Journal of Applied Glycoscience
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Volume 56 , Issue 2
Showing 1-12 articles out of 12 articles from the selected issue
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Regular Paper
  • Isao Hanashiro, Takuji Wakayama, Mari Hasegawa, Toshiyuki Higuchi, Kim ...
    Volume 56 (2009) Issue 2 Pages 65-70
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Structural changes caused by the introduction of the Wxα transgene, which encodes granule-bound starch synthase I (GBSSI), into the non-transgenic wx cultivar Musashimochi were examined and compared with the previous results (Plant Cell Physiol., 49, 925-933 (2008), using a transgenic line (WAB3-5) established independently from those used previously. Increases of amylose content of starch and extra-long chain (ELC) content of amylopectin were observed in the line WAB3-5 (actual amylose, 15.2%; ELC, 11.6% by weight). An HPSEC profile (size distribution) of WAB3-5 amylose showed characteristics in-between of those of Wxα and Wxb cultivars, and WAB3-5 amylose had a comparably high degree of branching, which was indicated by the molar fraction of branched molecules (MFB, 0.38) and number of chains of branched molecules (NCB, 10.5). Amylose from cv. Yumetoiro (Wxα) had a comparable degree of branching (MFB, 0.35; NCB, 13.7) with the WAB3-5 amylose. Chain-length distribution of WAB3-5 amylopectin showed a slightly higher amount of B2+B3 chains than cv. Musashimochi. These results are consistent with those of the previous study, providing additional evidence for the proposed role of GBSSI in both amylose and ELC synthesis in rice endosperm. ELCs of amylopectins from line WAB3-5 and cvs. IR36 and Yumetoiro showed size distributions that were basically similar to each other but distinct from those of their amylose counterpart. ELCs were hydrolyzed by β-amylolysis of amylopectins with different extents of trimming, 66.3, 92.0 and 77.0% for line WAB3-5, cv. IR36 and cv. Yumetoiro, respectively, suggesting the branched structure of the ELCs is different among the three amylopectins.
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Note
  • Takumi Takeda, Mitsuru Nikaido, Kazuhide Totani, Mitsuhiro Obara, Yuki ...
    Volume 56 (2009) Issue 2 Pages 71-76
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Recently, demand for the bio-fuel (e.g. ethanol) production to overcome green house gas emission has been overwhelmingly accepted in many countries. The majority of bio-ethanol is being produced from sucrose (e.g. sugarcane) and starch (e.g. corn), simultaneously causing the problem of food shortage and the rise of commodity prices. Nevertheless, demand for bio-ethanol as an alternative fuel will increase in the near future. To satisfy such demand, sustainable agriculture and new technology for effective bio-ethanol production from cellulosic materials are urgently needed. Here, we report the effective saccharification of several plant materials, which are mechano-chemically prepared using a high intensive ball mill. Enzymatic digestion of rice straw, sugarbeet and sugarcane with cellulase and xylanase was enhanced by treatment with the high intensive ball mill. Analysis of the digests with cellulase and xylanase revealed an increase in the amount of glucose and xylose residues as compositional sugars. The results might be caused by the destruction of the physical structure of cell walls and non-crystallization of cellulose and depolymerization of wall polysaccharides, because of the occurrence of cello-oligosaccharides from cellulose paper and solubilization of the polysaccharides from plant materials after treatment with the high intensive ball mill. We concluded that treatment with a high intensive ball mill as pre-treatment for enzymatic hydrolysis leads to the effective saccharification of plant cell walls.
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Proceedings of the Symposium on Amylases and Related Enzymes, 2008
  • Hitomi Kajiwara, Toshiki Mine, Takeshi Yamamoto
    Volume 56 (2009) Issue 2 Pages 77-82
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Sialyltransferases transfer N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) to the acceptor substrate. Up to the present, many sialyltransferases have been cloned from mammalian and bacterial sources. All the sialyltransferases have been classified into five families in the CAZy (carbohydrate-active enzymes) database (families 29, 38, 42, 52 and 80), and all of the marine bacterial sialyltransferases are classified into the family 80. During the course of our study, we have isolated several marine bacteria producing sialyltransferases. Many of them were identified as the bacteria which were classified into genera Photobacterium and Vibrio. Furthermore, we have also demonstrated that these marine bacterial sialyltransferases have unique acceptor substrate specificity, compared with those of mammalian sialyltransferases. N-Acetylneuraminic acid is usually linked to the terminal position of glycan moiety of glycoconjugates, including glycoprotein and glycolipid. Enzymatic sialylation using sialyltransferase is a single-step process with high positional and anomer selectivity and high reaction yield under mild reaction conditions. Therefore, sialyltransferase is believed to be one of the most important enzymes in the field of glycotechnology and to be a powerful tool for the study of glycobiology.
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  • Tomonari Tanaka, Atsushi Kobayashi, Masato Noguchi, Kei-ichi Kimura, K ...
    Volume 56 (2009) Issue 2 Pages 83-88
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Various 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) glycosides (DMT-glycosides) have been synthesized from the corresponding free sugars in water by using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) as a dehydrative condensing agent. The resulting DMT-glycosides were found to be recognized as substrates for glycosyl hydrolases and could be utilized as novel glycosyl donors for chemo-enzymatic glycosylations. DMT-glycosides will be efficient and general glycosyl donors for glycosidase-catalyzed transglycosylation reaction in the field of carbohydrate chemistry.
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  • Yoshinori Misawa, Ryuichi Masaka, Megumi Yano, Takeomi Murata, Taichi ...
    Volume 56 (2009) Issue 2 Pages 89-95
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Efficient enzymatic synthesis of spacer-linked divalent glycosides, which were designed as glycomimetics, was developed by using transglycosylation of chitinolytic enzyme from Amycolatopsis orientalis. The enzyme catalyzed the synthesis of target divalent glycoside hexan-1,6-diyl bis-(2-acetamido-2-deoxy-β-D-glucopyranoside) (4) through N-acetylglucosaminyl transfer from (GlcNAc)4 to 1,6-hexanediol. When a series of primary diols with different number of carbon atoms were used as acceptor, the corresponding spacer-linked divalent glycosides carrying GlcNAc (1-6) on both sides were obtained. The mechanism of formation of spacer-linked divalent glycosides was further revealed by the enzyme purification. It was specified that N-acetylhexosaminidase itself is directly concerned with transglycosylation of GlcNAc residues to hydroxyl groups on both sides. The interaction of wheat germ agglutinin with a series of spacer-linked divalent glycosides was studied by precipitation analysis and a biosensor based on surface plasmon resonance. Our results demonstrated that a series of spacer-linked divalent glycosides are capable of precipitating WGA as divalent ligands.
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  • Takayuki Ohnuma, Shoko Onaga, Katsuyoshi Murata, Tamo Fukamizo, Toki T ...
    Volume 56 (2009) Issue 2 Pages 97-104
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    We describe here the expression, purification and characterization of two recombinant LysM domains, LysM tandem (Asp1-Lys107) and LysM single (Cys60-Lys107), derived from Pteris ryukyuensis chitinase-A (PrChi-A). Using isothermal titration calorimetry and NMR spectroscopy, we determined their binding affinities to various GlcNAc oligomers and characterized the carbohydrate binding site. We found that the stoichiometry of (GlcNAc)n/LysM domain binding is 1:1, and that the binding affinities of the LysM domain for (GlcNAc)4 and (GlcNAc)5 were in the millimolar range. The binding process was enthalpically driven with an unfavorable change in entropy. (GlcNAc)5 titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc)5 binding. The docking study of (GlcNAc)4 with the modeled structure of LysM single supported that the binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments confirmed the critical involvement of Tyr72 in (GlcNAc)n/LysM domain binding. The study is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.
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  • Ryuichiro Suzuki, Takane Katayama, Shinya Fushinobu, Motomitsu Kitaoka ...
    Volume 56 (2009) Issue 2 Pages 105-110
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Endo-α-N-acetylgalactosaminidase (endo-α), a member of glycoside hydrolase (GH) family 101, catalyzes the hydrolysis of O-glycosidic α linkages of mucin-type O-glycan. Endo-α can be used in the synthesis of various glycoconjugates because of its transglycosylation activity. Therefore, it is important to elucidate the structure-function relationship of this enzyme. The gene encoding endo-α from Bifidobacterium longum JCM1217 has been cloned, and its gene product (EngBF) has been characterized in detail. EngBF releases a galacto-N-biose (Galβ1-3GalNAc, GNB) from glycoconjugates without damaging either the glycan or the core protein. This study presents the crystal structure of EngBF at 2.25 Å resolution. The catalytic domain of EngBF resembles the TIM barrel fold of GH13 α-amylase family. Based on structural comparison with α-amylase family, the catalytic nucleophile and acid/base catalyst residues of EngBF are determined to be Asp789 and Glu822, respectively. Moreover, the structural basis of substrate recognition by EngBF was predicted by automated docking and mutational studies, and was compared with endo-α from Clostridium perfringens strain 13 (EngCP). The difference in substrate specificities between EngBF and EngCP is attributed to variations in amino acid sequences in the regions forming the substrate binding pocket. Results of our present study provide insights into both the reaction and substrate recognition mechanisms of endoglycosidases that liberate mucin-type O-glycan from glycoconjugates.
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  • Hironori Hondoh, Hiroaki Otsuka-Rachi, Wataru Saburi, Haruhide Mori, M ...
    Volume 56 (2009) Issue 2 Pages 111-117
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    In glycoside hydrorase family (GH) 13, α-glucosidase, oligo-1,6-glucosidase and dextran glucosidase, which hydrolyze the non-reducing end glucosidic linkages of maltooligo- and/or isomaltoolligosaccharides, are categorized as α-glucoside hydrolase. Despite a high similarity in the sequence and overall structure of those family enzymes, GH 13 α-glucoside hydrolases show a wide range of substrate specificity. Until now, three crystal structures of α-glucoside hydrolase, dextran glucosidase from Streptococcus mutans (DGase), oligo-1,6-glucosidase from Bacillus cereus (O16G), and α-glucosidase from Geobacillus sp. HTA-462 (GSJ) have been determined. In this study, we have performed the structural comparison of these α-glucoside hydrolases. Their overall structures are generally similar, and consist of three major domains A, B and C as found in many α-amylase family enzymes. The significant structural differences in these enzymes are mainly found in loop regions. GSJ has a shorter β→α loop 6 in a different orientation in addition to the disordered regions, whereas DGase and O16G show high similarity in their tertiary structures. Though these enzymes have the different substrate preference, they all possess the completely conserved configuration at subsite -1. Therefore, the substrate preference will be originated from the structure at subsite for the reducing end side of substrate. The substrate binding modes of these glucoside hydrolases were predicted by superimposing the substrate molecule of substrate-complex structures of DGase and α-amylase. DGase and O16G are thought to have a similar manner in substrate binding with conserved amino acid residues. The substrate recognition of GSJ at subsite -1 and +1 would be similar to that of α-amylases since the key residues in substrate binding are conserved in both primary and tertiary structures.
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  • Yuji Honda, Shinya Fushinobu, Masafumi Hidaka, Takayoshi Wakagi, Hirof ...
    Volume 56 (2009) Issue 2 Pages 119-125
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Reducing end xylose releasing exo-oligoxylanase (REX, EC 3.2.1.156) hydrolyzes the glycosidic bond at reducing end of xylooligosaccharide with anomeric inversion manor. REX catalyzed “Hehre resynthesis-hydrolysis” reaction in the presence of α-xylobiosyl fluoride (α-X2F) and X1. We found that nine catalytic base mutants (D263→G, A, V, T, L, A, C, P or S) catalyzed glycosynthase reaction using α-X2F and X1 as the donor and acceptor substrates. D263C was found to be the best glycosynthase among these mutants. However, the mutant had a little hydrolytic activity, which produced X1 and X2 from glycosynthase reaction product, X3. To obtain mutant with more efficient the glycosynthase activity, we designed new glycosynthase based on the active site structure. We found that the nucleophilic water molecule activated by D263 was supported by Y198 with a hydrogen bond at its phenolic oxygen in the 3D structure of REX. To break the interaction between Y198 and water molecule, the residue was replaced with F residue (Y198F). Y198F mutant expressed a drastic decrease in the hydrolytic activity and a small increase in F- releasing activity from α-X2F in the presence of xylose. To create glycosynthase from inverting GHs, we conclude that an amino acid residue holding the nucleophilic water molecule was better target for mutation than catalytic base residue.
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  • Tetsuya Mori, Tomoyuki Nishimoto, Kazuhisa Mukai, Hikaru Watanabe, Tak ...
    Volume 56 (2009) Issue 2 Pages 127-136
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide from starch. This oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate α-1,4 and α-1,6 linkages. The cyclic tetrasaccharide, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}, was designated cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as α-amylase, β-amylase, glucoamylase, isoamylase, pullulanase, maltogenic α-amylase and α-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. A glycosyltransferase involved in the synthesis of CMM from starch was purified to homogeneity from the culture supernatant of A. globiformis M6. The enzyme acted on maltooligosaccharides that have degrees of polymerization more than 3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan and dextran. The CMM-forming enzyme catalyzed both intermolecular and intramolecular α-1,6-maltosyl transfer reaction and found to be a novel maltosyltransferase (6MT). To reveal the degradation pathway of CMM, we identified two enzymes, CMM hydrolase (CMMase) and α-glucosidase, as the responsible enzymes from the cell-free extract of the strain. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates; however, it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the α-1,6-linkage of CMM. α-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates; however, it did not degrade CMM. Furthermore, when CMMase and α-glucosidase existed simultaneously in the reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by synergistic action of CMMase and α-glucosidase. The genes for 6MT, CMMase and α-glucosidase were cloned from the genomic library of A. globiformis M6. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, CMMase and α-glucosidase, indicating that these enzymes should be assigned to this family. In the cloning experiments, three other open reading frames (ORFs) were found. These ORFs were expected to encode proteins concerned with incorporation of CMM via cell membrane. The genes for CMMase and α-glucosidase and three ORFs were located downstream of the gene for 6MT, and expected to form gene cluster. The results of gene analysis suggested that A. globiformis M6 has a unique starch utilization pathway via CMM.
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  • Hisashi Ashida, Toshihiko Kato, Akihito Kawahara, Yuki Tanaka, Midori ...
    Volume 56 (2009) Issue 2 Pages 137-143
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Free oligosaccharides (FOS) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. We characterized the enzymes involved in generation and degradation of FOS in the nematode Caenorhabditis elegans. 1) Peptide: N-glycanase (PNGase) from Caenorhabditis elegans, which releases FOS from misfolded glycoproteins in the cytosol, was shown to be a unique bifunctional enzyme having both deglycosylation and protein disulfide reductase activities. 2) Endo-β-N-acetylglucosaminidase was proven to release a single GlcNAc residue at the reducing end of FOS in vivo. 3) Luminal class 1 α-mannosidases, probably Golgi α-mannosidase I, was involved in generation of M5A´ isoform of Man5GlcNAc1 that is specific to C. elegans.
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  • Teruko Konishi, Tadashi Ishii
    Volume 56 (2009) Issue 2 Pages 145-150
    Released: October 15, 2009
    JOURNALS FREE ACCESS
    Plant cell walls undergo dynamic changes during plant growth and development. Although the cell wall remodeling is an essential feature of plant growth and development, the biosynthesis mechanisms are poorly understood. Arabinan is a pectic polysaccharide which is linked by arabinofuranosyl (Araƒ) residues with α-(1,5) linkage. Arabinofuranosyl residues are a quantifiably important constituent of plant primary and secondary cell walls. Plants use UDP-arabinofuranose (UDP-Araƒ) to synthesize Araƒ regions of the polysaccharides containing Araƒ residues including proteoglycans and glycoproteins. However, it is unknown how UDP-Araƒ is synthesized in plant cells. We succeeded to, for the first time, determine UDP-arabinopyranose mutase (UAM) activity and clone the gene of the enzyme from rice seedlings. UAM catalyzed the interconversion of UDP-arabinopyranose (UDP-Arap) to UDP-Araƒ. Microbial UDP-galactose mutases require reduced FAD for activity, however, the plant UAM do not require the cofactor. Thus, the plant mutase must have different catalytic mechanism. Three proteins were identified from partial amino acid sequence of UAM, which are encoded by Os03g40270, Os04g56520 and Os07g41360. These proteins have more than 80% sequence identity with reversibly glycosylated polypeptide. UAM genes are present in Chlamydomonas, Physcomitrella and pine, suggesting that UAM is widespread in green plants.
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