The Experts below are selected from a list of 969 Experts worldwide ranked by ideXlab platform

Hiroshi Kawaide - One of the best experts on this subject based on the ideXlab platform.

  • Characterization and evolutionary analysis of Ent-Kaurene Synthase like genes from the wild rice species Oryza rufipogon.
    Biochemical and biophysical research communications, 2016
    Co-Authors: Tomonobu Toyomasu, Hiroshi Kawaide, Chizu Sugawara, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Kiyotaka Horie, Morifumi Hasegawa, Masaru Chuba, Wataru Mitsuhashi
    Abstract:

    Cultivated rice (Oryza sativa) possesses various labdane-related diterpene Synthase genes, homologs of ent-copalyl diphosphate Synthase (CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene Synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene Synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins.

  • hormonal diterpenoids derived from ent kaurenoic acid are involved in the blue light avoidance response of physcomitrella patens
    Plant Signaling & Behavior, 2015
    Co-Authors: Sho Miyazaki, Masatoshi Nakajima, Hiroshi Kawaide
    Abstract:

    Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via Ent-Kaurene, but it does not produce GA. Disruption of the Ent-Kaurene Synthase gene in P. patens suppressed caulonemal differentiation. Application of Ent-Kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in Ent-Kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the Ent-Kaurene oxidase gene-disrupted mutant.

  • blue light irradiation up regulates the ent kaurene Synthase gene and affects the avoidance response of protonemal growth in physcomitrella patens
    Planta, 2014
    Co-Authors: Sho Miyazaki, Masatoshi Nakajima, Hikaru Toyoshima, Masahiro Natsume, Hiroshi Kawaide
    Abstract:

    Main conclusion We report a novel physiological response to blue light in the mossPhyscomitrella patens. Blue light regulatesEnt-Kaurene biosynthesis and avoidance response to protonemal growth.

  • Isoprenoid biosynthetic pathways and known inhibitors in various organisms.
    2012
    Co-Authors: Tomoko Toyama, Hiroshi Kawaide, Michiru Tahara, Kisaburo Nagamune, Kenji Arimitsu, Yoshio Hamashima, Nirianne M. Q. Palacpac, Toshihiro Horii, Kazuyuki Tanabe
    Abstract:

    Broken arrows indicate blocks in the biosynthesis due to specific inhibitors. “R” indicates various functional groups specific to individual compounds. CPPS, copalyl-diphosphate Synthase (EC 5.5.1.13); KO, Ent-Kaurene oxidase (EC 1.14.13.78); CAS, cycloartenol Synthase (EC 5.4.99.8); LS, lanosterol Synthase (EC 5.4.99.7); KS, Ent-Kaurene Synthase (EC 4.2.3.19); PP, pyrophosphate.

  • endogenous diterpenes derived from ent kaurene a common gibberellin precursor regulate protonema differentiation of the moss physcomitrella patens
    Plant Physiology, 2010
    Co-Authors: Kenichiro Hayashi, Keisuke Horie, Tamotsu Nakashima, Yuji Hiwatashi, Shinjiro Yamaguchi, Atsushi Hanada, Hiroshi Kawaide, Lewis N. Mander, Masatoshi Nakajima, Hisakazu Yamane
    Abstract:

    Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from Ent-Kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces Ent-Kaurene, a common precursor for GAs, and possesses a functional Ent-Kaurene Synthase, PpCPS/KS. To assess the biological role of Ent-Kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate Ent-Kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an Ent-Kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of Ent-Kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of Ent-Kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA9 methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA3 and GA4, both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from Ent-Kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.

Tomonobu Toyomasu - One of the best experts on this subject based on the ideXlab platform.

  • characterization of diterpene Synthase genes in the wild rice species oryza brachyatha provides evolutionary insight into rice phytoalexin biosynthesis
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Tomonobu Toyomasu, Wataru Mitsuhashi, Hisakazu Yamane, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Chisato Goda, Shiho Tomiyama, Nori Kurata, Kazunori Okada
    Abstract:

    Cultivated rice (Oryza sativa; Os) produces a variety of labdane-related diterpenoids; not only phytohormone gibberellins (GAs) but also phytoalexins for defense including phytocassanes, momilactones and oryzalexins. Their carbon skeleton diterpenes are constructed from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP) or its diastereomer syn-CDP. These two-step reactions are successively catalyzed by homologs of the two diterpene Synthases, ent-CDP Synthase (ent-CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of GAs; e.g. OsCPS4 and OsKSL8 that are involved in the biosynthesis of oryzalexin S, a rice phytoalexin. Oryza brachyantha (Ob) is the most distant wild rice species from Os among the Oryza genus. We previously reported that the Ob genome contains ObCPS_11g, ObKSL8-a, ObKSL8-b and ObKSL8-c for specialized metabolism at a locus similar to the OsKSL8 locus on chromosome 11. These Ob genes are closely related to OsCPS4 and OsKSL8, respectively. We herein characterize the diterpene Synthase genes in Ob, using functional analyses and expression analysis. Recombinant OsKSL8 and ObKSL8-a showed the same in vitro function when syn-CDP or normal-CDP were used as substrates. Nonetheless, our results suggest that Ob produces normal-CDP-related diterpenoid phytoalexins, presumably via ObKSL8-a, while Os produces a syn-CDP-related phytoalexin, oryzalexin S, via OsKSL8. This difference must be due to the kinds of CPS that are present in each species; Os has OsCPS4 encoding syn-CPS, while Ob has ObCPS_11g encoding normal-CPS. Thus, we propose the evolutionary history underlying oryzalexin S biosynthesis: the gain of a syn-CPS was a critical event allowing the biosynthesis of oryzalexin S.

  • Characterization and evolutionary analysis of Ent-Kaurene Synthase like genes from the wild rice species Oryza rufipogon.
    Biochemical and biophysical research communications, 2016
    Co-Authors: Tomonobu Toyomasu, Hiroshi Kawaide, Chizu Sugawara, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Kiyotaka Horie, Morifumi Hasegawa, Masaru Chuba, Wataru Mitsuhashi
    Abstract:

    Cultivated rice (Oryza sativa) possesses various labdane-related diterpene Synthase genes, homologs of ent-copalyl diphosphate Synthase (CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene Synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene Synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins.

  • functional characterization of wheat ent kaurene like Synthases indicates continuing evolution of labdane related diterpenoid metabolism in the cereals
    Phytochemistry, 2012
    Co-Authors: Ke Zhou, Meimei Xu, Mollie S Tiernan, Chizu Sugawara, Masami Usui, Makiko Chono, Tomonobu Toyomasu, Wataru Mitsuhashi, Peter M Chandler
    Abstract:

    Abstract Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of Ent-Kaurene Synthase-like (KSL) genes found in rice the (OsKSLs). Herein reported is the biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond Ent-Kaurene Synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying paper describing the wheat CPP Synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/elasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.

  • fusicoccins are biosynthesized by an unusual chimera diterpene Synthase in fungi
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Tomonobu Toyomasu, Tohru Dairi, Wataru Mitsuhashi, Mai Tsukahara, Akane Kaneko, Rie Niida, Nobuo Kato, Takeshi Sassa
    Abstract:

    Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H+-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C20 precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C5 isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C5 isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene Synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi Ent-Kaurene Synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.

  • cloning of a full length cdna encoding ent kaurene Synthase from gibberella fujikuroi functional analysis of a bifunctional diterpene cyclase
    Bioscience Biotechnology and Biochemistry, 2000
    Co-Authors: Tomonobu Toyomasu, Hiroshi Kawaide, Wataru Mitsuhashi, Minoru Otsuka, Atsuko Ishizaki, Shoko Shinoda, Takeshi Sassa
    Abstract:

    We report here the nucleotide sequence of a full-length cDNA encoding Ent-Kaurene Synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and Ent-Kaurene Synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and Ent-Kaurene.

Wataru Mitsuhashi - One of the best experts on this subject based on the ideXlab platform.

  • characterization of diterpene Synthase genes in the wild rice species oryza brachyatha provides evolutionary insight into rice phytoalexin biosynthesis
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Tomonobu Toyomasu, Wataru Mitsuhashi, Hisakazu Yamane, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Chisato Goda, Shiho Tomiyama, Nori Kurata, Kazunori Okada
    Abstract:

    Cultivated rice (Oryza sativa; Os) produces a variety of labdane-related diterpenoids; not only phytohormone gibberellins (GAs) but also phytoalexins for defense including phytocassanes, momilactones and oryzalexins. Their carbon skeleton diterpenes are constructed from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP) or its diastereomer syn-CDP. These two-step reactions are successively catalyzed by homologs of the two diterpene Synthases, ent-CDP Synthase (ent-CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of GAs; e.g. OsCPS4 and OsKSL8 that are involved in the biosynthesis of oryzalexin S, a rice phytoalexin. Oryza brachyantha (Ob) is the most distant wild rice species from Os among the Oryza genus. We previously reported that the Ob genome contains ObCPS_11g, ObKSL8-a, ObKSL8-b and ObKSL8-c for specialized metabolism at a locus similar to the OsKSL8 locus on chromosome 11. These Ob genes are closely related to OsCPS4 and OsKSL8, respectively. We herein characterize the diterpene Synthase genes in Ob, using functional analyses and expression analysis. Recombinant OsKSL8 and ObKSL8-a showed the same in vitro function when syn-CDP or normal-CDP were used as substrates. Nonetheless, our results suggest that Ob produces normal-CDP-related diterpenoid phytoalexins, presumably via ObKSL8-a, while Os produces a syn-CDP-related phytoalexin, oryzalexin S, via OsKSL8. This difference must be due to the kinds of CPS that are present in each species; Os has OsCPS4 encoding syn-CPS, while Ob has ObCPS_11g encoding normal-CPS. Thus, we propose the evolutionary history underlying oryzalexin S biosynthesis: the gain of a syn-CPS was a critical event allowing the biosynthesis of oryzalexin S.

  • Characterization and evolutionary analysis of Ent-Kaurene Synthase like genes from the wild rice species Oryza rufipogon.
    Biochemical and biophysical research communications, 2016
    Co-Authors: Tomonobu Toyomasu, Hiroshi Kawaide, Chizu Sugawara, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Kiyotaka Horie, Morifumi Hasegawa, Masaru Chuba, Wataru Mitsuhashi
    Abstract:

    Cultivated rice (Oryza sativa) possesses various labdane-related diterpene Synthase genes, homologs of ent-copalyl diphosphate Synthase (CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene Synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene Synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins.

  • functional characterization of wheat ent kaurene like Synthases indicates continuing evolution of labdane related diterpenoid metabolism in the cereals
    Phytochemistry, 2012
    Co-Authors: Ke Zhou, Meimei Xu, Mollie S Tiernan, Chizu Sugawara, Masami Usui, Makiko Chono, Tomonobu Toyomasu, Wataru Mitsuhashi, Peter M Chandler
    Abstract:

    Abstract Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of Ent-Kaurene Synthase-like (KSL) genes found in rice the (OsKSLs). Herein reported is the biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond Ent-Kaurene Synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying paper describing the wheat CPP Synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/elasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.

  • fusicoccins are biosynthesized by an unusual chimera diterpene Synthase in fungi
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Tomonobu Toyomasu, Tohru Dairi, Wataru Mitsuhashi, Mai Tsukahara, Akane Kaneko, Rie Niida, Nobuo Kato, Takeshi Sassa
    Abstract:

    Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H+-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C20 precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C5 isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C5 isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene Synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi Ent-Kaurene Synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.

  • cloning of a full length cdna encoding ent kaurene Synthase from gibberella fujikuroi functional analysis of a bifunctional diterpene cyclase
    Bioscience Biotechnology and Biochemistry, 2000
    Co-Authors: Tomonobu Toyomasu, Hiroshi Kawaide, Wataru Mitsuhashi, Minoru Otsuka, Atsuko Ishizaki, Shoko Shinoda, Takeshi Sassa
    Abstract:

    We report here the nucleotide sequence of a full-length cDNA encoding Ent-Kaurene Synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and Ent-Kaurene Synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and Ent-Kaurene.

Sho Miyazaki - One of the best experts on this subject based on the ideXlab platform.

Hisakazu Yamane - One of the best experts on this subject based on the ideXlab platform.

  • characterization of diterpene Synthase genes in the wild rice species oryza brachyatha provides evolutionary insight into rice phytoalexin biosynthesis
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Tomonobu Toyomasu, Wataru Mitsuhashi, Hisakazu Yamane, Koji Miyamoto, Matthew R. Shenton, Arisa Sakai, Chisato Goda, Shiho Tomiyama, Nori Kurata, Kazunori Okada
    Abstract:

    Cultivated rice (Oryza sativa; Os) produces a variety of labdane-related diterpenoids; not only phytohormone gibberellins (GAs) but also phytoalexins for defense including phytocassanes, momilactones and oryzalexins. Their carbon skeleton diterpenes are constructed from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP) or its diastereomer syn-CDP. These two-step reactions are successively catalyzed by homologs of the two diterpene Synthases, ent-CDP Synthase (ent-CPS) and Ent-Kaurene Synthase (KS) that are responsible for the biosynthesis of GAs; e.g. OsCPS4 and OsKSL8 that are involved in the biosynthesis of oryzalexin S, a rice phytoalexin. Oryza brachyantha (Ob) is the most distant wild rice species from Os among the Oryza genus. We previously reported that the Ob genome contains ObCPS_11g, ObKSL8-a, ObKSL8-b and ObKSL8-c for specialized metabolism at a locus similar to the OsKSL8 locus on chromosome 11. These Ob genes are closely related to OsCPS4 and OsKSL8, respectively. We herein characterize the diterpene Synthase genes in Ob, using functional analyses and expression analysis. Recombinant OsKSL8 and ObKSL8-a showed the same in vitro function when syn-CDP or normal-CDP were used as substrates. Nonetheless, our results suggest that Ob produces normal-CDP-related diterpenoid phytoalexins, presumably via ObKSL8-a, while Os produces a syn-CDP-related phytoalexin, oryzalexin S, via OsKSL8. This difference must be due to the kinds of CPS that are present in each species; Os has OsCPS4 encoding syn-CPS, while Ob has ObCPS_11g encoding normal-CPS. Thus, we propose the evolutionary history underlying oryzalexin S biosynthesis: the gain of a syn-CPS was a critical event allowing the biosynthesis of oryzalexin S.

  • endogenous diterpenes derived from ent kaurene a common gibberellin precursor regulate protonema differentiation of the moss physcomitrella patens
    Plant Physiology, 2010
    Co-Authors: Kenichiro Hayashi, Keisuke Horie, Tamotsu Nakashima, Yuji Hiwatashi, Shinjiro Yamaguchi, Atsushi Hanada, Hiroshi Kawaide, Lewis N. Mander, Masatoshi Nakajima, Hisakazu Yamane
    Abstract:

    Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from Ent-Kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces Ent-Kaurene, a common precursor for GAs, and possesses a functional Ent-Kaurene Synthase, PpCPS/KS. To assess the biological role of Ent-Kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate Ent-Kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an Ent-Kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of Ent-Kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of Ent-Kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA9 methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA3 and GA4, both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from Ent-Kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.

  • molecular cloning and characterization of a cdna encoding the gibberellin biosynthetic enzyme ent kaurene Synthase b from pumpkin cucurbita maxima l
    Plant Journal, 1996
    Co-Authors: Shinjiro Yamaguchi, Hisakazu Yamane, Noboru Murofushi, Tamio Saito, Yuji Kamiya
    Abstract:

    Summary The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to Ent-Kaurene. Ent-Kaurene Synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to Ent-Kaurene by Ent-Kaurene Synthase B (KSBI. A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia colias a fusion protein, which demonstrated the KSB activity to cyclize I3H1CDP to [3H]Ent-Kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.