2021/10/11 更新

写真a

ニヒラ タカノリ
仁平 高則
NIHIRA Takanori
所属
教育研究院 自然科学系 特任助教
農学部 農学科 特任助教
職名
特任助教
外部リンク

代表的な業績

    • 【論文】 Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi.  2013年11月

    • 【論文】 Discovery of β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans.  2013年9月

    • 【論文】 2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate.  2014年

学位

  • 博士(工学) ( 2001年3月   静岡大学 )

研究キーワード

  • 酵素化学

  • 糖質科学

研究分野

  • ライフサイエンス / 食品科学

  • ライフサイエンス / 応用生物化学

経歴(researchmap)

  • 新潟大学

    2011年4月 - 現在

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  • 国立研究開発法人農業・食品産業技術総合研究機構

    2006年4月 - 2011年3月

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  • 東京工業大学

    2003年7月 - 2006年3月

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  • 国立研究開発法人科学技術振興機構

    2002年4月 - 2006年3月

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  • 東京農工大学

    2001年4月 - 2002年3月

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経歴

  • 新潟大学   農学部 農学科   特任助教

    2019年2月 - 2021年9月

  • 新潟大学   応用生物化学科   特任助教

    2015年4月 - 2018年3月

  • 新潟大学   応用生物化学科   研究員

    2014年10月 - 2015年3月

  • 新潟大学   経営戦略本部 若手研究者育成推進室   特任助教

    2014年7月 - 2014年9月

  • 新潟大学   応用生物化学科   研究員

    2014年4月 - 2014年6月

  • 新潟大学   経営戦略本部 若手研究者育成推進室   特任助教

    2013年8月 - 2014年3月

  • 新潟大学   応用生物化学科   研究員

    2013年4月 - 2013年7月

  • 新潟大学   応用生物化学科   研究員

    2011年4月 - 2013年3月

▶ 全件表示

学歴

  • 静岡大学   大学院電子科学研究科

    1998年4月 - 2001年3月

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  • 静岡大学   大学院教育学研究科

    1996年4月 - 1998年3月

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  • 静岡大学   教育学部

    1992年4月 - 1996年3月

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所属学協会

 

論文

  • Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH. 国際誌

    Kazuhiro Chiku, Riku Tsukasaki, Yu Teshima, Mitsuru Yoshida, Hiroki Aramasa, Takanori Nihira, Hiroyuki Nakai, Hiroshi Ono, Motomitsu Kitaoka

    Carbohydrate research   496   108129 - 108129   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via β-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α-d-glucopyranosyl-d-fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10-4 s-1, whereas that for turanose [(2.6 ± 0.2) × 10-4 s-1] was about 2.2 times higher.

    DOI: 10.1016/j.carres.2020.108129

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  • Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family. 国際誌

    Koichi Abe, Masahiro Nakajima, Tetsuro Yamashita, Hiroki Matsunaga, Shinji Kamisuki, Takanori Nihira, Yuta Takahashi, Naohisa Sugimoto, Akimasa Miyanaga, Hiroyuki Nakai, Takatoshi Arakawa, Shinya Fushinobu, Hayao Taguchi

    The Journal of biological chemistry   292 ( 18 )   7487 - 7506   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo-β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

    DOI: 10.1074/jbc.M116.762724

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  • Mechanistic insight into the substrate specificity of 1,2-β-oligoglucan phosphorylase from Lachnoclostridium phytofermentans. 国際誌

    Masahiro Nakajima, Nobukiyo Tanaka, Nayuta Furukawa, Takanori Nihira, Yuki Kodutsumi, Yuta Takahashi, Naohisa Sugimoto, Akimasa Miyanaga, Shinya Fushinobu, Hayao Taguchi, Hiroyuki Nakai

    Scientific reports   7   42671 - 42671   2017年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on β-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-β-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique β-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites -1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on β-1,2-glucooligosaccharides.

    DOI: 10.1038/srep42671

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  • Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua. 国際誌

    Tomohiro Tsuda, Takanori Nihira, Kazuhiro Chiku, Erika Suzuki, Takatoshi Arakawa, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    FEBS letters   589 ( 24 Pt B )   3816 - 21   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for β-mannosides. Here, we characterized β-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with β-1,2-linked mannooligosaccharides. β-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

    DOI: 10.1016/j.febslet.2015.11.034

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  • An inverting β-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans. 国際誌

    Takanori Nihira, Kazuhiro Chiku, Erika Suzuki, Mamoru Nishimoto, Shinya Fushinobu, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    FEBS letters   589 ( 23 )   3604 - 10   2015年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of β-D-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α-D-mannose 1-phosphate but it released α-D-mannose from β-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a β-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of β-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the β-D-mannosidic bond.

    DOI: 10.1016/j.febslet.2015.10.008

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  • Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes. 国際誌

    Young-Woo Nam, Takanori Nihira, Takatoshi Arakawa, Yuka Saito, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    The Journal of biological chemistry   290 ( 30 )   18281 - 92   2015年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

    DOI: 10.1074/jbc.M115.664664

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  • Structural basis for reversible phosphorolysis and hydrolysis reactions of 2-O-α-glucosylglycerol phosphorylase. 国際誌

    Kouki K Touhara, Takanori Nihira, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    The Journal of biological chemistry   289 ( 26 )   18067 - 75   2014年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of β-d-glucose 1-phosphate (βGlc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically produce GG. However, its structural basis has remained unclear. In this study, the crystal structures of GGP complexed with glucose and the glucose analog isofagomine and glycerol were determined. Subsite -1 of GGP is similar to those of other GH65 enzymes, maltose phosphorylase and kojibiose phosphorylase, whereas subsite +1 is largely different and is well designed for GG recognition. An automated docking analysis was performed to complement these crystal structures, βGlc1P being docked at an appropriate position. To investigate the importance of residues at subsite +1 in the bifunctionality of GGP, we constructed mutants at these residues. Y327F and K587A did not show detectable activities for either reverse phosphorolysis or βGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained βGlc1P hydrolysis. The mode of substrate recognition and the reaction mechanisms of GGP were proposed based on these analyses. Specifically, an extensive hydrogen bond network formed by Tyr-327, Tyr-572, Lys-587, and water molecules contributes to fixing the acceptor molecule in both reverse phosphorolysis (glycerol) and βGlc1P hydrolysis (water) for a glycosyl transfer reaction. This study will contribute to the development of a large scale production system of GG by facilitating the rational engineering of GGP.

    DOI: 10.1074/jbc.M114.573212

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  • Discovery of two β-1,2-mannoside phosphorylases showing different chain-length specificities from Thermoanaerobacter sp. X-514. 国際誌

    Kazuhiro Chiku, Takanori Nihira, Erika Suzuki, Mamoru Nishimoto, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    PloS one   9 ( 12 )   e114882   2014年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

    DOI: 10.1371/journal.pone.0114882

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  • 2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate. 国際誌

    Takanori Nihira, Yuka Saito, Ken'ichi Ohtsubo, Hiroyuki Nakai, Motomitsu Kitaoka

    PloS one   9 ( 1 )   e86548   2014年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s(-1). No carbohydrate acted as acceptor for the reverse phosphorolysis using β-D-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s(-1); moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 (18)O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-D-glucosylglycerol (GG) at the rate of 180 s(-1). Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s(-1). We propose 2-O-α-D-glucopyranosylglycerol: phosphate β-D-glucosyltransferase as the systematic name and 2-O-α-D-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

    DOI: 10.1371/journal.pone.0086548

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  • Potassium ion-dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10. 国際誌

    Takanori Nihira, Yuka Saito, Kazuhiro Chiku, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    FEBS letters   587 ( 21 )   3382 - 6   2013年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We discovered a potassium ion-dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing β-D-glucose 1-phosphate and D-glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.

    DOI: 10.1016/j.febslet.2013.08.038

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  • Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi. 国際誌

    Takanori Nihira, Yuka Saito, Mamoru Nishimoto, Motomitsu Kitaoka, Kiyohiko Igarashi, Ken'ichi Ohtsubo, Hiroyuki Nakai

    FEBS letters   587 ( 21 )   3556 - 61   2013年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-D-glucopyranosyl-D-gluconic acid: phosphate α-D-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-D-glucose 1-phosphate and D-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.

    DOI: 10.1016/j.febslet.2013.09.014

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  • Discovery of β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans. 国際誌

    Takanori Nihira, Erika Suzuki, Motomitsu Kitaoka, Mamoru Nishimoto, Ken'ichi Ohtsubo, Hiroyuki Nakai

    The Journal of biological chemistry   288 ( 38 )   27366 - 27374   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-D-mannosyl-N-acetyl-D-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of β-1,4-D-mannosyl-N-acetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-D-mannopyranosyl-N-acetyl-D-glucosamine:phosphate α-D-mannosyltransferase as the systematic name and β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033.

    DOI: 10.1074/jbc.M113.469080

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  • Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-β-D-glucosyl disaccharides. 国際誌

    Takanori Nihira, Yuka Saito, Motomitsu Kitaoka, Mamoru Nishimoto, Ken'ichi Otsubo, Hiroyuki Nakai

    Carbohydrate research   361   49 - 54   2012年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-D-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-β-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.

    DOI: 10.1016/j.carres.2012.08.006

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  • Identification of Bacillus selenitireducens MLS10 maltose phosphorylase possessing synthetic ability for branched α-D-glucosyl trisaccharides. 国際誌

    Takanori Nihira, Yuka Saito, Motomitsu Kitaoka, Ken'ichi Otsubo, Hiroyuki Nakai

    Carbohydrate research   360   25 - 30   2012年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for α-D-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with β-D-glucose 1-phosphate as the donor. Bsel2056 showed the flexibility for monosaccharide acceptors with alternative C2 substituent (2-amino-2-deoxy-D-glucose, 2-deoxy-D-arabino-hexose, 2-acetamido-2-deoxy-D-glucose, D-mannose), resulting in production of 1,4-α-D-glucosyl disaccharides with strict regioselectivity. In addition, Bsel2056 synthesized two maltose derivatives possessing additional D-glucosyl residue bound to C2 position of the D-glucose residue at the reducing end, 1,4-α-D-glucopyranosyl-[1,2-α-D-glucopyranosyl]-D-glucose and 1,4-α-D-glucopyranosyl-[1,2-β-D-glucopyranosyl]-D-glucose, from 1,2-α-D-glucopyranosyl-D-glucose (kojibiose) and 1,2-β-D-glucopyranosyl-D-glucose (sophorose), respectively, as the acceptors. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of D-glucose.

    DOI: 10.1016/j.carres.2012.07.014

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  • 3-O-α-D-glucopyranosyl-L-rhamnose phosphorylase from Clostridium phytofermentans. 国際誌

    Takanori Nihira, Hiroyuki Nakai, Motomitsu Kitaoka

    Carbohydrate research   350   94 - 7   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with β-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate β-d-glucosyltransferase as the systematic name and 3-O-α-d-glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase.

    DOI: 10.1016/j.carres.2011.12.019

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  • Discovery of nigerose phosphorylase from Clostridium phytofermentans. 国際誌

    Takanori Nihira, Hiroyuki Nakai, Kazuhiro Chiku, Motomitsu Kitaoka

    Applied microbiology and biotechnology   93 ( 4 )   1513 - 22   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of D-glucose and β-D-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k(cat) = 67 s(-1) and K(m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using D-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on D-xylose, 1,5-anhydro-D-glucitol, D-galactose, and methyl-α-D-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with D-xylose and methyl-α-D-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-D-glucosyl-D-glucose:phosphate β-D-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.

    DOI: 10.1007/s00253-011-3515-9

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  • Kinetic studies of dextransucrase enzyme reactions on a substrate- or enzyme-immobilized 27 MHz quartz crystal microbalance. 国際誌

    Takanori Nihira, Toshiaki Mori, Megumi Asakura, Yoshio Okahata

    Langmuir : the ACS journal of surfaces and colloids   27 ( 6 )   2107 - 11   2011年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Catalytic elongation by dextransucrase (DSase) was monitored directly on a dextran-acceptor- or DSase-immobilized 27 MHz quartz crystal microbalance (QCM). Kinetic parameters for the binding of the enzyme to the dextran acceptor (k(on), k(off), and K(d)) and enzymatic elongation in the presence of a sucrose monomer (K(m) for sucrose and k(cat)) were determined. The kinetic parameters obtained by both methods were consistent.

    DOI: 10.1021/la104550m

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  • Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124. 国際誌

    Masahiro Nakajima, Takanori Nihira, Mamoru Nishimoto, Motomitsu Kitaoka

    Applied microbiology and biotechnology   78 ( 3 )   465 - 71   2008年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galbeta1-->3GlcNAc, LNB) and galacto-N-biose (Galbeta1-->3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a kcat/Km value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar kcat/Km values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

    DOI: 10.1007/s00253-007-1319-8

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  • Colorimetric quantification of alpha-D-galactose 1-phosphate. 国際誌

    Takanori Nihira, Masahiro Nakajima, Kousuke Inoue, Mamoru Nishimoto, Motomitsu Kitaoka

    Analytical biochemistry   371 ( 2 )   259 - 61   2007年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance. 国際誌

    Takanori Nihira, Masahiro Mizuno, Takashi Tonozuka, Yoshiyuki Sakano, Toshiaki Mori, Yoshio Okahata

    Biochemistry   44 ( 27 )   9456 - 61   2005年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.

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  • Direct monitoring of enzymatic glucan hydrolysis on a 27-MHz quartz-crystal microbalance. 国際誌

    Hidekazu Nishino, Takanori Nihira, Toshiaki Mori, Yoshio Okahata

    Journal of the American Chemical Society   126 ( 8 )   2264 - 5   2004年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    By using a highly sensitive 27-MHz glucan-immobilized quartz-crystal microbalance, we could follow kinetically all processes (enzyme binding and release, kon and koff, and intramolecular hydrolysis rates, kcat) of glucan hydrolysis by glucoamylase by detecting directly the formation and decay of the enzyme-substrate complex as mass changes.

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受賞

  • 奨励賞

    2015年9月   日本応用糖質科学会   オリゴ糖合成に有用な糖質加リン酸分解酵素に関する研究

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共同研究・競争的資金等の研究

  • 加リン酸分解酵素が関与する酸化的多糖分解経路の解明

    研究課題/領域番号:18K05387  2018年4月 - 2021年3月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    仁平 高則

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    澱粉やキチンなどの糖質の分解・代謝は,加水分解酵素の反応を中心に考えられてきたが,セルロース分解同様,酸化還元酵素による酸化的分解反応が関与する可能性が示唆されている。本研究では,このような新規代謝経路の全貌を解明するために,酸化的分解の中間産物である酸性オリゴ糖を加リン酸分解する酵素ホスホリラーゼを,ゲノム情報やメタゲノムライブラリーを利用し網羅的に探索し,酵素学的,速度論的および構造学的特性の解明を目指す。
    当年度は,昨年度に引き続きゲノムライブラリーおよびメタゲノムライブラリー作製,ゲノム情報を基盤とした網羅的なホスホリラーゼの探索を行なった。またホスホリラーゼの逆反応(合成反応)を評価するための高感度リン酸定量法の確立について検討を加えた。その結果,ホスホリラーゼ遺伝子を多く持つ微生物のゲノムDNAおよび環境中から回収したヘテロなゲノムDNAを断片化,クローン化することでゲノムライブラリーおよびメタゲノムライブラリーが追加・作製できた。また,ゲノム情報を基に網羅的にホスホリラーゼ遺伝子を探索した結果,新規ホスホリラーゼと推察される数種の機能未知タンパク質遺伝子をクローニングするに至った。そのうちの数種については可溶性または封入体として機能未知タンパク質の取得に至った。リン酸の高感度定量法については,市販酵素を組み合わせることにより,従来法の数十倍の感度で,かつ連続定量可能の定量法の構築ができた。

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  • 腸内細菌に特徴的なATP消費抑制型N-グリカン代謝経路の解明

    研究課題/領域番号:26440022  2014年4月 - 2017年3月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    仁平 高則, 中井 博之

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    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

    数種の腸内細菌で見出される加リン酸分解酵素(ホスホリラーゼ)が関与するATP消費抑制型N-グリカン代謝経路を解明するために,本代謝経路の鍵酵素であるβ-1,4-マンノシル-N-アセチルグルコサミンホスホリラーゼの反応速度論的および構造学的解析を進めた。結晶が得られたBacteroides thetaiotaomicron由来の当該酵素について現在構造解析中である。また当該酵素遺伝子の近傍に存在し,N-グリカンの代謝に寄与することが推測される機能未知遺伝子について調査するために,当該遺伝子がコードするタンパク質を各種構造のN-グリカンに作用させたが,酵素反応は確認されず性質決定に至らなかった。

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