研究者データベース

井上 晶(イノウエ アキラ)
水産科学研究院 海洋応用生命科学部門 海洋生物工学分野
教授

基本情報

所属

  • 水産科学研究院 海洋応用生命科学部門 海洋生物工学分野

職名

  • 教授

学位

  • 水産学博士(北海道大学)

科研費研究者番号

  • 70396307

J-Global ID

研究キーワード

  • アワビ   アルギン酸   褐藻類   ミオシン   タンパク質   コンブ類   アルギン酸リアーゼ   ATP分解酵素   組み換え酵素   セルラーゼ   カルモジュリン   酵素   プロトプラスト   マコンブ   ATPase   アクチン   モータータンパク質   細胞運動   水産学   海藻多糖   代謝   解凍系   ラミナラン   解糖系   アメフラシ   糖代謝   多糖分解酵素   腹足類   オリゴ糖   フコイダン   

研究分野

  • ライフサイエンス / 水圏生命科学

職歴

  • 2021年09月 - 現在 北海道大学 大学院水産科学研究院 教授
  • 2005年04月 - 2021年08月 北海道大学 大学院水産科学研究院 准教授

所属学協会

  • マリンバイオテクノロジー学会   日本水産学会   

研究活動情報

論文

  • Ryuji Nishiyama, Takao Ojima, Yuki Ohnishi, Yasuhiro Kumaki, Tomoyasu Aizawa, Akira Inoue
    Communications Biology 4 1 1254  2021年11月02日 [査読有り][通常論文]
     
    AbstractAlginate-assimilating bacteria degrade alginate into an unsaturated monosaccharide, which is converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU). DEHU is reduced to 2-keto-3-deoxy-D-gluconate by a DEHU-specific reductase using NAD(P)H. This is followed by pyruvate production via the Entner-Doudoroff pathway. Previously, we identified FlRed as a DEHU reductase in an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. Here, we showed that FlRed can also catalyze the oxidation of DEHU with NAD+, producing 2-keto-3-deoxy-D-glucarate (KDGR). FlRed showed a predilection for NADH and NAD+ over NADPH and NADP+, respectively, and the Km value for NADH was approximately 2.6-fold less than that for NAD+. Furthermore, we identified two key enzymes, FlDet and FlDeg, for KDGR catabolism. FlDet was identified as an enzyme of the ribonuclease activity regulator A family, which converts KDGR to α-ketoglutaric semialdehyde (α-KGSA). FlDeg, a type II α-KGSA dehydrogenase, generated α-ketoglutaric acid by oxidizing the aldehyde group of α-KGSA using NAD(P)+. Consequently, unlike the conventional DEHU reduction pathway, DEHU can be directly converted to α-ketoglutaric acid without consuming NAD(P)H. Alginate upregulated the expression of not only FlRed and two enzymes of the DEHU-reduction pathway, but also FlDet and FlDeg. These results revealed dual pathways of DEHU metabolism involving reduction or oxidation by FlRed.
  • Akira Inoue, Takao Ojima
    Biochemical and Biophysical Research Communications 545 112 - 118 2021年03月 [査読有り]
  • Akira Inoue, Masataka Kudo, Elisa Werner, Takao Ojima
    Carbohydrate Polymers 117306 - 117306 2020年11月 [査読有り][通常論文]
  • Joemark Narsico, Akira Inoue, Seiko Oka, Takao Ojima
    Biochemical and Biophysical Research Communications 525 4 982 - 988 2020年05月14日 [査読有り][通常論文]
     
    Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(β-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides.
  • Kenta Arai, Akira Inoue, Takao Ojima
    Fisheries Science 86 2 385 - 394 2020年03月 [査読有り]
  • Takahiro Yamaguchi, Joemark Narsico, Takanori Kobayashi, Akira Inoue, Takao Ojima
    Journal of Bioscience and Bioengineering 128 2 203 - 208 2019年08月 [査読有り]
  • Akira Inoue, Toshiyuki Iwayama, Takao Ojima
    Fisheries Science 85 4 717 - 729 2019年07月 [査読有り]
  • Inoue Akira, Ojima Takao
    Scientific Reports 9 1 4937  2019年03月 [査読有り][通常論文]
  • Takao Ojima, Mohammad M. Rahman, Yuya Kumagai, Ryuji Nishiyama, Joemark Narsico, Akira Inoue
    Methods in Enzymology 605 457 - 497 2018年01月01日 [査読有り][招待有り]
     
    Seaweed polysaccharides have been widely used as viscosifier, gelling agents, and stabilizer in the various application fields, e.g., food, pharmaceutical, nutraceutical, and chemical industries. Applications of seaweed polysaccharides are further expanding to versatile directions, e.g., biofuels, bioactive compounds, and functional materials for medical and basic researches. Production of functional oligo- and monosaccharides by the use of specific enzymes is also expected to improve the value of seaweed polysaccharides. The enzymes that depolymerize seaweed polysaccharides are distributed largely among seaweed-associating organisms like marine invertebrates and bacteria. Among them, herbivorous marine gastropods such as abalone and sea hare are the most prominent producers of polysaccharide-degrading enzymes. To date, various kinds of polysaccharide-degrading enzymes have been isolated from the digestive fluid and hepatopancreas of these animals. Among them, alginate lyase, β-1,3-glucanase, mannanase, and cellulase are the major constituents of their digestive fluid. In this chapter, the authors describe the general methods for the preparation and activity assay of the gastropod polysaccharide-degrading enzymes and provide basic knowledge for their primary structures.
  • Akira Inoue
    Methods in Enzymology 605 499 - 524 2018年01月01日 [査読有り][招待有り]
     
    Alginate, an anionic heteropolysaccharide extracted from natural brown algae, has useful properties for the food, chemical, medical, and agricultural industries. Degradation of alginate by alginate lyase is a key process to produce unsaturated oligoalginate and unsaturated monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid. Alginate lyases belonging to the polysaccharide lyase family 7 have been found in, and isolated from, organisms thriving in various environments. Furthermore, research on their function and structure has also progressed well. Here, the preparation of native and recombinant PL-7 alginate lyases and the methods for evaluation of enzymatic activity are summarized. Examples of PL-7 alginate lyase applications are also described.
  • Hui-Min Qin, Takuya Miyakawa, Akira Inoue, Ryuji Nishiyama, Akira Nakamura, Atsuko Asano, Takao Ojima, Masaru Tanokura
    Chemical Communications 54 5 555 - 558 2018年01月 [査読有り][通常論文]
     
    FlAlyA is an endolytic enzyme with a preference for polymannuronate. The crystal structure and mutagenesis studies elucidated that the structural variations at outer uronate-binding subsites +2, +3 and -2 control the enzymatic properties of PL-7 family enzymes. Lys158 mutations changed the pH dependency and enhanced the production of mono- and disaccharides.
  • Hui-Min Qin, Takuya Miyakawa, Akira Inoue, Akira Nakamura, Ryuji Nishiyama, Takao Ojima, Masaru Tanokura
    Scientific Reports 7 1 11425  2017年12月 [査読有り]
     
    Laminarinase from Flavobacterium sp. strain UMI-01, a new member of the glycosyl hydrolase 16 family of a marine bacterium associated with seaweeds, mainly degrades β-1,3-glucosyl linkages of β-glucan (such as laminarin) through the hydrolysis of glycosidic bonds. We determined the crystal structure of ULam111 at 1.60-Å resolution to understand the structural basis for its thermostability and substrate specificity. A calcium-binding motif located on the opposite side of the β-sheet from catalytic cleft increased its degrading activity and thermostability. The disulfide bridge Cys31-Cys34, located on the β2-β3 loop near the substrate-binding site, is responsible for the thermostability of ULam111. The substrates of β-1,3-linked laminarin and β-1,3-1,4-linked glucan bound to the catalytic cleft in a completely different mode at subsite -3. Asn33 and Trp113, together with Phe212, formed hydrogen bonds with preferred substrates to degrade β-1,3-linked laminarin based on the structural comparisons. Our structural information provides new insights concerning thermostability and substrate recognition that will enable the design of industrial biocatalysts.
  • Mitsuya Daisuke, Yamamoto Masashi, Okai Masahiko, Inoue Akira, Suzuki Tomohiro, Ojima Takao, Urano Naoto
    Advances in Micobiology 7 5 387 - 403 2017年05月 [査読有り][通常論文]
  • Hui-Min Qin, Takuya Miyakawa, Akira Inoue, Ryuji Nishiyama, Akira Nakamura, Atsuko Asano, Yoriko Sawano, Takao Ojima, Masaru Tanokura
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 6 2182 - 2190 2017年02月 [査読有り][通常論文]
     
    Alginate is an abundant algal polysaccharide, composed of beta-D-mannuronate and its C5 epimer alpha-L-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a beta-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 angstrom resolution and the putative substrate-binding model show that the enzyme adopts a beta-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the beta-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.
  • Ryuji Nishiyama, Akira Inoue, Takao Ojima
    MARINE DRUGS 15 2 2017年02月 [査読有り][通常論文]
     
    Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-D-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%-25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%-68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium state. An in vitro alginate-metabolizing system constructed from recFlKin, recFlAld, and previously reported alginate lyases and DEH reductase of the strain UMI-01 could convert alginate to pyruvate and glyceraldehyde-3-phosphate with an efficiency of 38%.
  • Crystal structure of laminarinase from Flavobacterium sp. UMI-01 reveals distinct thermostability and substrate specificity from other GH16 family members.
    H-M. Qin, T. Miyakawa, A. Inoue, A. Nakamura, T. Ojima, M. Tanokura
    Sci. Rep. 7 11425 1 - 9 2017年 [査読有り][通常論文]
  • Akira Inoue, Ryuji Nishiyama, Takao Ojima
    Algal Research 19 355 - 362 2016年11月 [査読有り][通常論文]
     
    We recently reported the isolation of Flavobacterium sp. UMI-01 as a novel alginate-assimilating bacterium from decayed brown algae. In our previous study, FlAlyA was purified and characterized as an endolytic alginate lyase, producing alginate oligosaccharides (AOSs) from its polymer. Additionally, crude extracts from strain UMI-01 were found to be capable of producing of 4-deoxy-L-erythro-5-hexoseulose uronic acids (DEHs) through degradation of AOSs. Here, we report the enzymatic characteristics of four alginate lyases, i.e., FlAlyA, FlAlyB, FlAlyC, and FlAlex, found in the genomic sequence of UMI-01 using recombinant proteins. Our results demonstrated that the distinct roles of these enzymes facilitated complete degradation of alginate to DEH in strain UMI-01. First, FlAlyA attacked the alginate polymer in an endolytic manner, producing AOS. Recombinant FlAlyA could completely degraded alginate into AOS in vitro, and its activity was 20-52-fold higher than those of commercially available enzymes. Next, DEH was generated as a final degradation product through the functions of FlAlyB, FlAlyC, and FlAlex, which exhibited distinct substrate preferences; FlAlyB, FlAlyC, and FlAlex were identified as polyM-, polyMG-, and polyG-specific enzymes, respectively. Thus, our results confirmed that the combination of these four enzymes resulted in the most efficient degradation of alginate to DEH in vitro. (C) 2016 Elsevier B.V. All rights reserved.
  • Makoto Terauchi, Chikako Nagasato, Akira Inoue, Toshiaki Ito, Taizo Motomura
    PLANTA 244 2 361 - 377 2016年08月 [査読有り][通常論文]
     
    This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.
  • Akira Inoue, Moe Anraku, Satoshi Nakagawa, Takao Ojima
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 30 15551 - 15563 2016年07月 [査読有り][通常論文]
     
    Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of epsilon-Proteobacteria Nitratiruptor sp.SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli. Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 degrees C and around 6, respectively. Ahigh concentration of NaCl (0.8-1.4 M) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 degrees C for 30 min. Heat stability was decreased in the presence of 5 mM DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.
  • Akira Inoue, Aya Satoh, Mio Morishita, Yuko Tokunaga, Takuya Miyakawa, Masaru Tanokura, Takao Ojima
    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS 16 282 - 291 2016年06月 [査読有り][通常論文]
     
    Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, beta-D-mannuronic acid and alpha-L-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to -VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35 degrees C, 7.0-8.2, and 300 mM, respectively, using the Ca2+-induced gel-formation assay. In addition, Ca2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, H-1-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes. (C) 2016 Elsevier B.V. All rights reserved.
  • Shogo Mochizuki, Ryuji Nishiyama, Akira Inoue, Takao Ojima
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 52 30962 - 30974 2015年12月 [査読有り][通常論文]
     
    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of similar to 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 degrees C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, alpha-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.
  • Akira Inoue, Chieco Mashino, Toshiki Uji, Naotsune Saga, Koji Mikami, Takao Ojima
    Current Biotechnology 4 3 240 - 248 2015年08月 [査読有り][通常論文]
     
    BACKGROUND: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. METHODS: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. RESULTS: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. CONCLUSION: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.
  • Akira Inoue, Ryuji Nishiyama, Shogo Mochizuki, Takao Ojima
    MARINE DRUGS 13 1 493 - 508 2015年01月 [査読有り][通常論文]
     
    In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.
  • Akira Inoue, Kohei Takadono, Ryuji Nishiyama, Kenji Tajima, Takanori Kobayashi, Takao Ojima
    MARINE DRUGS 12 8 4693 - 4712 2014年08月 [査読有り][通常論文]
     
    A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of similar to 30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 degrees C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I-Escherichia coli BL21(DE3) expression system and similar to eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.
  • Mohammad M. Rahman, Akira Inoue, Takao Ojima
    FRONTIERS IN CHEMISTRY 2 2014年 [査読有り][通常論文]
     
    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40 degrees C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55 degrees C for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic beta-1,4-glucanase (EC 3.2.1.4). A cDNA of 1013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.
  • Yuya Kumagai, Takuya Satoh, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 167 1 - 7 2014年01月 [査読有り][通常論文]
     
    Endo-beta-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 degrees C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-beta-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences. (C) 2013 Elsevier Inc. All rights reserved.
  • Takuya Satoh, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 166 1 15 - 22 2013年09月 [査読無し][通常論文]
     
    Previously, we isolated two alpha-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains alpha-glucosidase-like enzymes together with the alpha-amylases. Thus, in the present study, we isolated this alpha-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97 kDa on SDS-PAGE, and optimal temperature and pH of 45 degrees C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an alpha-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl alpha - D-glucopyranoside (alpha-pNPG) and isomaltose. This enzyme was regarded as a maltase-like alpha-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone alpha-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian alpha-glucosidases belonging to glycoside-hydrolase-family31. (C) 2013 Elsevier Inc. All rights reserved.
  • Takuya Ueda, Yuka Nakamura, Caleb M. Smith, Bryan A. Copits, Akira Inoue, Takao Ojima, Satoko Matsunaga, Geoffrey T. Swanson, Ryuichi Sakai
    GLYCOBIOLOGY 23 4 412 - 425 2013年04月 [査読有り][通常論文]
     
    Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100 degrees C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.
  • Yuya Kumagai, Takuya Satoh, Akira Inoue, Takao Ojima
    Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology 164 2 80 - 88 2013年02月 [査読無し][通常論文]
     
    Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58. kDa and 82. kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30 °C and 6.7, and 30 °C and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, the activity toward maltotetraose was appreciably higher in HdAmy82 than HdAmy58. cDNAs encoding HdAmy58 and HdAmy82 were cloned and the amino-acid sequences of 511 and 694 residues for HdAmy58 and HdAmy82, respectively, were deduced. The putative catalytic domains of HdAmy58 and HdAmy82 were located in the 17-511th and 19-500th amino-acid regions, respectively, and they showed approximately 50% amino-acid identity to each other. These sequences also showed 62-99% amino-acid identity to the catalytic domains of known α-amylases that belong to glycoside-hydrolase-family 13. The difference in the molecular masses between HdAmy58 and HdAmy82 was ascribed to the extension of approximately 190 residues in the C-terminus of HdAmy82. This extended region showed 41-63% amino-acid identity with the ancillary domains of several α-amylases previously reported. © 2012 Elsevier Inc.
  • Mohammad Matiur Rahman, Ling Wang, Akira Inoue, Takao Ojima
    CARBOHYDRATE RESEARCH 360 69 - 77 2012年10月 [査読有り][通常論文]
     
    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate. (C) 2012 Elsevier Ltd. All rights reserved.
  • Ueda Takuya, Nakamura Yuka, Focia Pamela J, Smith Caleb, Inoue Akira, Ojima Takao, Matsunaga Satoko, Freymann Douglas M, Sakai Ryuichi, Swanson Geoffrey T
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 244 2012年08月19日 [査読有り][通常論文]
  • Ling Wang, Mohammad Matiur Rahman, Akira Inoue, Takao Ojima
    FISHERIES SCIENCE 78 4 889 - 896 2012年07月 [査読有り][通常論文]
     
    Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate lyases (Hata et al., Fish Sci 75:755-763, 2009). LbAly35 showed practically no similarity to other molluscan alginate lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45 A degrees C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093 bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate lyases, the catalytic domain of LbAly35 showed similar to 45 % identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate lyases despite the significant deviation of its N-terminal region.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 162 1-3 24 - 33 2012年05月 [査読有り][通常論文]
     
    A beta-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 degrees C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 degrees C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 degrees C with p-nitrophenyl beta-D-mannopyranoside were 0.10 mM and 3.75 mu mol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic beta-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) beta-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod. (C) 2012 Elsevier Inc. All rights reserved.
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    BIOCHIMIE 93 10 1720 - 1730 2011年10月 [査読有り][通常論文]
     
    Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed similar to 40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg: however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1. (C) 2011 Elsevier Masson SAS. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 159 4 227 - 235 2011年08月 [査読無し][通常論文]
     
    Previously we isolated an endo-beta-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392 bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96 Da. Comparison of the primary structure of AkMan with other beta-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 beta-1,4-mannanases indicated that AkMan together with other molluscan beta-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21 (DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells. (C) 2011 Elsevier Inc. All rights reserved.
  • Akira Inoue, Chieco Mashino, Teina Kodama, Takao Ojima
    MARINE BIOTECHNOLOGY 13 2 256 - 263 2011年04月 [査読有り][通常論文]
     
    Functional recombinant abalone alginate lyase (rHdAly) and beta-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30A degrees C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35A degrees C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (< 1 x 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 x 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 mu m, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 4 317 - 325 2010年12月 [査読有り][通常論文]
     
    Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40 degrees C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38 degrees C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed similar to 20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14. (C) 2010 Elsevier Inc. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 1 137 - 143 2010年09月 [査読有り][通常論文]
     
    A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear beta-1,4-mannan from green algae Codium fragile producing tri-and disaccharides. The optimal temperature of AkMan was 55 degrees C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 degrees C. AkMan retained high activity at pH 4.0-7.5 and was not inactivated in such acidic pH range by the incubation at 40 degrees C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear beta-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-beta-1,4-mannanase (EC 3.2.1.78) splitting internal beta-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared similar to 55% amino-acid identity to the corresponding sequences of an abalone beta-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 beta-1,4-mannanases. (C) 2010 Elsevier Inc. All rights reserved.
  • Chikako Nagasato, Akira Inoue, Masashi Mizuno, Kazuki Kanazawa, Takao Ojima, Kazuo Okuda, Taizo Motomura
    PLANTA 232 2 287 - 298 2010年07月 [査読有り][通常論文]
     
    During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.
  • Mami Hata, Yuya Kumagai, Mohammad Matiur Rahman, Satoru Chiba, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    FISHERIES SCIENCE 75 3 755 - 763 2009年05月 [査読有り][通常論文]
     
    Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via beta-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.
  • Sayo Yamamoto, Takehiko Sahara, Daisuke Sato, Kosei Kawasaki, Satoru Ohgiya, Akira Inoue, Takao Ojima
    ENZYME AND MICROBIAL TECHNOLOGY 43 6 396 - 402 2008年11月 [査読有り][通常論文]
     
    Alginate lyase is an enzyme that degrades alginate chains via beta-elimination and has been used for the production of alginate oligosaccharides and protoplasts from brown algae. Previously, we deduced the amino-acid sequence of an abalone alginate lyase, HdAly, from its cDNA sequence and, through multiple amino-acid sequence alignment, found that several basic amino-acid residues were highly conserved among the polysaccharide-lyase family 14 (PL-14) enzymes including HdAly. In the present study, we assessed the functional importance of the conserved basic amino-acid residues in HdAly by using site-directed mutants that were produced with a cold-inducible yeast expression system consisting of an expression vector pLTex321sV5H and a host Saccharomyces cerevisiae BY4743. At first, we prepared wildtype HdAly with the yeast expression system and confirmed that this recombinant possesses nearly the same properties as native HdAly with respect to specific activity (1300 U/mg), optimal pH (7.8), optimal temperature (35 C), and protoplast-producing ability from the brown alga Laminaria japonica. Then, we prepared a series of site-directed mutants by replacing the conserved basic amino-acid residues of HdAly with Ala and determined the alginate-degrading activity of these mutants. As a result, we found that the replacement of Lys95 caused complete inactivation of HdAly and those of Arg92, Arg110, and Arg119 caused 65% or more inactivation. These results indicate that the region spanning Arg92 to Arg119 is closely related to the catalytic activity of HdAly. (C) 2008 Elsevier Inc. All rights reserved.
  • Akira Inoue, Mayu Kagaya, Takao Ojima
    JOURNAL OF APPLIED PHYCOLOGY 20 5 633 - 640 2008年10月 [査読有り][通常論文]
     
    Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg2+ and Sr2+ in addition to Ca2+ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 mu m, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.
  • Yuya Kumagai, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    FISHERIES SCIENCE 74 5 1127 - 1136 2008年10月 [査読有り][通常論文]
     
    The mid-gut gland of scallop Patinopecten yessoensis has been discarded in scallop processing factories as a fishery waste and various attempts have been made to turn the waste into valuable resources. In the present study, we tried to use mid-gut gland drips from scallop as a source of beta-1,3-glucanase. The mid-gut gland drips were collected in a local fishery factory in Yubetsu-cho, Hokkaido Prefecture. beta-1,3-Glucanase was purified from the mid-gut gland drips by ammonium sulfate fractionation followed by successive chromatography on Toyopearl Phenyl-650M and Toyopearl DEAE-650M. The scallop beta-1,3-glucanase, named PyLam38 in the present study, showed a molecular mass of approximately 38 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and hydrolyzed laminarin, a beta-1,3-glucan from Laminaria sp., producing laminaribiose and glucose with an optimal pH and temperature of 6.0 and 45 degrees C, respectively. PyLam38 exhibited high transglycosylation activity toward various accepter substrates such as monosaccharides, alcohols and xylooligosaccharides. Thus, PyLam38 was found to be useful for the production of various novel heterooligosaccharides consisting of laminarioligosaccharides and various accepters.
  • Yukiko Nishida, Ken-ichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    BIOCHIMIE 89 8 1002 - 1011 2007年08月 [査読有り][通常論文]
     
    Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases. (c) 2007 Elsevier Masson SAS. All rights reserved.
  • Shuuji Ootsuka, Naotsune Saga, Ken-ichi Suzuki, Akira Inoue, Takao Ojima
    JOURNAL OF BIOTECHNOLOGY 125 2 269 - 280 2006年09月 [査読有り][通常論文]
     
    An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650 M, hydroxyapatite, and TOYOPEARL HW50F The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes. (c) 2006 Elsevier B.V. All rights reserved.
  • Harumasa Suzuki, Ken-ichi Suzuki, Akira Inoue, Takao Ojima
    CARBOHYDRATE RESEARCH 341 11 1809 - 1819 2006年08月 [査読有り][通常論文]
     
    We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res. 2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate Iyase belonging to PL14. (c) 2006 Elsevier Ltd. All rights reserved.
  • M Nishikawa, S Nishikawa, A Inoue, AH Iwane, T Yanagida, M Ikebe
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 343 4 1159 - 1164 2006年05月 [査読有り][通常論文]
     
    It has been puzzled that in spite of its single-headed structure, myosin-IX shows the typical character of processive motor in multimolecule in vitro motility assay, because this cannot be explained by hand-over-hand mechanism of the two-headed processive myosins. Here, we show direct evidence of the processive movement of rnyosin-IX using two different single molecule techniques. Using optical trap nanometry, we found that rnyosin-IX takes several large (similar to 20 nm) steps before detaching from an actin filament. Furthermore, we directly visualized the single myosin-IX molecules moving on actin filaments for several hundred nanometers without dissociating from actin filament. Since myosin-IX processively moves without anchoring the neck domain, the result suggests that the neck tilting is not involved for the processive movement of myosin-IX. We propose that the rnyosin-IX head moves processively along all actin filament like an inchworm via a unique long and positively charged insertion in the loop 2 region of the head. (c) 2006 Elsevier Inc. All rights reserved.
  • T Doi, A Satoh, H Tanaka, A Inoue, F Yumoto, M Tanokura, Obtsuki, I, K Nishita, T Ojima
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 436 1 83 - 90 2005年04月 [査読有り][通常論文]
     
    Ca2+-binding sites I and II in the N-terminal lobe of Molluscan troponin C (TnC) have lost the ability to bind Ca2+ due to substitutions of the amino acid residues responsible for Ca2+ liganding. To evaluate the functional importance of the Ca2+-deficient N-terminal lobe in the Ca2+-regulatory function of Molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using Ca-45(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca2+/mol and 1 mol Ca2+/Mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin 1, they showed different Ca2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca2+ sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca2+ sensitivity. Our findings indicate that the N-terminal lobe of Molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca2+. (c) 2005 Elsevier Inc. All rights reserved.
  • O Sato, HD White, A Inoue, B Belknap, R Ikebe, M Ikebe
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 28 28844 - 28854 2004年07月 [査読有り][通常論文]
     
    The missense mutation of Cys(442) to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20 - 40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase (similar to 10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (< 10(-7) M) calcium but is calcium-sensitive with a 1.6-fold increase at high ( &SIM;10(-4) M) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI.
  • A Inoue, T Ojima, K Nishita
    JOURNAL OF BIOCHEMISTRY 136 1 107 - 114 2004年07月 [査読有り][通常論文]
     
    Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar a-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca2+-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca2+-regulation together with troponin.
  • TM Watanabe, H Tanaka, AH Iwane, S Maki-Yonekura, K Homma, A Inoue, R Ikebe, T Yanagida, M Ikebe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 26 9630 - 9635 2004年06月 [査読有り][通常論文]
     
    Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large (approximate to36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (approximate to32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.
  • S Komaba, A Inoue, S Maruta, H Hosoya, M Ikebe
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 24 21352 - 21360 2003年06月 [査読有り][通常論文]
     
    The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actin-based motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin ( 20- fold) with K-actin of 112 muM and V-max of 0.34 s(-1), indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity ( 0.11 +/- 0.05 mum/ s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C- terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin- based motor protein having a protein kinase activity.
  • A Inoue, M Ikebe
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 7 5478 - 5487 2003年02月 [査読有り][通常論文]
     
    Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg2+-ATPase activity that was markedly activated by actin with K-actin of 33 mum and V-max of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K-actin of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.
  • M Ikebe, A Inoue, S Nishikawa, K Homma, H Tanaka, AH Iwane, E Katayama, R Ikebe, T Yanagida
    MOLECULAR AND CELLULAR ASPECTS OF MUSCLE CONTRACTION 538 143 - 157 2003年 [査読有り][通常論文]
  • Y Iwamni, T Ojima, A Inoue, K Nishita
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 133 2 257 - 267 2002年10月 [査読有り][通常論文]
     
    The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5994 bp, including 5814 bp of translated region deducing an amino acid sequence of 1937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region. (C) 2002 Elsevier Science Inc, All.rights reserved.
  • A Inoue, J Saito, R Ikebe, M Ikebe
    NATURE CELL BIOLOGY 4 4 302 - 306 2002年04月 [査読有り][通常論文]
     
    Myosin is an actin-based molecular motor that constitutes a diverse superfamily(1,2). In contrast to conventional myosin, which binds to actin for only a short time during cross-bridge cycling, recent studies have demonstrated that class V myosin moves along actin filaments for a long distance without dissociating(3,4). This would make it suitable for supporting cargo movement in cells. Because myosin V has a two-headed structure with an expanded neck domain, it has been postulated to 'walk' along the 36-nm helical repeat of the actin filament, with one head attached to the actin and leading the other head to the neighbouring helical pitch(5). Here, we report that myosin IXb, a single-headed myosin, moves processively on actin filaments. Furthermore, we found that myosin IXb is a minus-end-directed motor. In addition to class VI myosin, this is the first myosin superfamily member identified that moves in the reverse directions. The processive movement of the single-headed myosin IXb cannot be explained by a 'hand-over-hand' mechanism. This suggests that an alternative mechanism must be operating for the processive movement of single-headed myosin IXb.
  • A Inoue, O Sato, K Homma, M Ikebe
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 292 2 300 - 307 2002年03月 [査読有り][通常論文]
     
    Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells. Here we found that DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain. DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain. The C-terminal 122 amino acid residues of DOC-2/ DAB2, containing the Grb2 binding site, is identified to be critical for the binding to myosin VI. Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule. The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site. The present findings suggest that myosin VI plays a role in transporting DOC-2/ DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways. (C) 2002 Elsevier Science (USA).
  • M Yoshimura, K Homma, J Saito, A Inoue, R Ikebe, M Ikebe
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 43 39600 - 39607 2001年10月 [査読有り][通常論文]
     
    Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity was highly activated by actin with K-actin of 9 mum. A predominant amount of myosin VI bound to actin in the presence of ATP unlike conventional myosins. K-ATP was much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggested that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylated myosin VI, and the site was identified as Thr(406). The phosphorylation of myosin VI significantly facilitated the actin-translocating activity of myosin VI. On the other hand, Ca2+ diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca2+. Calmodulin was not dissociated from the heavy chain at high Ca2+, suggesting that a conformational change of calmodulin upon Ca2+ binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results revealed the dual regulation of myosin VI by phosphorylation and Ca2+ binding to calmodulin light chain.
  • Human myosin IX is a single headed processive motor.
    Inoue A, Saito J, Nishikawa S, Iwane AH, Yanagida T, Mitsuko Ikebe
    Molecular Biology of the Cell 12 426  2001年 [査読無し][通常論文]
  • T Ojima, N Koizumi, K Ueyama, A Inoue, K Nishita
    JOURNAL OF BIOCHEMISTRY 128 5 803 - 809 2000年11月 [査読有り][通常論文]
     
    Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, Ii. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regulation was investigated using a site-directed mutant with an inactivated site TV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(C)). Equilibrium dialysis using Ca-45(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca2+. The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca2+-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnC(N). These results indicate that Ca2+ binds only to site IV and that Ca2+-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N) were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and Tnc, conferred Ca2+-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2+-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca2+-sensitivity. Thus, the major functions of scallop TnC, such as Ca2+-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca2+-regulatory function requires the presence of the N-terminal domain.
  • A Inoue, T Ojima, K Nishita
    FISHERIES SCIENCE 65 5 772 - 776 1999年10月 [査読有り][通常論文]
     
    cDNA clones encoding tropomyosin of akazara scallop Chlamys plipponensis akazara striated adductor muscle were isolated and sequenced. The largest cDNA obtained is composed of 1,995 bp including an open reading frame of 852 'bp at nucleotide positions 27-878, which deduced amino acid sequence of 284 residues with a calculated molecular weight of 32,540. According to database searches on NBRF-PIR 49.0 and GenBank, the amino acid sequence showed higher homology of 73% and 700ibto bloodfluke planorb tropomyosin and mussel anterior byssus retractor muscle tropomyosin, respectively, and considerably high homology of 53% and 51% to rabbit alpha- and beta-tropomyosins, respectively. The sequences corresponding to the critical region for actin-binding (residues 1-9) and troponin-T-binding region (near residues 150-180) of rabbit alpha-tropomyosin are conserved also in akazara scallop tropomyosin. As the nine residues at both of the N-terminus and C-terminus are generally regarded to form a head-to-tail interaction region, the residues at the N-terminus of akazara scallop tropomyosin show high sequence homology to those of various muscle tropomyosins. However, those at the C-terminus show lower sequence homology than those of vertebrate tropomyosin. Thus, head-to-tail interaction of akazara scallop tropomyosin may be different from rabbit tropomyosin on account of low homology of the C-terminal sequence.
  • T Ojima, N Kawashima, A Inoue, A Amauchi, M Togashi, S Watabe, K Nishita
    FISHERIES SCIENCE 64 5 812 - 819 1998年10月 [査読有り][通常論文]
     
    Primary structure of heavy meromyosin region of walleye pollack Theragra chalcogramma myosin heavy chain was determined by cDNA cloning. By using one PCR product and five cDNA clones isolated from a lambda gt11-cDNA library for the pollack dorsal muscle, a nucleotide sequence of 3,923 bp comprising 60 bp of 5'-untranslational region and 3,863 bp of coding region was determined. The deduced sequence of 1,287 amino acids showed considerably high homology to the corresponding regions of carp myosin (83%) and chicken and rabbit myosins (both 79%). The sequences of the regions for the putative ATP-binding, actin-binding, and regulatory light chain-binding were well conserved among the pollack, carp, chicken, and rabbit myosins (83-100% homology). On the other hand, relatively low sequence homologies were seen in the essential light chain-binding site (52-78%), junctions between 20-kDa and 50-kDa domains (27-33%) and 25-kDa and 50-kDa domains (53-57%) of subfragment-1.
  • A Inoue, T Ojima, K Nishita
    FISHERIES SCIENCE 64 3 459 - 463 1998年06月 [査読有り][通常論文]
     
    A cDNA clone encoding troponin-rr of ezo-giant scallop Patinopecten yessoensis striated adductor muscle has been isolated and sequenced. The cDNA is composed of 1,939 bp with a translational region of 975 bp at the nucleotide positions 111-1,085. It deduced 325 amino acid residues with a molecular weight of 38,438. Ezo-giant scallop troponin-T showed high homology with akazara scallop troponin-rr (92%). However, it possesses 11 amino acid residues more than akazara scallop troponin-T and substitutions of 26 residues. According to sequence comparison with rabbit troponin-T, the residues 39-126 and 180-233 of ezo-giant scallop troponin-T appears to be tropomyosin-interacting regions and the residues 206-210 to be troponin-I-interacting region. Moreover, ezo-giant scallop troponin-rr was assumed to have close phylogenetic relations to troponin-Ts of akazara scallop, nematode Caenorhabditis elegans, and fruit fly Drosophila melanogaster.
  • A Inoue, T Ojima, K Nishita
    FISHERIES SCIENCE 64 1 164 - 165 1998年02月 [査読有り][通常論文]
  • K Nishita, T Ojima, A Takahashi, A Inoue
    JOURNAL OF BIOCHEMISTRY 121 3 419 - 424 1997年03月 [査読有り][通常論文]
     
    Troponin which can confer Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity has been prepared from the smooth adductor muscle of Ezo-giant scallop (Patinopecten yessoensis). The troponin comprises 40-, 20-, and 19-kDa components, In order to characterize the components, they were separated from each other by CM-Toyopearl column chromatography in the presence of BM urea, Consequently, the 20-kDa component was identified as troponin C, based on the Ca2+-binding ability, The amount of Ca2+ bound to the troponin C was estimated to be 0.75 mol/mol at 10(-4) M Ca2+ by the equilibrium dialysis method, The 19-kDa component was identified as troponin I on the basis of not only its inhibitory effect on rabbit actomyosin Mg-ATPase activity along with the smooth adductor tropomyosin, but also the releasing effect of the smooth adductor troponin C on the inhibition, On the other hand, the 40-kDa component was regarded as troponin T on the basis that it bound to F-actin-tropomyosin filament and was indispensable for conferring Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity, along with troponin C and troponin I, The above assignments were confirmed by both amino acid analysis and immunoblotting using rabbit antisera raised against counterparts of scallop striated muscle troponin.
  • T Ojima, M Maita, A Inoue, K Nishita
    FISHERIES SCIENCE 63 1 137 - 141 1997年02月 [査読有り][通常論文]
     
    To investigate structure-function relationship for akazara scallop troponin C (TnC), we constructed a bacterial expression system using a cDNA clone which we isolated previously (Ojima, T., Tanaka, H., and Nishita, K. Arch. Biochem. Biophys., 311, 272-276 (1994)). The cDNA for akazara scallop TnC was subcloned into the expression plasmid pET-16b and expressed in Escherichia coli BL21 (DE3) by the induction with isopropyl-beta-D(-)-thiogalactopyranoside. Amount of the expressed TnC increased up to approximately 10% of total protein during 4 h incubation. The TnC was then extracted from the bacterial pellet and purified by subsequent column chromatographies on DEAE-Toyopearl 650M and Phenyl-Sepharose CL-4B. Although the expressed TnC possessed an extra moiety of 6 amino acids at N-terminus, which was derived from adaptor DNA of lambda gt11 and 5'-untranslated region of the cDNA clone, it showed basically the same properties as those of native TnC with respect to the Ca2+-induced difference UV-absorption spectrum and Ca2+-regulatory ability when reconstituted with native troponin T and troponin I.
  • A Inoue, T Ojima, K Nishita
    JOURNAL OF BIOCHEMISTRY 120 4 834 - 837 1996年10月 [査読有り][通常論文]
     
    A cDNA clone encoding troponin T of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle has been isolated and sequenced, The complete sequence deduced consists of 314 amino acid residues with a molecular weight of 37,206, Akazara scallop troponin T contains 55 amino acid residues more and 82 residues fewer than rabbit skeletal muscle troponin T and Drosophila melanogaster troponin T, respectively, showing almost the lowest sequence homology with rabbit troponin T (26%) but the highest homology with Drosophila troponin T (33%), Further, high sequence homology was seen in the functional regions: residues 33-120 and 174-227, corresponding respectively to residues 71-158 and 197-250 of rabbit troponin T (tropomyosin-binding regions); and residues 200-204, corresponding to 223-227 of rabbit troponin T (troponin I-binding region), In residues 1-70 (tropomyosin-binding region), however, only six residues are identical with rabbit troponin T.
  • Kazunaga Yazawa, Keiko Araki, Kazuo Watanabe, Chikako Ishikawa, Akira Inoue, Kiyosi Kondo, Shugo Watabe, Kanehisa Hashimoto
    Nippon Suisan Gakkaishi (Japanese Edition) 54 10 1835 - 1838 1988年 [査読有り][通常論文]
     
    Attempts were made to find eicosapentaenoic acid(EPA)-producing microorganisms. Out of some 7,000 bacterial strains screened, 112 of them showed more or less this activity. Most of them were isolated from intestinal contents of marine fishes which were known to contain high levels of EPA in their body lipid. A bacterial strain isolated from the Pacific mackerel Pneumatophorus japonicus showed a high productivity of EPA (3.8 to 10.8 mg/g dry cells) and also a high ratio of EPA to total fatty acids (27.8 to 36.3%). The strain was Gram-negative, motile, obligate aerobic and short rod-shaped, which we judge it to be a new species close to genus Alteromonas. © 1988, The Japanese Society of Fisheries Science. All rights reserved.

書籍

  • Akira Inoue (担当:分担執筆範囲:Degradation and Modification of Alginate by Enzymes in Marine Organisms (Chapter 85))
    WILEY 2020年10月 (ISBN: 9781119143772)
  • 公益社団法人日本水産学会, 竹内俊郎, 佐藤 實, 渡部終五, 林孝治, 多田千佳, 中野和典, 荒川久幸, 池田吉用, 和泉充, 潮秀樹, 北澤大輔, 水野英則, 佐藤陽一, 阿部知子, 福西暢尚, 浦野直人, 宮川拓也, 井上晶, 宮下和夫, 鈴木徹, 婁小波 
    恒星社厚生閣 2017年03月 (ISBN: 4769916019) 140

講演・口頭発表等

その他活動・業績

特許

受賞

  • 2019年 日本学術振興会 特別研究員等審査会専門委員(書面担当)の表彰
     
    受賞者: 井上 晶
  • 2016年 北海道 北海道科学技術奨励賞
     
    受賞者: 井上 晶
  • 2009年 日本水産学会 日本水産学会論文賞
     
    受賞者: 畑舞美;熊谷祐也;Mohammad Matiur Rahman;千葉智;田中啓之;井上晶;尾島孝男

教育活動情報

主要な担当授業

  • Introduction to Fisheries Sciences Ⅱ(水産科学汎論Ⅱ)
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 水産科学院
    キーワード : 水産科学,海洋生物学,生物多様性,生物資源, 環境, 資源探査, 漁業技術, 持続可能性, 環境保全, 養殖, 遺伝学, 生命工学, 微生物学, 化学,食糧科学,生物安全性
  • 海洋生物工学特論Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 水産科学院
    キーワード : 生化学、微生物学、細胞工学、魚病学、微生物生態学、分子生物学、酵素化学
  • 細胞生物学
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 細胞、DNA、RNA、分子モーター、細胞分裂、減数分裂、遺伝子発現、細胞極性、魚類、性分化
  • 水圏生化学実験
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : タンパク質、糖質、卵形成、生理学、生化学、免疫化学、筋肉
  • 基礎生命科学実験
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 組織 器官 解剖 切片 基本操作 緩衝液 吸光度分析

大学運営

委員歴

  • 2017年 - 現在   日本水産学会   編集委員


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.