understanding of structure-activity relationship of the enzymes involved in ent-kaurene biosynthesis between fungi and plants. To understand the catalytic sites of FCPS/KS, more detail studies will be required. Comparison of FCPS/KS with diterpene cyclases from G. fujikuroi and other fungi will be necessary for understanding the evolution of GA biosynthesis in fungi. We report the isolation and characterization of a cDNA encoding the fungal ent-kaurene synthase, FCPS/KS, fromPhaeosphaeria sp. L487. The genes for the fungal sesquiterpene cyclases, trichodiene synthase from Fusarium sporotrichoides (28Horn T.M. Beremand P.D. Gene ( Amst .). 1989; 79: 131-138Crossref PubMed Scopus (174) Google Scholar) and aristolochene synthase fromPenicillium roqueforti, have been characterized (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). However, although many fungal species produce interesting bioactive diterpene compounds, such as the GAs and fusicoccins (36Turner W. Aldridge D. Fungal Metabolites II. Academic Press, London1983: 272-302Google Scholar), none of the genes encoding diterpene cyclases have been isolated from fungi. Thus, this is the first report of the cloning of a diterpene cyclase of fungal origin. We initially focused on the cDNA cloning of fungal CPS, since the information about the sequence of CPS was obtained from various plants. The predicted amino acid sequence of FCPS/KS isolated from Phaeosphaeria sp. L487 has homology with the plant CPS and KS. The phylogenetic tree (Fig. 5) indicates that FCPS/KS has a significant evolutional relationship with plant CPS. However, whereas both CPS and KS have a transit peptide in their amino-terminal regions, this is absent in FCPS/KS. FCPS/KS contains neither membrane translocation sequences or membrane spanning regions. Therefore, FCPS/KS is likely to be localized in the cytoplasm, as are the fungal sesquiterpene cyclases (29Proctor R.H. Hohn T.M. J. Biol. Chem. 1993; 268: 4543-4548Abstract Full Text PDF PubMed Google Scholar). This is also consistent with the presence of enzyme activity in the soluble fraction of the cell-free preparation from Phaeosphaeria sp. L487. In plants, all GGDP-derived carbon skeletons are synthesized within plastids and sesquiterpene hydrocarbons within the cytoplasm or endoplasmic reticulum (37Kleinig H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 39-59Crossref Google Scholar). We revealed that FCPS/KS is a bifunctional cyclase having both activities of CPS and KS in plants. FCPS/KS catalyzes the two-step cyclization of GGDP to ent-kaurene via CDP with a single polypeptide. Amo-1618 inhibits CPS activity in FCPS/KS at low concentrations. In contrast, it does not inhibit the KS activity, even at high concentrations. Similar inhibition characteristics are observed in enzyme preparations of G. fujikuroi and Marah macrocarpus (35Hedden P. Phinney B.O. MacMillan J. Sponsel V.M. Phytochemistry. 1977; 16: 1913-1917Crossref Scopus (23) Google Scholar). It has been suggested that, in plants, CPS and KS may interact with each other to synthesize ent-kaurene from GGDP (6Shechter I. West C.A. J. Biol. Chem. 1969; 244: 3200-3209Abstract Full Text PDF PubMed Google Scholar). This suggests that the catalytic sites for the two cyclizations are separated. Furthermore, the similar modes of action of Amo-1618 on ent-kaurene biosynthesis in fungi and plants indicate that FCPS/KS of Phaeosphaeria sp. L487 and CPS-KS complex of plants have similar structure. At present, the two active sites responsible for CPS and KS activities within the FCPS/KS cannot be determined by sequence comparison. In contrast, AS is also a bifunctional enzyme which catalyzes the cyclization of GGDP to abietadiene via labdadienyl diphosphate (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The amino-terminal region of AS, containing the DXDDTA motif, resembles CPS, whereas the carboxyl-terminal region, containing the DDXXD motif, resembles KS (Fig. 2). The two active sites within the AS suggested a fusion of elements of CPS-type enzymes and KS-type enzymes (25Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, there are some examples where biosynthetic genes are separate in one organism but linked in another (38Yourno J. Kohno T. Roth J.R. Nature. 1970; 228: 820-824Crossref PubMed Scopus (69) Google Scholar, 39Kirschner K. Bisswanger H. Annu. Rev. Biochem. 1976; 45: 143-166Crossref PubMed Scopus (152) Google Scholar, 40Samson S.M. Dotzlaf J.E. Slisz M.L. Becker G.W. Van Frank R.M. Veal L.E. Yeh W.-K. Millar J.R. Queener S.W. Ingolia T.D. Bio/Technology. 1987; 5: 1207-1214Crossref Scopus (139) Google Scholar). FCPS/KS is an attractive model for understanding of structure-activity relationship of the enzymes involved in ent-kaurene biosynthesis between fungi and plants. To understand the catalytic sites of FCPS/KS, more detail studies will be required. Comparison of FCPS/KS with diterpene cyclases from G. fujikuroi and other fungi will be necessary for understanding the evolution of GA biosynthesis in fungi. We are grateful to Dr. Peter Hedden (IACR-Long Ashton Research Station, University of Bristol) and Prof. Tai-ping Sun (Duke University) for their critical readings of the manuscript. We thank Drs. Shinjiro Yamaguchi and Tahar Ait-Ali (Frontier Research Program, RIKEN) for providing KS and CPS recombinant proteins, respectively, and Yukiji Tachiyama for expert technical assistance on DNA sequencing." @default.
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• W2047532982 title "ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487" @default.
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