The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Hiroshi Kawaide - One of the best experts on this subject based on the ideXlab platform.
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Characterization of moss Ent-Kaurene oxidase (CYP701B1) using a highly purified preparation.
Journal of biochemistry, 2017Co-Authors: Chisato Noguchi, Hiroshi Kawaide, Sho Miyazaki, Osamu Gotoh, Yuzo Yoshida, Yuri AoyamaAbstract:CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the Ent-Kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of Ent-Kaurene to ent-kaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with Ent-Kaurene, and apparent Kd of Ent-Kaurene estimated by the spectral change caused by this spin-state shift was 2.5 μM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to Ent-Kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than Ent-Kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of Ent-Kaurene. The P450 monooxygenase adapted for Ent-Kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.
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Molecular evolution of the substrate specificity of Ent-Kaurene synthases to adapt to gibberellin biosynthesis in land plants.
The Biochemical journal, 2014Co-Authors: Manami Shimane, Kenichiro Hayashi, Hiroshi Nozaki, Masahiro Natsume, Yohei Ueno, Keiko Morisaki, Shingo Oogami, Hiroshi KawaideAbstract:Ent-Kaurene is a key intermediate in the biosynthesis of the plant hormone gibberellin. In Ent-Kaurene biosynthesis in flowering plants, two diterpene cyclases (DTCs), ent-copalyl diphosphate (ent-CDP) synthase (ent-CPS) and Ent-Kaurene synthase (KS), catalyse the cyclization of geranylgeranyl diphosphate to ent-CDP and ent-CDP to Ent-Kaurene, respectively. In contrast, the moss Physcomitrella patens has a bifunctional ent-CPS/KS (PpCPS/KS) that catalyses both cyclization reactions. To gain more insight into the functional diversity of Ent-Kaurene biosynthetic enzymes in land plants, we focused on DTCs in the lycophyte Selaginella moellendorffii. The present paper describes the characterization of two S. moellendorffii DTCs (SmKS and SmDTC3) in vitro. SmDTC3 converted ent-CDP into ent-16α-hydroxykaurane and also used other CDP stereoisomers as substrate. Remarkably, SmKS, which produces Ent-Kaurene from ent-CDP, showed similar substrate selectivity: both SmKS and SmDTC3 synthesized sandaracopimaradiene from normal CDP. Therefore, the diversity of substrate recognition among KSs from other plants was investigated. PpCPS/KS could use normal CDP and syn-CDP as well as ent-CDP as substrate. In contrast, lettuce KS showed high specificity for ent-CDP, and rice KS recognized only ent-CDP. Our studies imply that ancient KS having low substrate specificity has evolved to be specific for ent-CDP to the biosynthesis of gibberellin.
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Blue-light irradiation up-regulates the Ent-Kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens
Planta, 2014Co-Authors: Sho Miyazaki, Masahiro Natsume, Masatoshi Nakajima, Hikaru Toyoshima, Hiroshi KawaideAbstract:Main conclusion We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth. Abstract Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent -kaurenoic acid via ent -kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent -kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent -copalyl diphosphate synthase/ ent -kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent -kaurene and ent -kaurenoic acid. This indicates that derivatives of ent -kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent -kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent -kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent -kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.
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Isoprenoid biosynthetic pathways and known inhibitors in various organisms.
2012Co-Authors: Tomoko Toyama, Hiroshi Kawaide, Michiru Tahara, Kisaburo Nagamune, Kenji Arimitsu, Yoshio Hamashima, Nirianne M. Q. Palacpac, Toshihiro Horii, Kazuyuki TanabeAbstract: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.
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The CYP701B1 of Physcomitrella patens is an Ent-Kaurene oxidase that resists inhibition by uniconazole-P.
FEBS letters, 2011Co-Authors: Sho Miyazaki, Masahiro Natsume, Takumi Katsumata, Hiroshi KawaideAbstract:The moss Physcomitrella patens produces both Ent-Kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as Ent-Kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding Ent-Kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from Ent-Kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the Ent-Kaurene oxidase inhibitor uniconazole-P (IC50 64 μM), even though the activity of Arabidopsis Ent-Kaurene oxidase (CYP701A3) was sensitive (IC50 0.26 μM).
Yuji Kamiya - One of the best experts on this subject based on the ideXlab platform.
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Emission of Ent-Kaurene, a Diterpenoid Hydrocarbon Precursor for Gibberellins, into the Headspace from Plants
Plant & cell physiology, 2004Co-Authors: Minoru Otsuka, Takeshi Sassa, Yuji Kamiya, Kazunori Okada, Hiromichi Kenmoku, Mikihiro Ogawa, Wataru Mitsuhashi, Tomonobu Toyomasu, Shinjiro YamaguchiAbstract:;Ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing entkaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and Ent-Kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor Ent-Kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the Ent-Kaurene overproducers in a closed environment, the airborne entkaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in Ent-Kaurene oxidase). These results suggest that Ent-Kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that Ent-Kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.
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Overexpression of AtCPS and AtKS in Arabidopsis Confers Increased Ent-Kaurene Production But No Increase in Bioactive Gibberellins
Plant physiology, 2003Co-Authors: Christine M. Fleet, Shinjiro Yamaguchi, Atsushi Hanada, Hiroshi Kawaide, Yuji Kamiya, Charles J. David, Tai-ping SunAbstract:The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and Ent-Kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/Ent-Kaurene synthase overexpressors have greatly increased levels of the early intermediates Ent-Kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for Ent-Kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.
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Antisense and chemical suppression of the nonmevalonate pathway affects Ent-Kaurene biosynthesis in Arabidopsis.
Planta, 2002Co-Authors: Kazunori Okada, Hiroshi Kawaide, Tomohisa Kuzuyama, Haruo Seto, Ian S. Curtis, Yuji KamiyaAbstract:Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of Ent-Kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of Ent-Kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that Ent-Kaurene is mainly produced through the nonmevalonate pathway in the plastid.
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Functional analysis of the two interacting cyclase domains in Ent-Kaurene synthase from the fungus Phaeosphaeria sp. L487 and a comparison with cyclases from higher plants
The Journal of biological chemistry, 2000Co-Authors: Hiroshi Kawaide, Takeshi Sassa, Yuji KamiyaAbstract:We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria Ent-Kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). Ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant Ent-Kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of Ent-Kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.
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The GA2 Locus of Arabidopsis thaliana Encodes Ent-Kaurene Synthase of Gibberellin Biosynthesis
Plant physiology, 1998Co-Authors: Shinjiro Yamaguchi, Hiroshi Kawaide, Tai-ping Sun, Yuji KamiyaAbstract:The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to Ent-Kaurene, which is catalyzed by Ent-Kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]Ent-Kaurene. The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant. Taken together, our results show that the GA2 locus encodes KS.
Reuben J. Peters - One of the best experts on this subject based on the ideXlab platform.
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A pair of threonines mark Ent-Kaurene synthases for phytohormone biosynthesis.
Phytochemistry, 2021Co-Authors: Reid Brown, Meirong Jia, Reuben J. PetersAbstract:All land plants (embryophytes) must contain an Ent-Kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KSs have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of Ent-Kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.
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18O2 labeling experiments illuminate the oxidation of Ent-Kaurene in bacterial gibberellin biosynthesis
Organic & biomolecular chemistry, 2017Co-Authors: Raimund Nagel, Reuben J. PetersAbstract:Bacteria can produce gibberellin plant hormones. While the bacterial biosynthetic pathway is similar to that of plants, the individual enzymes are very distantly related and arose via convergent evolution. The cytochromes P450 (CYPs) that catalyze the multi-step oxidation of the alkane precursor Ent-Kaurene (1) to ent-kauren-19-oic acid (5), are called Ent-Kaurene oxidases (KOs), and in plants are from the CYP701 family, and share less than 19% amino acid sequence identity with those from bacteria, which are from the phylogenetically distinct CYP117 family. Here the reaction series catalyzed by CYP117 was examined by 18O2 labeling experiments, the results indicate successive hydroxylation of 1 to ent-kauren-19-ol (2) and then ent-kauren-19,19-diol (3) and most likely an intervening dehydration to ent-kauren-19-al (4) prior to the concluding hydroxylation to 5. Accordingly, the bacterial and plant KOs converged on catalysis of the same series of reactions, despite their independent evolutionary origin.
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Extending a Single Residue Switch for Abbreviating Catalysis in Plant Ent-Kaurene Synthases.
Frontiers in Plant Science, 2016Co-Authors: Reuben J. PetersAbstract:Production of Ent-Kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s) present in all land plants (i.e., embryophyta). The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP) and the subsequently acting class I diterpene synthase that produces Ent-Kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases (CPSs) and Ent-Kaurene synthases (KSs) generally observed. This evolutionary scenario implies that plant KSs should have conserved structural features uniquely required for production of Ent-Kaurene relative to related enzymes that have alternative function. Notably, substitution of threonine for a conserved isoleucine has been shown to “short-circuit” the complex bicyclization and rearrangement reaction catalyzed by KSs after initial cyclization, leading to predominant production of ent-pimaradiene, at least in KSs from angiosperms. Here this effect is shown to extend to KSs from earlier diverging plants (i.e., bryophytes), including a bifunctional CPS/KS. In addition, attribution of the dramatic effect of this single residue “switch” on product outcome to electrostatic stabilization of the ent-pimarenyl carbocation intermediate formed upon initial cyclization by the hydroxyl introduced by threonine substitution has been called into question by the observation of similar effects from substitution of alanine. Here further mutational analysis and detailed product analysis is reported that supports the importance of electrostatic stabilization by a hydroxyl or water.
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An Ent-Kaurene-derived diterpenoid virulence factor from Xanthomonas oryzae pv. oryzicola.
The New phytologist, 2014Co-Authors: David M. Hershey, Li Wang, Adam J. Bogdanove, Reuben J. PetersAbstract:Summary Both plants and fungi produce Ent-Kaurene as a precursor to the gibberellin plant hormones. A number of rhizobia contain functionally conserved, sequentially acting ent-copalyl diphosphate and Ent-Kaurene synthases (CPS and KS, respectively), which are found within a well-conserved operon that may lead to the production of gibberellins. Intriguingly, the rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) contains a homologous operon. Here, we report biochemical characterization of the encoded CPS and KS, and the impact of insertional mutagenesis on virulence and the plant defense response for these genes, as well as that for one of the cytochromes P450 (CYP112) found in the operon. Activity of the CPS and KS found in this phytopathogen was verified – that is, Xoc is capable of producing Ent-Kaurene. Moreover, knocking out CPS, KS or CYP112 led to mutant Xoc that exhibited reduced virulence. Investigation of the effect on marker gene transcript levels suggests that the Xoc diterpenoid affects the plant defense response, most directly that mediated by jasmonic acid (JA). Xoc produces an Ent-Kaurene-derived diterpenoid as a virulence factor, potentially a gibberellin phytohormone, which is antagonistic to JA, consistent with the recent recognition of opposing effects for these phytohormones on the microbial defense response.
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CYP701A8: A Rice Ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism
Plant physiology, 2012Co-Authors: Qiang Wang, Matthew L. Hillwig, Reuben J. PetersAbstract:All higher plants contain an Ent-Kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the Ent-Kaurene C4α-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3α position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of Ent-Kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3α-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.
Tai-ping Sun - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of AtCPS and AtKS in Arabidopsis Confers Increased Ent-Kaurene Production But No Increase in Bioactive Gibberellins
Plant physiology, 2003Co-Authors: Christine M. Fleet, Shinjiro Yamaguchi, Atsushi Hanada, Hiroshi Kawaide, Yuji Kamiya, Charles J. David, Tai-ping SunAbstract:The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and Ent-Kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/Ent-Kaurene synthase overexpressors have greatly increased levels of the early intermediates Ent-Kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for Ent-Kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.
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The GA2 Locus of Arabidopsis thaliana Encodes Ent-Kaurene Synthase of Gibberellin Biosynthesis
Plant physiology, 1998Co-Authors: Shinjiro Yamaguchi, Hiroshi Kawaide, Tai-ping Sun, Yuji KamiyaAbstract:The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to Ent-Kaurene, which is catalyzed by Ent-Kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]Ent-Kaurene. The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant. Taken together, our results show that the GA2 locus encodes KS.
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Regulation and cellular localization of ent‐kaurene synthesis
Physiologia Plantarum, 1997Co-Authors: Tai-ping Sun, Yuji KamiyaAbstract:701-708. The two-step cyclization reaction of Ent-Kaurene synthesis from geranylgeranyl diphosphate is the first committed step in the biosynthetic pathway of the plant hor- mone gibberellin. Recent molecular cloning and characterization of the genes encod- ing the two corresponding enzymes, copalyl diphosphate synthase (CPS) and ent-kau- rene synthase (KS), have demonstrated that Ent-Kaurene synthesis is localized in the plastids and is highly regulated in specific tissues and cell types during plant develop- ment. In addition to occurring in actively growing tissues, Ent-Kaurene synthesis also takes place in fully expanded leaves. Therefore mature leaves may produce gibberel- lin intermediates or bioactive gibberellins for transport to responsive tissues. DNA se- quence analyses have revealed a conserved aspartate-rich motif, D(W)DDTA among CPS and other protonation-initiated terpene cyclases, while KS contains a highly con- served DDXXD motif which was proposed to function as a divalent metal ion-diphos- phate complex binding site in ionization-initiated terpene cyclases and prenyltrans- ferases. Key words - Cell-type specific regulation, Ent-Kaurene synthesis, plastid localization, terpene cyclases. 2-p. Sun (corresponding authol; e-mail tps@acpub.duke.edu), Developmental, Cell and Molecular Biology Group, Dept of Botany, Box
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The LS locus of pea encodes the gibberellin biosynthesis enzyme Ent-Kaurene synthase A
The Plant journal : for cell and molecular biology, 1997Co-Authors: Tahar Ait-ali, James B. Reid, Tai-ping Sun, Stephen M. Swain, Yuji KamiyaAbstract:Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to Ent-Kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases Ent-Kaurene synthase A (KSA) and Ent-Kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to Ent-Kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.
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The Arabidopsis GA1 locus encodes the cyclase Ent-Kaurene synthetase A of gibberellin biosynthesis.
The Plant cell, 1994Co-Authors: Tai-ping Sun, Yuji KamiyaAbstract:The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to Ent-Kaurene catalyzed by Ent-Kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of Ent-Kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme Ent-Kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.
Hiroshi Nozaki - One of the best experts on this subject based on the ideXlab platform.
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Molecular evolution of the substrate specificity of Ent-Kaurene synthases to adapt to gibberellin biosynthesis in land plants.
The Biochemical journal, 2014Co-Authors: Manami Shimane, Kenichiro Hayashi, Hiroshi Nozaki, Masahiro Natsume, Yohei Ueno, Keiko Morisaki, Shingo Oogami, Hiroshi KawaideAbstract:Ent-Kaurene is a key intermediate in the biosynthesis of the plant hormone gibberellin. In Ent-Kaurene biosynthesis in flowering plants, two diterpene cyclases (DTCs), ent-copalyl diphosphate (ent-CDP) synthase (ent-CPS) and Ent-Kaurene synthase (KS), catalyse the cyclization of geranylgeranyl diphosphate to ent-CDP and ent-CDP to Ent-Kaurene, respectively. In contrast, the moss Physcomitrella patens has a bifunctional ent-CPS/KS (PpCPS/KS) that catalyses both cyclization reactions. To gain more insight into the functional diversity of Ent-Kaurene biosynthetic enzymes in land plants, we focused on DTCs in the lycophyte Selaginella moellendorffii. The present paper describes the characterization of two S. moellendorffii DTCs (SmKS and SmDTC3) in vitro. SmDTC3 converted ent-CDP into ent-16α-hydroxykaurane and also used other CDP stereoisomers as substrate. Remarkably, SmKS, which produces Ent-Kaurene from ent-CDP, showed similar substrate selectivity: both SmKS and SmDTC3 synthesized sandaracopimaradiene from normal CDP. Therefore, the diversity of substrate recognition among KSs from other plants was investigated. PpCPS/KS could use normal CDP and syn-CDP as well as ent-CDP as substrate. In contrast, lettuce KS showed high specificity for ent-CDP, and rice KS recognized only ent-CDP. Our studies imply that ancient KS having low substrate specificity has evolved to be specific for ent-CDP to the biosynthesis of gibberellin.
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Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in Ent-Kaurene synthase of Physcomitrella patens.
The FEBS journal, 2010Co-Authors: Hiroshi Kawaide, Kenichiro Hayashi, Yuka Sakigi, Akihiko Matsuo, Ryo Kawanabe, Masahiro Natsume, Hiroshi NozakiAbstract:Ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, Ent-Kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and Ent-Kaurene synthase (KS). Recently, the moss Physcomitrella patens Ent-Kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional Ent-Kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both Ent-Kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional Ent-Kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce Ent-Kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only Ent-Kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to Ent-Kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.
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Identification and functional analysis of bifunctional Ent-Kaurene synthase from the moss Physcomitrella patens
FEBS Letters, 2006Co-Authors: Kenichiro Hayashi, Hiroshi Kawaide, Miho Notomi, Yuka Sakigi, Akihiko Matsuo, Hiroshi NozakiAbstract:Ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, Ent-Kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and Ent-Kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal Ent-Kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified Ent-Kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the Ent-Kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to Ent-Kaurene and ent-16α-hydroxykaurene).