Motomitsu Kitaoka

National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

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Publications (171)391.11 Total impact

  • Yuji Honda · Sachiko Arai · Kentaro Suzuki · Motomitsu Kitaoka · Shinya Fushinobu
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    ABSTRACT: Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting glycoside hydrolase (GH) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active site pocket, in contrast to the long glycan binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. D139, D143 and E555 in the active site were located near the β-O1 hydroxyl group of GlcN, with D139 and D143 holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at D139, we obtained D139E as the best glycosynthase. Compared with the wild type, the hydrolytic activity of D139E was significantly suppressed (< 0.1%) whereas the fluoride ion-releasing activity also decreased (< 3%). Therefore, the glycosynthase activity of D139E was lower than that of glycosynthases previously created from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of D139 might inevitably reduce fluoride ion-releasing activity.
    No preview · Article · Nov 2015 · Biochemical Journal
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    ABSTRACT: 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.
    No preview · Article · Nov 2015 · FEBS letters
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    ABSTRACT: 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.
    No preview · Article · Oct 2015 · FEBS letters
  • Motomitsu Kitaoka
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    ABSTRACT: Phosphorylases are useful catalysts for the practical preparation of various sugars. The number of known specificities was 13 in 2002 and is now 30. The drastic increase in available genome sequences has facilitated the discovery of novel activities. Most of these novel phosphorylase activities have been identified through the investigations of glycoside hydrolase families containing known phosphorylases. Here, the diversity of phosphorylases in each family is described in detail.
    No preview · Article · Aug 2015 · Applied Microbiology and Biotechnology
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    ABSTRACT: 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 (GH) 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 (GlcUA) 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 GlcUA. Structural and sequence comparisons with other GH94 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. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Jun 2015 · Journal of Biological Chemistry
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    ABSTRACT: Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and β-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.
    No preview · Article · May 2015 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
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    ABSTRACT: We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Mar 2015 · Carbohydrate research
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    ABSTRACT: The aerobic soil bacterium Cellvibrio vulgaris has a β-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-β-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and β-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for β-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed β-d-mannosyl-(1→4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-β-d-mannosyl-d-glucose phosphorylase.
    No preview · Article · Feb 2015 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: 1,2-β-Glucan was produced enzymatically from 1.0 M sucrose and 0.5 M glucose by the combination of sucrose phosphorylase and 1,2-β-oligoglucan phosphorylase in the presence of 100 mM inorganic phosphate. Accumulation of 1,2-β-glucan in 2 L of the reaction mixture reached over 800 mM (glucose equivalent). Sucrose, glucose and fructose were removed after the reaction by yeast treatment. 1,2-β-Glucan was precipitated with ethanol to obtain 167 g of 1,2-β-glucan from 1 L of the reaction mixture.
    Preview · Article · Jan 2015
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    ABSTRACT: 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.
    Preview · Article · Dec 2014 · PLoS ONE
  • Yuan Liu · Mamoru Nishimoto · Motomitsu Kitaoka
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    ABSTRACT: Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-d-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-d-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-d-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.
    No preview · Article · Oct 2014 · Carbohydrate Research
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    ABSTRACT: The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host derived β-galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β-galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 (BlGal42A). BlGal42A displays a preference for undecorated β1-6 and β1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted β1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite +1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite −1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino-acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1-6 and β1-3 galactosides likely reflects structural differences between these substrates and β1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.
    No preview · Article · Oct 2014 · Molecular Microbiology
  • Ryota Fujii · Motomitsu Kitaoka · Kiyoshi Hayashi
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    ABSTRACT: We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.
    No preview · Article · Jul 2014 · Methods in Molecular Biology
  • Ryota Fujii · Motomitsu Kitaoka · Kiyoshi Hayashi
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    ABSTRACT: Although proteins can be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult. Here we describe a simple method called random insertional-deletional strand exchange mutagenesis (RAISE). This method is based on gene shuffling and can be used to introduce a wide variety of insertions, deletions, and substitutions. RAISE involves three steps: DNA fragmentation, attachment of a random short sequence, and reconstruction. This yields unique mutants and can be a powerful technique for protein engineering.
    No preview · Article · Jul 2014 · Methods in Molecular Biology
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    ABSTRACT: 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 with the glucose analogue 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.
    No preview · Article · May 2014 · Journal of Biological Chemistry
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    ABSTRACT: 4-O-β-d-Mannosyl-d-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-β-d-mannosyl-d-glucose (Man-Glc) to α-d-mannosyl phosphate and d-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts β-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-β-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi–bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5–8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-d-glucose and d-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl β-d-glucoside and 1,5-anhydro-d-glucitol as acceptor substrates.
    No preview · Article · Apr 2014 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.
    Preview · Article · Mar 2014 · PLoS ONE
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    ABSTRACT: 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.
    Preview · Article · Jan 2014 · PLoS ONE
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    ABSTRACT: One-pot enzymatic production of nigerose was demonstrated from abundantly available sugar resources, including maltose, cellobiose, sucrose and starch. (i) 319 mM nigerose was generated from 500 mM maltose by the combined actions of maltose phosphorylase and nigerose phosphorylase, which share the same β-D-glucose 1-phosphate, in the presence of phosphate. The yield was 62% based on the concentration of maltose as the starting material. (ii) 129 mM nigerose was produced from 250 mM cellobiose by cellobiose phosphorylase and nigerose phosphorylase in the presence of phosphate, in combination with the enzymatic pathway to convert α-D-glucose 1-phosphate to β-D-glucose 1-phosphate via D-glucose 6-phosphate by the combined actions of α-phosphoglucomutase and β-phosphoglucomutase, resulting in a yield of 52%. (iii) 350 mM nigerose was produced from 500 mM sucrose by substituting cellobiose phosphorylase with sucrose phosphorylase and adding xylose isomerase, giving a yield of 67%. (iv) 270 mM nigerose was generated from 100 mg/mL starch and 500 mM D-glucose by the concomitant actions of glycogen phosphorylase, isoamylase, α-phosphoglucomutase, β-phosphoglucomutase and nigerose phosphorylase, in the presence of phosphate. In addition, 280 mM 3-O-α-D-glucopyranosyl-D-galactose was produced by substituting D-glucose with D-galactose. Based on the concentrations of D-glucose and D-galactose as the starting materials, the yields were calculated to be 52 and 56%, respectively. These one-pot enzymatic approaches can be extended to include practical production of a variety of oligosaccharides by substituting nigerose phosphorylase with other β-D-glucose 1-phosphate-forming phosphorylases together with various carbohydrate acceptors.
    No preview · Article · Jan 2014
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    ABSTRACT: We characterized two α-1,3-glucoside phosphorylases that belonged to glycoside hydrolase family 65 from Clostridium phytofermentans: Cphy_3313 and Cphy_3314. Cphy_3313 was a typical nigerose phosphorylase that phosphorolyzed nigerose into D-glucose and β-D-glucose 1-phosphate (βGlc1P). Cphy_3314 catalyzed the synthesis of a series of α-1,3-oligoglucans using nigerose as the acceptor and βGlc1P as the donor. Kinetic analyses of their phosphorolytic reactions with α-1,3-oligoglucans (DP = 3 and 4) revealed that Cphy_3314 utilized a typical sequential Bi Bi mechanism, while this enzyme did not exhibit any significant phosphorolytic activity for nigerose. These results suggest that Cphy_3314 is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of α-1,3-oligoglucans with DP of 3 or higher. In this study, we propose 3-O-α-D-oligoglucan: phosphate β-D-glucosyltransferase as the systematic name and α-1,3-oligoglucan phosphorylase as the short name for Cphy_3314.
    No preview · Article · Jan 2014

Publication Stats

3k Citations
391.11 Total Impact Points

Institutions

  • 2009-2015
    • National Agriculture and Food Research Organization
      Tsukuba, Ibaraki, Japan
    • The Ohio State University
      • Department of Chemistry and Biochemistry
      Columbus, Ohio, United States
  • 2000-2015
    • National Food Research Institute
      Ibaragi, Ōsaka, Japan
    • Yamagata University
      • Department of Material and Biological Chemistry
      Ямагата, Yamagata, Japan
  • 2013
    • Niigata University
      • Faculty of Agriculture
      Niahi-niigata, Niigata, Japan
  • 2008
    • Ishikawa Prefectural University
      Ноноичи, Ishikawa, Japan
  • 2004
    • The University of Tokyo
      • Department of Biotechnology
      Tōkyō, Japan