The Experts below are selected from a list of 1590 Experts worldwide ranked by ideXlab platform
Patrizia Aducci - One of the best experts on this subject based on the ideXlab platform.
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From plant physiology to pharmacology: Fusicoccin leaves the leaves
Planta, 2019Co-Authors: Lorenzo Camoni, Sabina Visconti, Patrizia Aducci, Mauro MarraAbstract:Main conclusion This review highlights 50 years of research on the fungal diterpene Fusicoccin, during which the molecule went from a tool in plant physiology research to a pharmacological agent in treating animal diseases. Fusicoccin is a phytotoxic glycosylated diterpene produced by the fungus Phomopsis amygdali , a pathogen of almond and peach plants. Widespread interest in this molecule started when it was discovered that it is capable of causing stomate opening in all higher plants, thereby inducing wilting of leaves. Thereafter, FC became, and still is, a tool in plant physiology, due to its ability to influence a number of fundamental processes, which are dependent on the activation of the plasma membrane H^+-ATPase. Molecular studies carried out in the last 20 years clarified details of the mechanism of proton pump stimulation, which involves the Fusicoccin-mediated irreversible stabilization of the complex between the H^+-ATPase and activatory 14-3-3 proteins. More recently, FC has been shown to influence cellular processes involving 14-3-3 binding to client proteins both in plants and animals. In this review, we report the milestones achieved in more than 50 years of research in plants and highlight recent advances in animals that have allowed this diterpene to be used as a 14-3-3 targeted drug.
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In vivo OS treatment inhibits the association of P. lunatus 14-3-3 proteins with the plasma membrane.
2018Co-Authors: Lorenzo Camoni, Patrizia Aducci, Francesca Barbero, Massimo E. MaffeiAbstract:A, SDS-PAGE analysis of plasma membranes of controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC) blotted onto nitrocellulose and probed with anti-H+-ATPase. B, SDS-PAGE analysis of plasma membranes of controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC) blotted onto nitrocellulose and probed with anti-14-3-3 antibodies. C, Densitometric analysis of H+-ATPase in controls (white bars) and OS-treated (black bars) plants without (-FC) and with the use of Fusicoccin (+FC). D, Densitometric analysis of 14-3-3 proteins in controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC). Data are the mean of four independent experiments. Metric bars indicate standard deviation. C and D, expressed as percentage of maximum densitometric value. Asterisk indicates significant (P< 0.05) differences.
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In vivo OS treatment inhibition of the of P. lunatus plasma membrane H+-ATPase activity.
2018Co-Authors: Lorenzo Camoni, Patrizia Aducci, Francesca Barbero, Massimo E. MaffeiAbstract:Apices of P. lunatus leaves were treated with either OS or FC and the vanadate-sensitive ATPase phosphohydrolitic activity was assayed as described in Materials and Methods. A. ATPase activity of whole leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC). B, ATPase activity of proximal half leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC). C, ATPase activity of plasma membranes purified from the distal half of the leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of Fusicoccin (+FC). Data given are the mean of four independent experiments. Metric bars indicate standard deviation. Asterisk indicates significant (P< 0.05) differences.
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In vivo OS inhibition of the interaction between H+-ATPase and 14-3-3 proteins.
2018Co-Authors: Lorenzo Camoni, Patrizia Aducci, Francesca Barbero, Massimo E. MaffeiAbstract:A, Autoradiography and overlay assay performed using plasma membranes purified from control (C) and OS-treated P. lunatus leaves without (-FC) and after the addition (+FC) of 10 μM FC. B, Densitometric analysis of H+-ATPase controls (white bars) and OS-treated leaves (black bars) without (-FC) and with the use of Fusicoccin (+FC). Data given are the mean of four independent experiments. Metric bars indicate standard deviation.
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the phytotoxin Fusicoccin promotes platelet aggregation via 14 3 3 glycoprotein ib ix v interaction
Biochemical Journal, 2011Co-Authors: Lorenzo Camoni, Cristina Di Lucente, Sabina Visconti, Patrizia AducciAbstract:The fungal toxin Fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that Fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as Fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate the present study to be a starting point for future therapeutic use of Fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of Fusicoccin as a drug directed to other 14-3-3-target complexes.
Claudia Oecking - One of the best experts on this subject based on the ideXlab platform.
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a plant plasma membrane h atpase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14 3 3 regulatory proteins in the absence of Fusicoccin
Journal of Biological Chemistry, 2000Co-Authors: Olivier Maudoux, Claudia Oecking, Marc Boutry, Henri Batoko, Kris Gevaert, Joel Vandekerckhove, Pierre MorsommeAbstract:The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with Fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of Fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.
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phosphorylation of thr 948 at the c terminus of the plasma membrane h atpase creates a binding site for the regulatory 14 3 3 protein
The Plant Cell, 1999Co-Authors: Fredrik Svennelid, Claudia Oecking, Christer Larsson, Anne Olsson, Markus Piotrowski, Magnus Rosenquist, Cristian Ottman, Marianne SommarinAbstract:The plant plasma membrane H 1 -ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H 1 -ATPase‐14-3-3 complex that can be stabilized by the fungal toxin Fusicoccin. A novel 14-3-3 binding motif, QQXYpT 948 V, at the C terminus of the H 1 -ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H 1 -ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H 1 -ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H 1 -ATPase that is obtained in the presence of recombinant 14-3-3 and Fusicoccin. Furthermore, binding of 14-3-3 to the H 1 -ATPase in the absence of Fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of Fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H 1 ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H 1 -ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H 1 -ATPase in vivo. Indeed, replacing Thr-948 in the plant H 1 -ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H 1 -ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H 1 -ATPase activity in the plant and thus for plant growth.
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modulation of plasma membrane h atpase activity differentially activates wound and pathogen defense responses in tomato plants
The Plant Cell, 1999Co-Authors: Andreas Schaller, Claudia OeckingAbstract:Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (Fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in Fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, Fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.
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complementation of the saccharomyces cerevisiae plasma membrane h atpase by a plant h atpase generates a highly abundant Fusicoccin binding site
Journal of Biological Chemistry, 1998Co-Authors: Markus Piotrowski, Pierre Morsomme, Marc Boutry, Claudia OeckingAbstract:Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme, The fungal phytotoxin Fusicoccin (FC) appears to stabilize this H+-ATPase 14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity Fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs, The existence of a (plant) PMA2. (yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase 14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs, Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.
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the Fusicoccin receptor of plants is a member of the 14 3 3 superfamily of eukaryotic regulatory proteins
FEBS Letters, 1994Co-Authors: Claudia Oecking, Christoph Eckerskorn, Elmar W. WeilerAbstract:The receptor for the wilt-inducing phytotoxin Fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9′-nor-8′-hydroxyFusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the Fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14-3-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14-3-3 isoform(s) constituting the Fusicoccin receptor are distinguishable from other cellular 14-3-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-114 phase separation experiments, they, as well as the target enzyme of Fusicoccin action, the H+-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of Fusicoccin action.
Nobuo Kato - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions
2015Co-Authors: Prakash Parvatkar, Nobuo Kato, Motonari Uesugi, Shin-ichi Sato, Junko OhkandaAbstract:Synthetic agents that disrupt intracellular protein–protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, Fusicoccin, which binds 14-3-3 protein with submicromolar affinity. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing Fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast, chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. Compound 3, but not 1 or 2, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell death is caused by inhibition of 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs
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a novel Fusicoccin derivative preferentially targets hypoxic tumor cells and inhibits tumor growth in xenografts
Anti-cancer Agents in Medicinal Chemistry, 2012Co-Authors: Koshi Kawakami, Nobuo Kato, Junko Ohkanda, Miho Hattori, Takatsugu Inoue, Yuriko Maruyama, Miki Tongu, Takaya Yamada, Miho Akimoto, Keizo TakenagaAbstract:Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel Fusicoccin derivative (ISIR-042), but not its parent or related compounds such as Fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.
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phosphopeptide dependent labeling of 14 3 3 ζ proteins by Fusicoccin based fluorescent probes
Angewandte Chemie, 2012Co-Authors: Michiko Takahashi, Nobuo Kato, Akie Kawamura, Tsuyoshi Nishi, Itaru Hamachi, Junko OhkandaAbstract:: Fluorescent combination: Cell-penetrating probes derived from the diterpene Fusicoccin can form ternary complexes with 14-3-3 proteins and phosphopeptide ligands, whereupon the probes site-specifically attach a fluorescent tag onto the surface of the 14-3-3 proteins.
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dioxygenases key enzymes to determine the aglycon structures of Fusicoccin and brassicicene diterpene compounds produced by fungi
Journal of the American Chemical Society, 2011Co-Authors: Yusuke Ono, Tomonobu Toyomasu, Nobuo Kato, Takeshi Sassa, Atsushi Minami, Motoyoshi Noike, Yusuke Higuchi, Tohru DairiAbstract:Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified Fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to Fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8β,16-diol. In this study, we repo...
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Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Tomonobu Toyomasu, Tohru Dairi, Wataru Mitsuhashi, Mai Tsukahara, Akane Kaneko, Rie Niida, Nobuo Kato, Takeshi SassaAbstract: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.
Takeshi Sassa - One of the best experts on this subject based on the ideXlab platform.
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dioxygenases key enzymes to determine the aglycon structures of Fusicoccin and brassicicene diterpene compounds produced by fungi
Journal of the American Chemical Society, 2011Co-Authors: Yusuke Ono, Tomonobu Toyomasu, Nobuo Kato, Takeshi Sassa, Atsushi Minami, Motoyoshi Noike, Yusuke Higuchi, Tohru DairiAbstract:Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified Fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to Fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8β,16-diol. In this study, we repo...
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Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Tomonobu Toyomasu, Tohru Dairi, Wataru Mitsuhashi, Mai Tsukahara, Akane Kaneko, Rie Niida, Nobuo Kato, Takeshi SassaAbstract: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.
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identification of phyllocladene sandaracopimaradiene and kaurene as new fungal metabolites from Fusicoccin producing phomopsis amygdali f6
Bioscience Biotechnology and Biochemistry, 2004Co-Authors: Hiromichi Kenmoku, Nobuo Kato, Mio Tanaka, Koichi Ogiyama, Takeshi SassaAbstract:A chemical analysis of the diterpene hydrocarbons produced by Fusicoccin-producing fungus Phomopsis amygdali F6 identified five phyllocladene-related tri- and tetracyclic diterpene hydrocarbons. The presence of (+)-phyllocladene, (−)-sandaracopimaradiene, (+)-isopimara-8,15-diene, and (+)-pimara-8(14),15-diene in the fungus was demonstrated by GC-MS, 1H-NMR, and [α]D measurements. (+)-Kaurene was also identified by GC-MS and chiral capillary GC. The possible biosynthetic relationship of these metabolites is discussed.
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fusicocca 3 16 10 14 diene and β and δ araneosenes new Fusicoccin biosynthesis related diterpene hydrocarbons from phomopsis amygdali
Bioscience Biotechnology and Biochemistry, 2004Co-Authors: Takeshi Sassa, Hiromichi Kenmoku, Kayoko Nakayama, Nobuo KatoAbstract:Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to Fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.
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novel Fusicoccins r and s and the Fusicoccin s aglycon phomopsiol from phomopsis amygdali niigata 2 a and their seed germination stimulating activity in the presence of abscisic acid
Bioscience Biotechnology and Biochemistry, 2004Co-Authors: Naoto Tajima, Nobuo Kato, Manabu Nukina, Takeshi SassaAbstract:Our search for new 3-hydroxyFusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy Fusicoccins, called Fusicoccins R and S, and the Fusicoccin S aglycon, called phomopsiol, together with known 3α-hydroxyFusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of Fusicoccins R and S were also determined to be those of 3′-deacetyl-3α-hydroxyFusicoccin A and 3β-hydroxy-3-epiFusicoccin H. The lettuce seed germination-stimulating activity of Fusicoccins R and S, phomopsiol and 3α-hydroxyFusicoccin J was examined in the presence of ABA; Fusicoccin R and 3α-hydroxyFusicoccin J were highly active, while Fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel Fusicoccins having a 3α- or 3β-hydroxy group in their diterpene moiety are briefly discussed.
Elmar W. Weiler - One of the best experts on this subject based on the ideXlab platform.
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the Fusicoccin receptor of plants is a member of the 14 3 3 superfamily of eukaryotic regulatory proteins
FEBS Letters, 1994Co-Authors: Claudia Oecking, Christoph Eckerskorn, Elmar W. WeilerAbstract:The receptor for the wilt-inducing phytotoxin Fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9′-nor-8′-hydroxyFusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the Fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14-3-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14-3-3 isoform(s) constituting the Fusicoccin receptor are distinguishable from other cellular 14-3-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-114 phase separation experiments, they, as well as the target enzyme of Fusicoccin action, the H+-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of Fusicoccin action.
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Fusicoccin action in cell suspension cultures of corydalis sempervirens pers
Planta, 1991Co-Authors: Simone Schulz, Edith Oelgemoller, Elmar W. WeilerAbstract:Mid-log-phase cell suspensions of Corydalis sempervirens Pers., when incubated in micromolar or submicromolar concentrations of Fusicoccin, strongly acidified the culture medium. High-affinity Fusicoccin-binding sites were found in microsomes prepared from these cells using the radioligand [3H]-9′-norFusicoccin-8′-alcohol. Binding was saturable with an apparent dissociation constant (Kd) of 2.8 nM, a pH optimum of 6.0, a temperature optimum of 35° C and was rapid (t1/2 = 8 min). The site abundance was 0.76±0.17 pmol · (mg of protein)−1. In the same membrane preparations, the K+, Mg2+-ATPase (EC 3.6.1.3) was characterized. The enzyme was highly vanadate-sensitive (IC50=6.5 μM) and nucleotide-specific (ATP≫NTP), had a pH optimum of 6.2, an apparent Km for ATP of 0.23±0.12 mM, and Vmax of 10.6±1.8 nkat (mg of protein)−1. Fusicoccin doubled Vmax and lowered, by a factor of 2, the apparent Km for ATP of the enzyme when the cells were incubated with the toxin for 30 min prior to homogenization of the cells. The stimulation of the enzyme was also pronounced when Fusicoccin was added to the homogenization medium just prior to homogenization of the cells, but was slight to zero when the toxin was added at the microsomal stage. The pronounced stimulatory effect of Fusicoccin on the ATPase was seen at pH 7.1, i.e. at a pH typical for the cytoplasmic compartment, but was not detectable at pH 6.2, the pH optimum of the enzyme. The implications of these findings for an understanding of Fusicoccin action are discussed.
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Characterization and purification of the Fusicoccin-binding complex from plasma membranes of Commelina communis.
European Journal of Biochemistry, 1991Co-Authors: Claudia Oecking, Elmar W. WeilerAbstract:The fungal phytotoxin Fusicoccin binds with high affinity to plasma membranes of the monocotyledonous plant, Commelina communis L. The sites bind the toxin with an apparent Kd of 5.2 nM and a pH optimum of 6.0. They occur at a level of approximately 6–8 pmol/mg plasma membrane protein. Photoaffinity labeling with the biologically active Fusicoccin derivative 9′-nor-8′-(4-azido[3,5-3H]benzoyl) diaminoethylFusicoccin identified a polypeptide of 31.5 kDa on SDS/PAGE which was strongly labeled. A second 32.5-kDa band was also consistently labeled, although not to the same extent. The binding sites were solubilized in functional form and a purification scheme was developed based on affinity and ion-exchange procedures. The purified fraction contains two polypeptides of apparent molecular masses of 30.5 kDa and 31.6 kDa. A detailed molecular analysis of the Fusicoccin-binding complex is now possible.
