33 results on '"Akiho Yokota"'
Search Results
2. Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon
- Author
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Akira Katoh, Kouhei Hanada, Kazuya Yoshimura, Masataka Kajikawa, Yoshihiko Nanasato, Rina Kosaka, Akiho Yokota, Kinya Akashi, Atsushi Kato, and Hisashi Tsujimoto
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0106 biological sciences ,0301 basic medicine ,Osmotic shock ,Cell division ,Arabidopsis ,Drought avoidance ,GTPase ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Plant Roots ,Analytical Chemistry ,Citrullus ,03 medical and health sciences ,Xerophyte ,Gene Expression Regulation, Plant ,Osmotic Pressure ,Botany ,Primordium ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,biology ,Dose-Response Relationship, Drug ,Organic Chemistry ,Lateral root ,fungi ,Water ,food and beverages ,General Medicine ,Wild watermelon ,biology.organism_classification ,Plants, Genetically Modified ,Droughts ,Ran-GTPase ,030104 developmental biology ,ran GTP-Binding Protein ,Root growth ,Ran ,010606 plant biology & botany ,Biotechnology - Abstract
Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions. AB - Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.
- Published
- 2016
3. Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO
- Author
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Hiroki Ashida, Antoine Danchin, Agnieszka Sekowska, Tomoko Sakiyama, Nicole Tandeau de Marsac, Yohtaro Saito, and Akiho Yokota
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Models, Molecular ,Protein Conformation ,Ribulose-Bisphosphate Carboxylase ,Molecular Sequence Data ,Enolase ,Bacillus subtilis ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Bacterial Proteins ,Amino Acid Sequence ,Photosynthesis ,Molecular Biology ,Phylogeny ,chemistry.chemical_classification ,Ribulose 1,5-bisphosphate ,Enzyme Catalysis and Regulation ,Sequence Homology, Amino Acid ,biology ,RuBisCO ,Carbon fixation ,Active site ,Cell Biology ,Carbon Dioxide ,biology.organism_classification ,Organophosphates ,Recombinant Proteins ,Amino acid ,Models, Chemical ,chemistry ,Phosphopyruvate Hydratase ,Mutagenesis, Site-Directed ,biology.protein ,Rhodopseudomonas palustris - Abstract
The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO2 fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg2+ for catalysis and, like RuBisCO, was stimulated by CO2. Four residues that are essential for the enolization reaction of RuBisCO, Lys175, Lys201, Asp203, and Glu204, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys123, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a Ki value of 103 μm. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.
- Published
- 2009
4. Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase fromBacillus subtilis
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Hiroki Ashida, Tsuyoshi Inoue, Akiho Yokota, Hiroyoshi Matsumura, Tomonori Yadani, Yohtaro Saito, Yasushi Kai, Haruka Tamura, and Shogo Koga
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Ribulose-Bisphosphate Carboxylase ,Molecular Sequence Data ,Enolase ,Biophysics ,Bacillus subtilis ,Crystallography, X-Ray ,Biochemistry ,law.invention ,Bacterial Proteins ,Structural Biology ,law ,PEG ratio ,Genetics ,Molecule ,Amino Acid Sequence ,Crystallization ,biology ,Chemistry ,Resolution (electron density) ,social sciences ,Condensed Matter Physics ,biology.organism_classification ,Organophosphates ,Recombinant Proteins ,Solvent ,Crystallography ,Crystallization Communications ,Phosphopyruvate Hydratase ,Protein Multimerization ,Monoclinic crystal system - Abstract
2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 A resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P2(1), with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 A, beta = 90.8 degrees. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V(M) value of 2.2 A(3) Da(-1) and a solvent content of 43%.
- Published
- 2009
5. Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon
- Author
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Kinya Akashi, Kentaro Takahara, and Akiho Yokota
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Ornithine ,Chloroplasts ,DNA, Complementary ,Light ,Arginine ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,Molecular Sequence Data ,Glutamic Acid ,Protein Sorting Signals ,Transfection ,Biochemistry ,Catalysis ,Amidohydrolases ,Citrullus ,chemistry.chemical_compound ,Biosynthesis ,Acetyltransferases ,Citrulline ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Plant Proteins ,chemistry.chemical_classification ,Dehydration ,Sequence Homology, Amino Acid ,biology ,Temperature ,Sequence Analysis, DNA ,Cell Biology ,Hydrogen-Ion Concentration ,Enzyme assay ,Amino acid ,Plant Leaves ,Chloroplast ,Kinetics ,Protein Transport ,Enzyme ,chemistry ,biology.protein ,Sequence Alignment - Abstract
Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.
- Published
- 2005
6. The Tomato Photomorphogenetic Mutant, aurea, is Deficient in Phytochromobilin Synthase for Phytochrome Chromophore Biosynthesis
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Philip J. Linley, Chitose Kami, Takuya Muramoto, Akiho Yokota, Takayuki Kohchi, Keiko Mukougawa, Hideo Kataoka, and Naoko Iwata
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Physiology ,Molecular Sequence Data ,Mutant ,Plant Science ,Biology ,Evolution, Molecular ,chemistry.chemical_compound ,Solanum lycopersicum ,Arabidopsis ,Amino Acid Sequence ,Bilin ,Peptide sequence ,Gene ,Phylogeny ,Genetics ,Expressed sequence tag ,Phytochrome ,food and beverages ,Cell Biology ,General Medicine ,biology.organism_classification ,Biochemistry ,chemistry ,Mutation ,Photomorphogenesis ,Oxidoreductases - Abstract
The aurea mutants of tomato have been widely used as phytochrome-deficient mutants for photomorphogenetic and photobiological studies. By expressed sequence tag (EST)-based screening of sequence databases, we found a tomato gene that encodes a protein homologous to Arabidopsis HY2 for phytochromobilin synthase catalyzing the last step of phytochrome chromophore biosynthesis. The tomato protein expressed in Escherichia coli showed phytochromobilin synthase activity. The corresponding loci in all aurea mutants tested have nucleotide substitutions, deletions or DNA rearrangements. These results indicate that aurea is a mutant of phytochromobilin synthase in tomato. We also discuss a phylogenetic analysis of phytochromobilin synthases in the bilin reductase family.
- Published
- 2005
7. Characterization of Arabidopsis ZIM, a member of a novel plant-specific GATA factor gene family
- Author
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Akiho Yokota, Yuko Matsuda, Kohei Ando, Takayuki Kohchi, Miho Takemura, Masahito Shikata, and Akiko Nishii
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Physiology ,Molecular Sequence Data ,Protein domain ,Plant Science ,Biology ,Conserved sequence ,chemistry.chemical_compound ,Arabidopsis ,Transcriptional regulation ,Brassinosteroid ,Gene family ,Amino Acid Sequence ,Cysteine ,Gene ,Transcription factor ,Conserved Sequence ,DNA Primers ,Genetics ,Base Sequence ,Sequence Homology, Amino Acid ,Arabidopsis Proteins ,Reverse Transcriptase Polymerase Chain Reaction ,food and beverages ,Zinc Fingers ,biology.organism_classification ,chemistry ,Multigene Family ,Sequence Alignment ,Transcription Factors - Abstract
The Arabidopsis gene ZIM encodes a putative transcription factor containing a novel GATA-type zinc-finger domain with a longer spacer between its two sets of conserved cysteine residues (C-X2-C-X20-C-X2-C). In Arabidopsis, ZIM and homologous proteins, ZML1 and ZML2, were identified as GATA factors containing the C-X2-C-X20-C-X2-C motif, a CCT domain, and an uncharacterized conserved domain. Proteins that possess this domain structure were found exclusively in plants, indicating that they belong to a novel family of plant-specific GATA-type transcription factors. When ZIM was overexpressed using a CaMV 35S promoter in Arabidopsis, hypocotyls and petioles were elongated. The elongation phenotype was observed under all wavelengths of light tested and even in the presence of biosynthetic inhibitors of either brassinosteroid or gibberellin. In ZIM-overexpressing plants, XTH33 which is predicted to function in cell wall modification was detected as an up-regulated gene by microarray analysis, and this could account for the elongation phenotype. Genes in ZIM-overexpressing plants were identified that were up-regulated in a tissue-specific manner, which suggests that transcriptional regulation by ZIM and its consequent effects are spatially controlled.
- Published
- 2004
8. Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices ofArabidopsis
- Author
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Akiho Yokota, Miho Takemura, Takayuki Kohchi, Jun-ichiro Hattan, and Hirosuke Kanamoto
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Green Fluorescent Proteins ,Meristem ,Molecular Sequence Data ,Arabidopsis ,Transfection ,Plant Roots ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Green fluorescent protein ,Gene Expression Regulation, Plant ,Auxin ,Two-Hybrid System Techniques ,Arabidopsis thaliana ,Amino Acid Sequence ,Molecular Biology ,Cell Proliferation ,chemistry.chemical_classification ,Indoleacetic Acids ,biology ,Arabidopsis Proteins ,Kinase ,fungi ,Organic Chemistry ,food and beverages ,General Medicine ,Meristem maintenance ,biology.organism_classification ,Cell biology ,chemistry ,Polar auxin transport ,Carrier Proteins ,Protein Kinases ,Sequence Alignment ,Protein Binding ,Signal Transduction ,Biotechnology - Abstract
Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown. A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI). IRKI is a novel but highly conserved protein found in higher plants. The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems. In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells. IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.
- Published
- 2004
9. Chemical modification of arginine alleviates the decline in activity during catalysis of spinach Rubisco
- Author
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M. Anwaruzzaman, Akiho Yokota, Eiichi Mizohata, Hiromi Okuno, Ken-ichi Tomizawa, Yasushi Kai, and Shigeru Shigeoka
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Models, Molecular ,Phenylglyoxal ,Oxygenase ,Arginine ,Protein Conformation ,Ribulose-Bisphosphate Carboxylase ,Biophysics ,Photosynthesis ,Peptide Mapping ,Biochemistry ,chemistry.chemical_compound ,Multienzyme Complexes ,Spinacia oleracea ,Amino Acid Sequence ,Molecular Biology ,Molecular Structure ,biology ,fungi ,digestive, oral, and skin physiology ,RuBisCO ,food and beverages ,Chemical modification ,Cell Biology ,biology.organism_classification ,Pyruvate carboxylase ,chemistry ,biology.protein ,Spinach ,Indicators and Reagents - Abstract
Arginine residues of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were chemically modified with phenylglyoxal (PhG). PhG inactivated Rubisco with a half-time of 20–25 min. An inclusion of a catalytic product, 3-phosphoglycerate (PGA), protected Rubisco from inactivation and delayed the half-time to 60–90 min. Peptide mapping and sequencing of Rubisco modified for 60 min with radiolabeled PhG in the presence of 10 mM PGA revealed that Arg187, Arg258, and Arg431 of the large subunit were modified. The extent and rate of the decline in activity during catalysis (fallover phenomenon) were reduced by the modification. This is the first report identifying PhG-modified arginine residues and to demonstrate the effect of the modification of arginine residues on the kinetics of fallover.
- Published
- 2003
10. Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5-bisphosphate
- Author
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Yasushi Kai, Ko Kato, Hiroyoshi Matsumura, Yousuke Okano, Maki Kumei, Tsuyoshi Inoue, Naoki Shibata, Hiroki Takuma, Akiho Yokota, Jun Onodera, and Eiichi Mizohata
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Models, Molecular ,Protein Folding ,Stereochemistry ,Ribulose-Bisphosphate Carboxylase ,Molecular Sequence Data ,Chlamydomonas reinhardtii ,Crystallography, X-Ray ,Photosynthesis ,Methylation ,Protein Structure, Secondary ,chemistry.chemical_compound ,Sugar Alcohols ,Protein structure ,Spinacia oleracea ,Structural Biology ,Animals ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,Pentosephosphates ,Binding Sites ,Ribulose 1,5-bisphosphate ,biology ,Chlamydomonas ,RuBisCO ,food and beverages ,Lyase ,biology.organism_classification ,Enzyme Activation ,Kinetics ,Protein Subunits ,chemistry ,Biochemistry ,biology.protein ,Protein folding ,RNA Editing ,Protein Processing, Post-Translational ,Sequence Alignment - Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO(2) and O(2), respectively, with ribulose-1,5-bisphosphate. Many photosynthetic organisms have form I rubiscos comprised of eight large (L) and eight small (S) subunits. The crystal structure of the complex of activated rubisco from the green alga Chlamydomonas reinhardtii and the reaction intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate (2-CABP) has been solved at 1.84 A resolution (R(cryst) of 15.2 % and R(free) of 18.1 %). The subunit arrangement of Chlamydomonas rubisco is the same as those of the previously solved form I rubiscos. Especially, the present structure is very similar to the activated spinach structure complexed with 2-CABP in the L-subunit folding and active-site conformation, but differs in S-subunit folding. The central insertion of the Chlamydomonas S-subunit forms the longer betaA-betaB loop that protrudes deeper into the solvent channel of rubisco than higher plant, cyanobacterial, and red algal (red-like) betaA-betaB loops. The C-terminal extension of the Chlamydomonas S-subunit does not protrude into the solvent channel, unlike that of the red algal S-subunit, but lies on the protein surface anchored by interactions with the N-terminal region of the S-subunit. Further, the present high-resolution structure has revealed novel post-translational modifications. Residue 1 of the S-subunit is N(alpha)-methylmethionine, residues 104 and 151 of the L-subunit are 4-hydroxyproline, and residues 256 and 369 of the L-subunit are S(gamma)-methylcysteine. Furthermore, the unusual electron density of residue 471 of the L-subunit, which has been deduced to be threonine from the genomic DNA sequence, suggests that the residue is isoleucine produced by RNA editing or O(gamma)-methylthreonine.
- Published
- 2002
11. Characterization of Ascorbate Peroxidases from Unicellular Red Alga Galdieria partita
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Masami Ueda, Norihide Kurano, Satoshi Sano, Shigetoh Miyachi, Chikahiro Miyake, Akiho Yokota, Sakihito Kitajima, Shigeru Shigeoka, and Toru Takeda
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Hot Temperature ,Euglena gracilis ,Physiology ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Plant Science ,Isozyme ,Euglena ,chemistry.chemical_compound ,Ascorbate Peroxidases ,Spinacia oleracea ,Enzyme Stability ,Botany ,Amino Acid Sequence ,Enzyme Inhibitors ,Heme ,Sequence Homology, Amino Acid ,biology ,ved/biology ,Chlamydomonas ,food and beverages ,Cell Biology ,General Medicine ,biology.organism_classification ,APX ,Peptide Fragments ,Isoenzymes ,Molecular Weight ,Kinetics ,Peroxidases ,chemistry ,Biochemistry ,Spectrophotometry ,Rhodophyta ,biology.protein ,Thermodynamics ,Sequence Alignment ,Peroxidase - Abstract
Galdieria partita, a unicellular red alga isolated from acidic hot springs and tolerant to sulfur dioxide, has at least two ascorbate peroxidase (APX) isozymes. This was the first report to demonstrate that two isozymes of APX are found in algal cells. Two isozymes were separated from each other at the hydrophobic chromatography step of purification and named APX-A and APX-B after the elution order in the chromatography. APX-B accounted for 85% of the total activity. Both isozymes were purified. APXs from Galdieria were monomers whose molecular weights were about 28,000, similar to stromal APX of higher plants. APX-A cross-reacted with monoclonal antibody raised against APX of Euglena gracilis in immunoblotting, but APX-B did not, although the antibody can recognize all other APXs tested. The amino-terminal sequences of APX-A and -B from Galdieria had some homology with each other but little homology with those from other sources. Their Km values for ascorbate and hydrogen peroxide were comparable with those of APX from higher plants. Unlike the green algal enzymes, the donor specificities of Galdieria APXs were as high as those of plant chloroplastic APX. On the contrary, these APXs reduced tertiary-butyl hydroperoxide as an electron acceptor as APXs from Euglena and freshwater Chlamydomonas do. The inhibition of APX-A and -B by cyanide and azide, and characteristics of their light absorbance spectra indicated that they were heme peroxidases.
- Published
- 2001
12. Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme
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Hyun-Chul Kim, Hiroki Ashida, Bitna Lim, Keun Ho Chun, Wonchull Kang, Yun Chan Lim, Jin Kuk Yang, Yong-Keun Jung, Akiho Yokota, Le Thi My Le, Na Yeon Kim, Hye Min Lee, Se Hoon Hong, Tae Yeon Kim, and Sang Soo Hah
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Programmed cell death ,Molecular Sequence Data ,Caspase 1 ,Apoptosis ,Methionine ,Catalytic Domain ,Neoplasms ,Humans ,Amino Acid Sequence ,Inflammation ,Multidisciplinary ,Cell Death ,Sequence Homology, Amino Acid ,biology ,Aldolase A ,Pyroptosis ,Lyase ,Salvage enzyme ,Caspase 9 ,Squamous carcinoma ,Molecular Docking Simulation ,PNAS Plus ,Biochemistry ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Apoptosis Regulatory Proteins ,Bacillus subtilis ,HeLa Cells - Abstract
APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 μM and a Vmax of 1.39 μmol min(-1) mg(-1). The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.
- Published
- 2013
13. Responses of Wild Watermelon to Drought Stress: Accumulation of an ArgE Homologue and Citrulline in Leaves during Water Deficits
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Shinji Kawasaki, Shinichiro Fujii, Takayuki Kohchi, Akiho Yokota, Chikahiro Miyake, and Masato Uchida
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DNA, Complementary ,Physiology ,Molecular Sequence Data ,Drought tolerance ,Acetylornithine deacetylase ,Plant Science ,Biology ,Amidohydrolases ,Disasters ,chemistry.chemical_compound ,Complementary DNA ,Citrulline ,Electrophoresis, Gel, Two-Dimensional ,Amino Acid Sequence ,Peptide sequence ,Plant Proteins ,chemistry.chemical_classification ,Base Sequence ,Sequence Homology, Amino Acid ,Water ,Cell Biology ,General Medicine ,Carboxypeptidase ,Amino acid ,Plant Leaves ,Enzyme ,Biochemistry ,chemistry ,Fruit ,biology.protein ,Peptides - Abstract
Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.
- Published
- 2000
14. Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ 70 factors of bacterial RNA polymerases in Arabidopsis thaliana
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Akiho Yokota, Kohki Yoshimoto, Hirokazu Kobayashi, Kyoichi Isono, Masanori Shimizu, Kimiyuki Satoh, and Yasuo Niwa
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Chloroplasts ,Molecular Sequence Data ,Arabidopsis ,Sigma Factor ,Genes, Plant ,Genome ,chemistry.chemical_compound ,Bacterial Proteins ,Transcription (biology) ,Sigma factor ,RNA polymerase ,Amino Acid Sequence ,Plastid ,Gene ,Polymerase ,Genetics ,Multidisciplinary ,biology ,Arabidopsis Proteins ,DNA-Directed RNA Polymerases ,Biological Sciences ,biology.organism_classification ,chemistry ,biology.protein ,Peptides ,Sequence Alignment - Abstract
Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1 , SIG2 , and SIG3 , for polypeptides possessing amino acid sequences for domains conserved in σ 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana . Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.
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- 1997
15. Potential involvement of N-terminal acetylation in the quantitative regulation of the ε subunit of chloroplast ATP synthase under drought stress
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Kazuo Yoshida, Akiho Yokota, Saki Hoshiyasu, Masayuki Fujiwara, Kaori Kohzuma, Yoichiro Fukao, and Kinya Akashi
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Gene isoform ,Protein subunit ,Molecular Sequence Data ,Applied Microbiology and Biotechnology ,Biochemistry ,Isozyme ,Aminopeptidases ,Analytical Chemistry ,Citrullus ,chemistry.chemical_compound ,Adenosine Triphosphate ,Stress, Physiological ,Chloroplast Proton-Translocating ATPases ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,biology ,Hydrolysis ,fungi ,Organic Chemistry ,food and beverages ,Acetylation ,General Medicine ,biology.organism_classification ,Droughts ,Chloroplast ,Isoenzymes ,Protein Subunits ,chemistry ,Adenosine triphosphate ,Biotechnology - Abstract
In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the e subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the e isoforms indicated that the structural difference between the e isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated e isoform preferentially decreased in drought, whereas the abundance of the acetylated e isoform was unchanged. Moreover, metalloprotease activity that decomposed the e subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated e subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the e subunit involves N-terminal acetylation and stress-induced proteases.
- Published
- 2013
16. Responses of the photosynthetic electron transport system to excess light energy caused by water deficit in wild watermelon
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Akiho Yokota, Satoko Sanda, Tadayuki Kawamura, Masayoshi Kuwano, Yuri Munekage, Kinya Akashi, and Kazuo Yoshida
- Subjects
Iron-Sulfur Proteins ,Photoinhibition ,Light ,Physiology ,Molecular Sequence Data ,Dehydrogenase ,Plant Science ,Biology ,Photosynthesis ,Thylakoids ,Citrullus ,Electron Transport ,Soil ,Stress, Physiological ,Genetics ,medicine ,Electrophoresis, Gel, Two-Dimensional ,Dehydration ,Amino Acid Sequence ,Photosystem ,Plant Proteins ,food and beverages ,Membrane Proteins ,Water ,Cell Biology ,General Medicine ,medicine.disease ,Electron transport chain ,Droughts ,Plant Leaves ,Kinetics ,Isoelectric point ,Biochemistry ,Solubility ,NAD+ kinase ,Sequence Alignment - Abstract
In plants, drought stress coupled with high levels of illumination causes not only dehydration of tissues, but also oxidative damage resulting from excess absorbed light energy. In this study, we analyzed the regulation of electron transport under drought/high-light stress conditions in wild watermelon, a xerophyte that shows strong resistance to this type of stress. Under drought/high-light conditions that completely suppressed CO(2) fixation, the linear electron flow was diminished between photosystem (PS) II and PS I, there was no photoinhibitory damage to PS II and PS I and no decrease in the abundance of the two PSs. Proteome analyses revealed changes in the abundance of protein spots representing the Rieske-type iron-sulfur protein (ISP) and I and K subunits of NAD(P)H dehydrogenase in response to drought stress. Two-dimensional electrophoresis and immunoblot analyses revealed new ISP protein spots with more acidic isoelectric points in plants under drought stress. Our findings suggest that the modified ISPs depress the linear electron transport activity under stress conditions to protect PS I from photoinhibition. The qualitative changes in photosynthetic proteins may switch the photosynthetic electron transport from normal photosynthesis mode to stress-tolerance mode.
- Published
- 2011
17. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA
- Author
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Kenji, Nishimura, Hiroki, Ashida, Taro, Ogawa, and Akiho, Yokota
- Subjects
Chloroplasts ,Genes, Essential ,RNA, Chloroplast ,Sequence Homology, Amino Acid ,Arabidopsis Proteins ,Ribulose-Bisphosphate Carboxylase ,Blotting, Western ,Green Fluorescent Proteins ,Molecular Sequence Data ,Arabidopsis ,Blotting, Northern ,Recombinant Proteins ,DEAD-box RNA Helicases ,RNA, Ribosomal, 23S ,Mutation ,Amino Acid Sequence ,Protein Binding - Abstract
In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.
- Published
- 2010
18. Crystal structure of 5-methylthioribose 1-phosphate isomerase product complex from Bacillus subtilis: Implications for catalytic mechanism
- Author
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Yohtaro Saito, Akiho Yokota, Haruka Tamura, Tsuyoshi Inoue, Hiroyoshi Matsumura, Yasushi Kai, and Hiroki Ashida
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Molecular Sequence Data ,Bacillus subtilis ,Isomerase ,Crystallography, X-Ray ,Biochemistry ,Article ,Conserved sequence ,Protein structure ,Bacterial Proteins ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Aldose-Ketose Isomerases ,Conserved Sequence ,Binding Sites ,biology ,Chemistry ,Substrate (chemistry) ,Active site ,Protein superfamily ,biology.organism_classification ,biology.protein - Abstract
The methionine salvage pathway (MSP) plays a crucial role in recycling a sulphahydryl derivative of the nucleoside. Recently, the genes and reactions in MSP from Bacillus subtilis have been identified, where 5-methylthioribose 1-phosphate isomerase (M1Pi) catalyzes a conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). Herein, we report the crystal structures of B. subtilis M1Pi (Bs-M1Pi) in complex with its product MTRu-1-P, and a sulfate at 2.4 and 2.7 A resolution, respectively. The electron density clearly shows the presence of each compound in the active site. The structural comparison with other homologous proteins explains how the substrate uptake of Bs-M1Pi may be induced by an open/closed transition of the active site. The highly conserved residues at the active site, namely, Cys160 and Asp240 are most likely to be involved in catalysis. The structural analysis sheds light on its catalytic mechanism of M1Pi.
- Published
- 2008
19. Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2
- Author
-
Sakihito, Kitajima, Taise, Shimaoka, Miyo, Kurioka, and Akiho, Yokota
- Subjects
Models, Molecular ,Binding Sites ,Chloroplasts ,Molecular Sequence Data ,Tryptophan ,Heme ,Hydrogen Peroxide ,Recombinant Proteins ,Ascorbate Peroxidases ,Cross-Linking Reagents ,Peroxidases ,Catalytic Domain ,Mutation ,Tobacco ,Amino Acid Sequence ,Apoproteins ,Plant Proteins ,Protein Binding - Abstract
Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.
- Published
- 2007
20. A new rubisco-like protein coexists with a photosynthetic rubisco in the planktonic cyanobacteria Microcystis
- Author
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Elke Dittmann, Agnieszka Sekowska, Philippe Quillardet, Hiroki Ashida, Yohtaro Saito, Akiho Yokota, Isabelle Callebaut, Christiane Bouchier, Alyssa Carré-Mlouka, Nicole Tandeau de Marsac, Annick Méjean, Génétique et biochimie des microorganismes (GBM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences, Nara Institute of Science and Technology, Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche Université de Hong-Kong-Pasteur, Réseau International des Instituts Pasteur (RIIP), Institute of Biology, Humboldt-Universität zu Berlin, Génomique (Plate-Forme) - Genomics Platform, and Institut Pasteur [Paris]
- Subjects
Models, Molecular ,Cyanobacteria ,Microcystis ,Low protein ,Subfamily ,Transcription, Genetic ,Ribulose-Bisphosphate Carboxylase ,Molecular Sequence Data ,Photosynthesis ,Biochemistry ,03 medical and health sciences ,Bacterial Proteins ,Microcystis aeruginosa ,Amino Acid Sequence ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,RuBisCO ,Cell Biology ,biology.organism_classification ,Chloroplast ,Genes, Bacterial ,biology.protein ,Sequence Alignment ,Genome, Bacterial - Abstract
Two genes encoding proteins related to large subunits of Rubisco were identified in the genome of the planktonic cyanobacterium Microcystis aeruginosa PCC 7806 that forms water blooms worldwide. The rbcL(I) gene belongs to the form I subfamily typically encountered in cyanobacteria, green algae, and land plants. The second and newly discovered gene is of the form IV subfamily and widespread in the Microcystis genus. In M. aeruginosa PCC 7806 cells, the expression of both rbcL(I) and rbcL(IV) is sulfur-dependent. The purified recombinant RbcL(IV) overexpressed in Escherichia coli cells did not display CO(2) fixation activity but catalyzed enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate, and the rbcL(IV) gene rescued a Bacillus subtilis MtnW-deficient mutant. Therefore, the Microcystis RbcL(IV) protein functions both in vitro and in vivo and might be involved in a methionine salvage pathway. Despite variations in the amino acid sequences, RbcL(IV) shares structural similarities with all members of the Rubisco superfamily. Invariant amino acids within the catalytic site may thus represent the minimal set for enolization, whereas variations, especially located in loop 6, may account for the limitation of the catalytic reaction to enolization. Even at low protein concentrations in vitro, the recombinant RbcL(IV) assembles spontaneously into dimers, the minimal unit required for Rubisco forms I-III activity. The discovery of the coexistence of RbcL(I) and RbcL(IV) in cyanobacteria, the ancestors of chloroplasts, enlightens episodes of the chaotic evolutionary history of the Rubiscos, a protein family of major importance for life on Earth.
- Published
- 2006
21. An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation
- Author
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Akiho Yokota, Shigeru Shigeoka, Sakihito Kitajima, and Ken-ichi Tomizawa
- Subjects
Chloroplasts ,GPX3 ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Biochemistry ,chemistry.chemical_compound ,Ascorbate Peroxidases ,Cytosol ,Spinacia oleracea ,Tobacco ,Amino Acid Sequence ,Hydrogen peroxide ,Molecular Biology ,Heme ,Binding Sites ,biology ,Cytochrome c peroxidase ,Cell Biology ,Hydrogen Peroxide ,Molecular biology ,chemistry ,Peroxidases ,Thylakoid ,Rhodophyta ,biology.protein ,Sequence Alignment ,Peroxidase - Abstract
Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249–264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.
- Published
- 2006
22. Co-expression of cytochrome b561 and ascorbate oxidase in leaves of wild watermelon under drought and high light conditions
- Author
-
Kinya Akashi, Yoshihiko Nanasato, and Akiho Yokota
- Subjects
DNA, Complementary ,Citrullus lanatus ,Light ,Physiology ,Drought tolerance ,Molecular Sequence Data ,Plant Science ,Green fluorescent protein ,Citrullus ,Disasters ,Amino Acid Sequence ,Cloning, Molecular ,Phylogeny ,DNA Primers ,Cytochrome b561 ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,food and beverages ,Cell Biology ,General Medicine ,biology.organism_classification ,Cytochrome b Group ,Apoplast ,Chloroplast ,Plant Leaves ,Light intensity ,Biochemistry ,Ascorbate Oxidase ,Electrophoresis, Polyacrylamide Gel ,Subcellular Fractions - Abstract
Despite carrying out C3 photosynthesis, wild watermelon (Citrullus lanatus sp.) exhibits exceedingly good tolerance to severe drought at high light intensities. However, the mechanism(s) by which this plant protects itself from photodamage has yet to be elucidated. In this study, we characterized wild watermelon cytochrome b561 (cyt b561), which potentially mediates regeneration of apoplastic ascorbate by transferring electrons from cytosolic ascorbate across the plasma membrane. Two cDNA species for wild watermelon cyt b561, designated CLb561A and CLb561B, were isolated. Levels of both CLb561A mRNA and protein were significantly elevated in the leaves during drought at a light intensity of 700 micromol photons m(-2) s(-1). The transcript of CLb561B was detected to a much lesser extent, but no CLb561B protein was produced under any condition used in this study. A transient expression assay with the CLb561A::green fluorescent protein fusion construct showed clear fluorescence on the plasma membrane of onion epidermal cells. The CLb561A protein was enriched in the plasma membrane fraction in leaves of transgenic tobacco expressing CLb561A. Moreover, the high activity of apoplastic ascorbate oxidase (AO), which was able to dispose of cyt b561-transferred reducing equivalents, increased in leaves of wild watermelon grown at high light intensity, but not lower light intensities. Taken together, these observations suggest the occurrence of a novel pathway for excess light energy dissipation in wild watermelon leaves, where excessive energy absorbed by chloroplasts can be transported to and dissipated safely in the apoplasts through the cooperative action of cyt b561 and AO.
- Published
- 2005
23. Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon
- Author
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Noriyuki Nishimura, Yoshinori Ishida, Kinya Akashi, and Akiho Yokota
- Subjects
Antioxidant ,DNA, Complementary ,Time Factors ,Citrullus lanatus ,medicine.medical_treatment ,Radical ,Drought tolerance ,Molecular Sequence Data ,Biophysics ,Biology ,medicine.disease_cause ,Biochemistry ,Catalysis ,Citrullus ,Disasters ,chemistry.chemical_compound ,Botany ,medicine ,Metallothionein ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Molecular Biology ,Phylogeny ,Plant Proteins ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,Arabidopsis Proteins ,Hydroxyl Radical ,Gene Expression Profiling ,Cell Biology ,DNA ,biology.organism_classification ,Recombinant Proteins ,Oxidative Stress ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,RNA ,Hydroxyl radical ,Oxidative stress - Abstract
Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions.
- Published
- 2004
24. A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO
- Author
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Akiho Yokota, Kazuo Kobayashi, Yohtaro Saito, Naotake Ogasawara, Chojiro Kojima, and Hiroki Ashida
- Subjects
Magnetic Resonance Spectroscopy ,Operon ,Protein subunit ,Ribulose-Bisphosphate Carboxylase ,Mutant ,Molecular Sequence Data ,Bacillus subtilis ,Photosynthesis ,Rhodospirillum rubrum ,Catalysis ,Methionine ,Bacterial Proteins ,Amino Acid Sequence ,Phylogeny ,Multidisciplinary ,biology ,RuBisCO ,biology.organism_classification ,Recombinant Proteins ,Biochemistry ,Genes, Bacterial ,Thioglycosides ,Mutation ,biology.protein ,Sequence Alignment ,Bacteria - Abstract
The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis , and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We found that such a RuBisCO-like protein (RLP) from B. subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCOfrom the photosynthetic bacterium Rhodospirillum rubrum . Thus, the photosynthetic RuBisCO from R. rubrum retains the ability to function in the methionine salvage pathway in B. subtilis .
- Published
- 2003
25. Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts
- Author
-
Taise, Shimaoka, Chikahiro, Miyake, and Akiho, Yokota
- Subjects
Oxidative Stress ,Chloroplasts ,Sequence Homology, Amino Acid ,Spinacia oleracea ,Blotting, Western ,Molecular Sequence Data ,Escherichia coli ,Mutagenesis, Site-Directed ,Electrophoresis, Polyacrylamide Gel ,Amino Acid Sequence ,Oxidoreductases ,Catalysis ,Recombinant Proteins - Abstract
Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The Km value for DHA and the summed Km value for GSH were determined to be 53 +/- 12 micro m and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s-1. Incubation of 10 microm DHAR with 1 mm DHA and 10 microm GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.
- Published
- 2003
26. Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants
- Author
-
Akiho Yokota, Masami Ueda, Ken-ichi Tomizawa, Takayuki Kohchi, Sakihito Kitajima, Satoshi Sano, Chikahiro Miyake, and Shigeru Shigeoka
- Subjects
Chloroplasts ,DNA, Complementary ,Immunoblotting ,Molecular Sequence Data ,Ascorbic Acid ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Ascorbate Peroxidases ,Cytosol ,L-ascorbate peroxidase ,Chlorophyta ,Enzyme Stability ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Plant Proteins ,chemistry.chemical_classification ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Organic Chemistry ,food and beverages ,Active site ,General Medicine ,Plants ,APX ,Recombinant Proteins ,Amino acid ,Chloroplast ,Isoenzymes ,Kinetics ,chemistry ,Peroxidases ,Spectrophotometry ,Rhodophyta ,biology.protein ,Sequence Alignment ,Biotechnology ,Peroxidase - Abstract
Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.
- Published
- 2003
27. Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves
- Author
-
Akiho Yokota, Taise Shimaoka, and Chikahiro Miyake
- Subjects
Chloroplasts ,DNA, Complementary ,Physiology ,Trypsin inhibitor ,Molecular Sequence Data ,Plant Science ,Photosynthesis ,Spinacia oleracea ,Complementary DNA ,Amino Acid Sequence ,Plastid ,Cloning, Molecular ,Peptide sequence ,chemistry.chemical_classification ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,food and beverages ,Cell Biology ,General Medicine ,biology.organism_classification ,Molecular biology ,Chloroplast ,Isoenzymes ,Plant Leaves ,Kinetics ,Enzyme ,Biochemistry ,chemistry ,Spinach ,Electrophoresis, Polyacrylamide Gel ,Oxidoreductases ,Chromatography, Liquid - Abstract
Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had K(m) values of 70 micromolar and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 micromol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.
- Published
- 2001
28. Characterization of a novel gene encoding a putative single zinc-finger protein, ZIM, expressed during the reproductive phase in Arabidopsis thaliana
- Author
-
Masahito Shikata, Akiko Nishii, Hidetomo Fujita, Akiho Yokota, Miho Takemura, and Takayuki Kohchi
- Subjects
DNA, Complementary ,DNA, Plant ,Molecular Sequence Data ,Arabidopsis ,Gene Expression ,Biology ,Genes, Plant ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Complementary DNA ,Gene expression ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,Gene ,Gene Library ,Plant Proteins ,Genetics ,Zinc finger ,Cell Nucleus ,Base Sequence ,Sequence Homology, Amino Acid ,cDNA library ,Arabidopsis Proteins ,Organic Chemistry ,Nucleic acid sequence ,Zinc Fingers ,General Medicine ,Nuclear localization sequence ,Genome, Plant ,Biotechnology ,Transcription Factors - Abstract
By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.
- Published
- 2000
29. The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase
- Author
-
Takayuki Kohchi, Howard M. Goodman, Akiho Yokota, Inhwan Hwang, and Takuya Muramoto
- Subjects
Oxygenase ,Swine ,Mutant ,Molecular Sequence Data ,Arabidopsis ,Plant Science ,Green fluorescent protein ,chemistry.chemical_compound ,Gene Expression Regulation, Plant ,Transit Peptide ,Animals ,Humans ,Amino Acid Sequence ,Plastids ,Cloning, Molecular ,Heme ,Biliverdin ,biology ,food and beverages ,Cell Biology ,biology.organism_classification ,Plants, Genetically Modified ,Heme oxygenase ,chemistry ,Biochemistry ,Heme Oxygenase (Decyclizing) ,Mutation ,Phytochrome ,Research Article - Abstract
The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.
- Published
- 1999
30. Distribution of fallover in the carboxylase reaction and fallover-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms
- Author
-
Akiho Yokota, Jin Hamada, Koichi Uemura, Hiroshi Tokai, Hiroshi Murayama, Yukito Enomoto, Shoko Fujiwara, Takashi Higuchi, and Hiroshi Yamamoto
- Subjects
Oxygenase ,Euglena gracilis ,Physiology ,Ribulose-Bisphosphate Carboxylase ,ved/biology.organism_classification_rank.species ,Plant Science ,Biology ,Photosynthesis ,Catalysis ,chemistry.chemical_compound ,Species Specificity ,Spinacia oleracea ,Animals ,Amino Acid Sequence ,Cells, Cultured ,Phylogeny ,Ribulose 1,5-bisphosphate ,Bacteria ,Sequence Homology, Amino Acid ,ved/biology ,Ribulose ,fungi ,RuBisCO ,food and beverages ,Eukaryota ,Cell Biology ,General Medicine ,Plants ,Pyruvate carboxylase ,Kinetics ,Biochemistry ,chemistry ,Suicide inhibition ,Enzyme Induction ,biology.protein ,Sequence Alignment - Abstract
The biphasic reaction course, fallover, of carboxylation catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gonatozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.
- Published
- 1998
31. MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila
- Author
-
Hiroki Ashida, Toshihiro Nakano, Akiho Yokota, and Izuru Ohki
- Subjects
Protein Conformation ,Molecular Sequence Data ,Enzyme Metabolism ,Protein domain ,lcsh:Medicine ,Bacillus subtilis ,Gene mutation ,Biochemistry ,Microbiology ,Tetrahymena thermophila ,Ribulosephosphates ,chemistry.chemical_compound ,Methionine ,Model Organisms ,Dioxygenase ,Amino Acid Sequence ,lcsh:Science ,Biology ,Hydro-Lyases ,Phylogeny ,Microbial Metabolism ,Liquid Chromatography ,chemistry.chemical_classification ,Chromatography ,Multidisciplinary ,biology ,Protozoan Models ,lcsh:R ,Tetrahymena ,Proteins ,biology.organism_classification ,Recombinant Proteins ,Enzymes ,Chemistry ,Metabolism ,Enzyme ,chemistry ,Dehydratase ,lcsh:Q ,Metabolic Pathways ,Signal Transduction ,Research Article - Abstract
To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.
- Published
- 2013
32. The Arabidopsis HY2 Gene Encodes Phytochromobilin Synthase, a Ferredoxin-Dependent Biliverdin Reductase
- Author
-
Munehisa Masuda, J. Clark Lagarias, Akiho Yokota, Nicole Frankenberg, Keiko Mukougawa, and Takayuki Kohchi
- Subjects
Chromosomes, Artificial, Bacterial ,DNA, Plant ,Mutant ,Molecular Sequence Data ,Arabidopsis ,Plant Science ,Cyanobacteria ,Genes, Plant ,Green fluorescent protein ,chemistry.chemical_compound ,Transit Peptide ,Amino Acid Sequence ,Plastids ,Cloning, Molecular ,Ferredoxin ,Alleles ,Biliverdin ,Phytochrome ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Biliverdin reductase ,Chromosome Mapping ,Cell Biology ,biology.organism_classification ,Plants, Genetically Modified ,Recombinant Proteins ,chemistry ,Biochemistry ,Mutation ,Oxidoreductases ,Research Article - Abstract
Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P Phi B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in P Phi B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P Phi B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX alpha to P Phi B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria.
- Published
- 2001
33. Thioredoxin peroxidase in the Cyanobacterium Synechocystis sp. PCC 6803
- Author
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Hiroshi Yamamoto, Akiho Yokota, Ken-ichi Tomizawa, Karl-Josef Dietz, Norio Murata, and Chikahiro Miyake
- Subjects
Photosystem II ,Molecular Sequence Data ,Biophysics ,Gene Expression ,medicine.disease_cause ,Cyanobacteria ,Biochemistry ,Electron Transport ,Open Reading Frames ,tert-Butylhydroperoxide ,Structural Biology ,Genetics ,medicine ,Escherichia coli ,Amino Acid Sequence ,Photosynthesis ,Molecular Biology ,Peptide sequence ,Thioredoxin peroxidase ,DNA Primers ,chemistry.chemical_classification ,Synechocystis sp. PCC 6803 ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Synechocystis ,Cell Biology ,Peroxiredoxins ,biology.organism_classification ,Hydrogen peroxide ,Recombinant Proteins ,Amino acid ,Neoplasm Proteins ,Open reading frame ,chemistry ,Peroxidases ,Genes, Bacterial ,biology.protein ,Thioredoxin ,Chlorophyll fluorescence ,Cyanobacterium ,Peroxidase - Abstract
The amino acid sequence deduced from the open reading frame designated sll0755 in Synechocystis sp. PCC 6803 is similar to the amino acid sequences of thioredoxin peroxidases from other organisms, In the present study, we found that a recombinant SLL0755 protein that was expressed in Escherichia coli mas able to reduce H2O2 and tertiary butyl hydroperoxide with thioredoxin from E. coli as the electron donor, Targeted disruption of open reading frame sll0755 in Synechocystis sp, PCC 6803 cells completely eliminated the H2O2-dependent and tertiary butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II. These results indicate that the product of open reading frame sll0755 is a thioredoxin peroxidase whose activities are coupled to the photosynthetic electron transport system in Synechocystis sp. PCC 6803, (C) 1999 Federation of European Biochemical Societies.
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