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Master

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Applied Bioscience

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Applied Bioscience

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Degree

  • Ph. D. (S. Chiba) (Hokkaido University)（Hokkaido University）

Profile and Settings

  • Name (Japanese)

    Okuyama

  • Name (Kana)

    Masayuki

  • Name

    200901065754657289

Alternate Names

Achievement

Research Interests

  • 糖質分解酵素   分子進化   基質認識   α-グルコシダーゼ   立体構造解析   触媒メカニズム   α-グルコシダーゼII   糖転移反応   自殺基質   タンパク質工学   タンパク質の分子進化   小胞体グルコシダーゼ   触媒機構   基質特異性   糖質加水分解酵素   デキストラナーゼ   部位特異的変異   糖転移   国際情報交換   蜂蜜生成   蛋白質工学   アジア原産ミツバチ   アミノ酸置換   多糖合成酵素   タイ:韓国   構造因子   酵素反応   グリコシダーゼ   合成酵素   オリゴ糖合成   分子酵素学   protein engineering   molecular enzymology   

Research Areas

  • Life sciences / Applied molecular and cellular biology
  • Life sciences / Applied biochemistry

Research Experience

  • 2022/04 - Today 北海道大学大学院農学研究院 基盤研究部門応用生命科学分野 教授
  • 2014 Hokkaido University
  • 2007/04 - 2012/03 北海道大学大学院 農学研究科助 教
  • 2002/06 - 2007/03 北海道大学大学院 農学研究科 助手

Education

  • 1998/04 - 2001/03  北海道大学大学院
  • 1996/04 - 1998/03  北海道大学大学院
  • 1993/04 - 1996/03  Hokkaido University  Faculty of Agriculture  Department of Applied Bioscience

Awards

  • 2012 Committee of Plant and Seaweed Polysaccharides Workshop Committee of Plant and Seaweed Polysaccharides Workshop, Poster prize
     
    受賞者: OKUYAMA Masayuki
  • 2010 農芸化学奨励賞
  • 2007 CBM7 7th Carbohydrate Bioengineering Meeting Poster prize
  • 2003 日本農芸化学会 日本農芸化学会BBB論文賞
     
    受賞者: 奥山 正幸

Published Papers

  • Wilaiwan Kaenying, Takayoshi Tagami, Eukote Suwan, Chariwat Pitsanuwong, Sinchai Chomngam, Masayuki Okuyama, Palangpon Kongsaeree, Atsuo Kimura, Prachumporn T. Kongsaeree
    Heliyon 9 (11) e21923 - e21923 2405-8440 2023/11
  • Weeranuch Lang, Takayoshi Tagami, Yuya Kumagai, Seiya Tanaka, Hye-Jin Kang, Masayuki Okuyama, Wataru Saburi, Haruhide Mori, Tohru Hira, Chaehun Lee, Takuya Isono, Toshifumi Satoh, Hiroshi Hara, Takayuki Kurokawa, Nobuo Sakairi, Yoshiaki Yuguchi, Atsuo Kimura
    Carbohydrate Polymers 319 121185 - 121185 0144-8617 2023/11 [Refereed][Not invited]
  • Weeranuch Lang, Takayoshi Tagami, Hye-Jin Kang, Masayuki Okuyama, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 307 120629 - 120629 0144-8617 2023/05
  • Weeranuch Lang, Sarote Sirisansaneeyakul, Takayoshi Tagami, Hye-Jin Kang, Masayuki Okuyama, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 305 120565 - 120565 0144-8617 2023/04 [Refereed]
  • Weeranuch Lang, Debashish Mondol, Aphichat Trakooncharoenvit, Takayoshi Tagami, Masayuki Okuyama, Tohru Hira, Nobuo Sakairi, Atsuo Kimura
    Food Hydrocolloids 137 108430 - 108430 0268-005X 2023/04 [Refereed]
  • Wilaiwan Kaenying, Khuanjarat Choengpanya, Takayoshi Tagami, Pakorn Wattana-Amorn, Weeranuch Lang, Masayuki Okuyama, Yaw-Kuen Li, Atsuo Kimura, Prachumporn T. Kongsaeree
    Applied Microbiology and Biotechnology 107 (7-8) 2335 - 2349 0175-7598 2023/03/06 [Refereed][Not invited]
  • Rikako Shishiuchi, Hyejin Kang, Takayoshi Tagami, Yoshitaka Ueda, Weeranuch Lang, Atsuo Kimura, Masayuki Okuyama
    ACS Omega 7 (50) 47411 - 47423 2470-1343 2022/12/09 [Refereed]
  • Weeranuch Lang, Yuya Kumagai, Shinji Habu, Juri Sadahiro, Takayoshi Tagami, Masayuki Okuyama, Shinichi Kitamura, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 291 119562 - 119562 0144-8617 2022/09
  • Yuya Kumagai, Hideki Kishimura, Weeranuch Lang, Takayoshi Tagami, Masayuki Okuyama, Atsuo Kimura
    Marine Drugs 20 (4) 250 - 250 2022/03/31 
    The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases.
  • Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Wataru Saburi, Rakrudee Sarnthima, Takayoshi Tagami, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Doman Kim, Atsuo Kimura
    Applied microbiology and biotechnology 106 (2) 689 - 698 2022/01 
    Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.
  • Takayoshi Tagami, Minghao Chen, Yuta Furunaga, Asako Kikuchi, Juri Sadahiro, Weeranuch Lang, Masayuki Okuyama, Yoshikazu Tanaka, Tomohito Iwasaki, Min Yao, Atsuo Kimura
    The FEBS journal 289 (4) 1118 - 1134 2021/10/19 
    Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a β-glucose, cryoprotectant and hydrolysis product, respectively. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.
  • Masayuki Okuyama, Ryo Serizawa, Masanari Tanuma, Asako Kikuchi, Juri Sadahiro, Takayoshi Tagami, Weeranuch Lang, Atsuo Kimura
    The Journal of biological chemistry 296 100398 - 100398 2021/02/08 [Refereed]
     
    Glycoside hydrolase family 68 (GH68) enzymes catalyze β-fructosyltransfer from sucrose to another sucrose, so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, β-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the β-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and β-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the β-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, β-D-fructofuranosyl α-D-mannopyranoside, by β-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.
  • Min Ma, Masayuki Okuyama, Takayoshi Tagami, Asako Kikuchi, Patcharapa Klahan, Atsuo Kimura
    Journal of agricultural and food chemistry 67 (12) 3380 - 3388 0021-8561 2019/03/27 [Refereed][Not invited]
     
    α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3II- O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.
  • Patcharapa Klahan, Masayuki Okuyama, Kohei Jinnai, Min Ma, Asako Kikuchi, Yuya Kumagai, Takayoshi Tagami, Atsuo Kimura
    Bioscience, biotechnology, and biochemistry 82 (9) 1480 - 1487 0916-8451 2018/09 [Refereed][Not invited]
     
    Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL-1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL-1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the -3 and -4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the -4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL-1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732.
  • Asako Kikuchi, Masayuki Okuyama, Koji Kato, Shohei Osaki, Min Ma, Yuya Kumagai, Kana Matsunaga, Patcharapa Klahan, Takayoshi Tagami, Min Yao, Atsuo Kimura
    Biochimie 142 41 - 50 0300-9084 2017/11 [Refereed][Not invited]
     
    Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, α-glucoside hydrolase and α-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 α-glucoside hydrolase and α-galactosidase, were characterized in this study. BT3664 was identified to be an α-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both β-l-arabinopyranoside and α-d-galactopyranoside, and thus we designate BT3661 as a β-l-arabinopyranosidase/α-d-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 α-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears Glu361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The kcat/Km value for the hydrolysis of p-nitrophenyl α-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl β-l-arabinopyranoside.
  • Masayuki Okuyama, Masashi Miyamoto, Ichiro Matsuo, Shogo Iwamoto, Ryo Serizawa, Masanari Tanuma, Min Ma, Patcharapa Klahan, Yuya Kumagai, Takayoshi Tagami, Atsuo Kimura
    Bioscience, biotechnology, and biochemistry 81 (8) 1503 - 1511 0916-8451 2017/08 [Refereed][Not invited]
     
    The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc2Man3-Dansyl was faster than that of Glc1Man3-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2) although the activity was low.
  • Min Ma, Masayuki Okuyama, Megumi Sato, Takayoshi Tagami, Patcharapa Klahan, Yuya Kumagai, Haruhide Mori, Atsuo Kimura
    Applied microbiology and biotechnology 101 (16) 6399 - 6408 0175-7598 2017/08 [Refereed][Not invited]
     
    Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1-4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1-4)Glc(α1-4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1-6)Glc(α1-4)Glc], isomaltose [Glc(α1-6)Glc], and isomaltotriose [Glc(α1-6)Glc(α1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).
  • Masayuki Okuyama, Kana Matsunaga, Ken-Ichi Watanabe, Keitaro Yamashita, Takayoshi Tagami, Asako Kikuchi, Min Ma, Patcharapa Klahan, Haruhide Mori, Min Yao, Atsuo Kimura
    The FEBS journal 284 (5) 766 - 783 1742-464X 2017/03 [Refereed][Not invited]
     
    The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α-glycosidases can give poor yields of desired products because they require generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer from α-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, β-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, β-lactosyl α-d-galactoside, was solved to elucidate the ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2 and 3 of the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside recognition is conserved among GH27, 31, 36 and 97 α-galactosidases. DATABASE: The atomic coordinates (code: 5E1Q) have been deposited in the Protein Data Bank.
  • Yang-Hsin Hsu, Takayoshi Tagami, Kana Matsunaga, Masayuki Okuyama, Takashi Suzuki, Naonobu Noda, Masahiko Suzuki, Hanako Shimura
    The Plant journal : for cell and molecular biology 89 (2) 325 - 337 0960-7412 2017/01 [Refereed][Not invited]
     
    Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3'5'-O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.
  • Yuya Kumagai, Misugi Uraji, Kun Wan, Masayuki Okuyama, Atsuo Kimura, Tadashi Hatanaka
    FEBS letters 590 (17) 2862 - 9 0014-5793 2016/09 [Refereed][Not invited]
     
    Streptomyces thermolilacinus mannanase (StMan), which requires Ca(2+) for its enhanced thermal stability and hydrolysis activity, possesses two Ca(2+) -binding sites in loop6 and loop7. We evaluated the function of the Ca(2+) -binding site in loop7 and the hydrogen bond between residues Ser247 in loop6 and Asp279 in loop7. The Ca(2+) -binding in loop7 was involved only in thermal stability. Mutations of Ser247 or Asp279 retained the Ca(2+) -binding ability; however, mutants showed less thermal stability than StMan. Phylogenetic analysis indicated that most glycoside hydrolase family 5 subfamily 8 mannanases could be stabilized by Ca(2+) ; however, the mechanism of StMan thermal stability was found to be quite specific in some actinomycete mannanases.
  • Julan Liao, Masayuki Okuyama, Keigo Ishihara, Yoshinori Yamori, Shigeo Iki, Takayoshi Tagami, Haruhide Mori, Seiya Chiba, Atsuo Kimura
    Bioscience, biotechnology, and biochemistry 80 (9) 1747 - 52 0916-8451 2016/09 [Refereed][Not invited]
     
    The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.
  • Takayoshi Tagami, Eri Miyano, Juri Sadahiro, Masayuki Okuyama, Tomohito Iwasaki, Atsuo Kimura
    The Journal of biological chemistry 291 (32) 16438 - 47 0021-9258 2016/08/05 [Refereed][Not invited]
     
    The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of β-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s(-1) mm(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.
  • Yuya Kumagai, Masayuki Okuyama, Atsuo Kimura
    Carbohydrate polymers 146 396 - 401 0144-8617 2016/08/01 [Refereed][Not invited]
     
    Biologically active β-(1,3)-glucan oligosaccharides were prepared from curdlan using GH64 enzyme (KfGH64). KfGH64 showed low activity toward native curdlan; thereby pretreatment conditions of curdlan were evaluated. KfGH64 showed the highest activity toward curdlan with heat treatment. The most efficient pretreatment (90°C for 0.5h) converted approximately 60% of curdlan into soluble saccharides under the optimized enzyme reaction conditions (pH 5.5, 37°C, 100rpm mixing speed, 24h, and 10μg of KfGH64/1g of curdlan). The resulting products were predominantly laminaripentaose and a small amount of β-(1,3)-glucans with an average degree of polymerization (DP) of 13 and 130. The products did not contain small oligosaccharides (DP<5), indicating that the hydrolysis of heat-treated curdlan by KfGH64 is a suitable method for the production of biologically active β-(1,3)-glucan oligosaccharides.
  • Rungarun Suthangkornkul, Pornpisut Sriworanun, Hiroyuki Nakai, Masayuki Okuyama, Jisnuson Svasti, Atsuo Kimura, Saengchan Senapin, Dumrongkiet Arthan
    Phytochemistry 127 4 - 11 0031-9422 2016/07 [Refereed][Not invited]
     
    Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-β-glucosides, torvosides A and H. Furostanol glycoside 26-O-β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 β-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 β-glucosidase that hydrolyzes furostanol 26-O-β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.
  • Masayuki Okuyama, Wataru Saburi, Haruhide Mori, Atsuo Kimura
    Cellular and molecular life sciences : CMLS 73 (14) 2727 - 51 1420-682X 2016/07 [Refereed][Not invited]
     
    α-Glucosidases (AGases) and α-1,4-glucan lyases (GLases) catalyze the degradation of α-glucosidic linkages at the non-reducing ends of substrates to release α-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific α-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the α-(1 → 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose.
  • Structural and biochemical studies of plant α-glucosidases with a series of long-chain inhibitors.
    Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A
    Bull Appl Glycosci 6 (2) 103 - 108 2016 [Refereed][Not invited]
  • Ga-Hyun Joe, Midori Andoh, Aki Shinoki, Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Masayuki Okuyama, Atsuo Kimura, Hidehisa Shimizu, Hiroshi Hara, Satoshi Ishizuka
    Biomedical research (Tokyo, Japan) 37 (3) 179 - 86 0388-6107 2016 [Refereed][Not invited]
     
    The term "megalo-saccharide" is used for saccharides with ten or more saccharide units, whereas the term "oligo-saccharide" is used for saccharides containing fewer than ten monosaccharide units. Megalo-type α-1,6-glucosaccharide (M-IM) is a non-digestible saccharide and not utilized by intestinal bacteria, suggesting that ingested M-IM may encounter ileum Peyer's patches that contains immune cells such as macrophages. Macrophages are responsible for antigen incorporation and presentation during the initial step of immune responses. We investigated whether M-IMs modulate macrophage functions such as cytokine production, nitric oxide production, cell viability, and phagocytosis. Primary macrophages collected from the rats were cultured with the existence of M-IM or lipopolysaccharides (LPS). M-IM and LPS induced the production of tumor necrosis factor α (TNFα), interleukin 6 (IL6), and nitric oxide in the primary macrophages. The gene expression profile of inflammatory factors including TNFα, IL6, and ILlβ in M-IM-stimulated cells was similar to that of LPS-stimulated cells. The M-IM did not affect phagocytosis in the primary macrophages. The M-IM-induced TNFα production was suppressed in the cells treated with a tolllike receptor 4 (TLR4) inhibitor called TAK-242. In conclusion, the M-IM modulates cytokine expression via TLR4 signaling and may play a role in the modulation of immune responses.
  • Yasushi Masuda, Masayuki Okuyama, Takahisa Iizuka, Hiroyuki Nakai, Wataru Saburi, Taro Fukukawa, Janjira Maneesan, Takayoshi Tagami, Tetsushi Naraoka, Haruhide Mori, Atsuo Kimura
    Bioscience, biotechnology, and biochemistry 80 (3) 479 - 85 0916-8451 2016 [Refereed][Not invited]
     
    Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest Vmax/Km value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.
  • Yuya Kumagai, Keitaro Yamashita, Takayoshi Tagami, Misugi Uraji, Kun Wan, Masayuki Okuyama, Min Yao, Atsuo Kimura, Tadashi Hatanaka
    The FEBS journal 282 (20) 4001 - 14 1742-464X 2015/10 [Refereed][Not invited]
     
    Endo-β-1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III) ,6(IV) -α-d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.
  • Wataru Saburi, Hiroaki Rachi-Otsuka, Hironori Hondoh, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    FEBS letters 589 (7) 865 - 9 0014-5793 2015/03/24 [Refereed][Not invited]
     
    Glycoside hydrolase family 13 contains exo-glucosidases specific for α-(1→4)- and α-(1→6)-linkages including α-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The α-(1→6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to α-(1→4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher kcat/Km for maltose than wild-type, but its kcat/Km for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher kcat/Km for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the α-(1→6)-glucosidic linkage.
  • Momoko Kobayashi, Wataru Saburi, Daichi Nakatsuka, Hironori Hondoh, Koji Kato, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Min Yao
    FEBS letters 589 (4) 484 - 9 0014-5793 2015/02/13 [Refereed][Not invited]
     
    Streptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form α-(1→6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4Å resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG.
  • Juri Sadahiro, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura
    Biochemical and biophysical research communications 456 (1) 500 - 5 0006-291X 2015/01/02 [Refereed][Not invited]
     
    Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium.
  • Sadahiro J, Mori H, Saburi W, Okuyama M, Kimura A
    Biochem Biophys Res Commun 456 (1) 500 - 505 2015 [Refereed][Not invited]
  • Wataru Saburi, Masayuki Okuyama, Yuya Kumagai, Atsuo Kimura, Haruhide Mori
    BIOCHIMIE 108 140 - 148 0300-9084 2015/01 [Refereed][Not invited]
     
    alpha-Glucosidases are ubiquitous enzymes that hydrolyze the alpha-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an alpha-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant B5pAG31A, produced in Escherichia colt, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This is the first report of an alpha-glucosidase with high activity toward neotrehalose. The transglucosylation products, nigerose, kojibiose, isomaltose, and neotrehalose, were generated from 440 mm maltose. Substitution of Tyr268, situated on the beta -> alpha loop 1 of B5pAG31A, with Trp increased hydrolytic activity toward isomaltose. This mutation reduced the hydrolytic activity toward maltooligosaccharides more than toward kojibiose, nigerose, and neotrehalose. Analysis of the Y173A mutant of B5pAG31A showed that Tyr173, situated on the N-terminal domain loop, is associated with the formation of subsite +2. In Y173A, the k(cad)/K-m for maltooligosaccharides slightly decreased with an increasing degree of polymerization compared with wild type. Among the amino acid residues surrounding the substrate binding site, Va1543 and Glu545 of B5pAG31A were different from the corresponding residues of Bacillus thermoamyloliquefaciens alpha-glucosidase II, which has higher activity toward isomaltose than B5pAG31A. The E545G mutation slightly enhanced isomaltase activity without a large reduction of hydrolytic activities toward other substrates. V543A showed 1.8-3.5-fold higher hydrolytic activities toward all substrates other than neotrehalose compared with wild type, although its preference for isomaltose was unchanged. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.
  • Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 (3) 1796 - 1803 0021-9258 2015/01 [Refereed][Not invited]
     
    The alpha-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower K-m and higher k(cat)/K-m for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligo-saccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of alpha-glycosidases. The clear correlation obtained between log K-i of the acarviosyl-maltooligosaccharides and log(K-m/k(cat)) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the K-m for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.
  • Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Janjira Maneesan, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Atsuo Kimura
    BIORESOURCE TECHNOLOGY 169 518 - 524 0960-8524 2014/10 [Refereed][Not invited]
     
    Intermolecular interaction of linear-type alpha-(1 -> 6)-glucosyl megalosaccharide rich (L-IMS) and water-insoluble anionic ethyl red was firstly characterized in a comparison with inclusion complexation by cyclodextrins (CDs) to overcome the problem of poor solubility and bioavailability. Phase solubility studies indicated an enhancement of 3- and 9-fold over the solubility in water upon the presence of L-IMS and beta-CD, respectively. H-1 NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the beta-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by beta-CD showed an uncompetitive inhibitory-type effect to azoreductase, particularly with high water content that did not promote dye liberation. Addition of the solid dye dispersed into coupled-enzyme reaction system supplied by L-IMS as the dye solubilizer provided usual degradation rate. The dye intermission in series exhibited successful removal with at least 5 cycles was economically feasible. (C) 2014 Elsevier Ltd. All rights reserved.
  • Masayuki Okuyama, Takuya Yoshida, Hironori Hondoh, Haruhide Mori, Min Yao, Atsuo Kimura
    FEBS LETTERS 588 (17) 3213 - 3217 0014-5793 2014/08 [Refereed][Not invited]
     
    The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of k(cat)/K-m values in the presence or absence of calcium ion indicated that the calcium ion lowered the pK(a) of the base catalyst. The significant decreases in k(cat)/K-m for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Weeranuch Lang, Sarote Sirisansaneeyakul, Ligia O. Martins, Lukana Ngiwsara, Nobuo Sakairi, Wasu Pathom-aree, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    JOURNAL OF ENVIRONMENTAL MANAGEMENT 132 155 - 164 0301-4797 2014/01 [Refereed][Not invited]
     
    This study reports the characterization of the ability of Dermacoccus spp. isolated from the deepest point of the world's oceans for azo dye decolorization. A detailed investigation of Dermacoccus abyssi MT1.1(T) with respect to the azoreductase activity and enzymatic mechanism as well as the potential role of the bacterial strain for biocleaning of industrial dye baths is reported. Resting cells with oxygen-insensitive azoreductase resulted in the rapid decolorization of the polysulfonated dye Brilliant Black BN (BBN) which is a common food colorant. The highest specific decolorization rate (nu(s)) was found at 50 degrees C with a moderately thermal tolerance for over 1 h. Kinetic analysis showed the high rates and strong affinity of the enzymatic system for the dye with a V-max = 137 mg/g cell/h and a Km = 19 mg/L. The degradation of BBN produces an initial orange intermediate, 8-amino-5-((4-sulfonatophenyl)diazenyl)naphthalene-2-sulfonic acid, identified by mass spectrometry which is later converted to 4-aminobenzene sulfonic acid. Nearly 80% of the maximum nu(s) is possible achieved in resting cell treatment with the salinity increased up to 5.0% NaCl in reaction media. Therefore, this bacterial system has potential for dye decolorization bioprocesses occurring at high temperature and salt concentrations e.g. for cleaning dye-containing saline wastewaters. (C) 2013 Elsevier Ltd. All rights reserved.
  • Weeranuch Lang, Sarote Sirisansaneeyakul, Lukana Ngiwsara, Sonia Mendes, Ligia O. Martins, Masayuki Okuyama, Atsuo Kimura
    BIORESOURCE TECHNOLOGY 150 298 - 306 0960-8524 2013/12 [Refereed][Not invited]
     
    This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 mu M at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles. (C) 2013 Elsevier Ltd. All rights reserved.
  • Wataru Saburi, Momoko Kobayashi, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (44) 31670 - 31677 0021-9258 2013/11 [Refereed][Not invited]
     
    Dextran glucosidase from Streptococcus mutans (SmDG) catalyzes the hydrolysis of an alpha-1,6-glucosidic linkage at the non-reducing end of isomaltooligosaccharides and dextran. This enzyme has an Asp-194 catalytic nucleophile and two catalytically unrelated Cys residues, Cys-129 and Cys-532. Cys-free SmDG was constructed by replacement with Ser (C129S/C532S (2CS), the activity of which was the same as that of the wild type, SmDG). The nucleophile mutant of 2CS was generated by substitution of Asp-194 with Cys (D194C-2CS). The hydrolytic activity of D194C-2CS was 8.1 x 10(-4) % of 2CS. KI-associated oxidation of D194C-2CS increased the activity up to 0.27% of 2CS, which was 330 times higher than D194C-2CS. Peptide-mapping mass analysis of the oxidized D194C-2CS (Ox-D194C-2CS) revealed that Cys-194 was converted into cysteine sulfinate. Ox-D194C-2CS and 2CS shared the same properties (optimum pH, pI, and substrate specificity), whereas Ox-D194C-2CS had much higher transglucosylation activity than 2CS. This is the first study indicating that a more acidic nucleophile (-SOO-) enhances transglycosylation. The introduction of cysteine sulfinate as a catalytic nucleophile could be a novel approach to enhance transglycosylation.
  • Kyung-Mo Song, Masayuki Okuyama, Mariko Nishimura, Takayoshi Tagami, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (8) 1759 - 1765 0916-8451 2013/08 [Refereed][Not invited]
     
    The specificity for the alpha-1,4- and alpha-1,6-glucosidic linkages varies among glycoside hydrolase family 31 alpha-glucosidases. This difference in substrate specificity has been considered to be due to the difference in an aromatic residue on beta ->alpha loop 1 in the catalytic domain with a (beta/alpha)(8) barrel fold; i.e., the enzymes having Tyr and Trp on beta ->alpha loop 1 were respectively described as alpha-1,4-specific and alpha-1,6-specific alpha-glucosidases. Schwanniomyees oceidentalis alpha-glucosidase, however, prefers the alpha-1,4-glucosidic linkage, although the enzyme possesses Trp324 at the corresponding position. The mutation of Trp324 to Tyr decreased the ability for hydrolysis of the alpha-1,6-glucosidic linkage and formation of the alpha-1,6-glucosidic linkage in transglycosylation, indicating Trp324 to be closely associated with alpha-1,6 specificity, even if the enzyme preferred the alpha-1,4-glucosidic linkage. The mutant enzyme was found to catalyze the production of the branched oligosaccharide, 2,4-di-O-(alpha-D-glucopyranosyl)-D-glucopyranose, more efficiently than the wild-type enzyme.
  • Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (26) 19296 - 19303 0021-9258 2013/06 [Refereed][Not invited]
     
    Sugar beet alpha-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k(cat)/K-m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific alpha-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 angstrom resolution. The active site pocket of SBG is formed by a (beta/alpha)(8) barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (beta/alpha)(8) barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.
  • Takayoshi Tagami, Yoshiyuki Tanaka, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (2) 312 - 319 0916-8451 2013/02 [Refereed][Not invited]
     
    Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mm acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. H-1 and C-13 NMR spectra indicated that AC5-AC10 were alpha-acarviosyl-(1 -> 4)maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.
  • Takayoshi Tagami, Masayuki Okuyama, Hiroyuki Nakai, Young-Min Kim, Haruhide Mori, Kazunori Taguchi, Birte Svensson, Atsuo Kimura
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1834 (1) 329 - 335 1570-9639 2013/01 [Refereed][Not invited]
     
    Glycoside hydrolase family 31 alpha-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger alpha-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K-m for maltotriose, while sugar beet alpha-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K-m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K-m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K-m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AG5. (C) 2012 Elsevier B.V. All rights reserved.
  • Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara
    FOOD CHEMISTRY 136 (2) 293 - 296 0308-8146 2013/01 [Refereed][Not invited]
     
    The presence of an alpha-1,6-glucosaccharide enhances absorption of water-soluble quercetin glycosides, a mixture of quercetin-3-O-beta-D-glucoside (Q3G, 31.8%), mono (23.3%), di (20.3%) and more D-glucose adducts with alpha-1,4-linkage to a D-glucose moiety of Q3G, in a ligated small intestinal loop of anesthetized rats. We prepared alpha-1,6-glucosaccharides with different degrees of polymerization (DP) enzymatically and separated them into a megalo-isomaltosaccharide-containing fraction (M-IM, average DP = 11.0) and an oligo-isomaltosaccharide-containing fraction (O-IM, average DP = 3.6). Luminal injection of either saccharide fraction promoted the absorption of total quercetin-derivatives from the small intestinal segment and this effect was greater for M-IM than O-IM addition. M-IM also increased Q3G, but not the quercetin aglycone, concentration in the water-phase of the luminal contents more strongly than O-IM. The enhancement of Q3G solubilization in the luminal contents may be responsible for the increases in the quercetin glucoside absorption promoted by alpha-1,6-glucosaccharides, especially that by M-IM. These results suggest that the ingestion of alpha-1,6-glucosaccharides promotes Q3G bioavailability. (C) 2012 Elsevier Ltd. All rights reserved.
  • Tagami T, Okuyama M, Nakai H, Kim YM, Mori H, Taguchi K, Svensson B, Kimura A
    Biochimica et biophysica acta 1834 (1) 329 - 335 0006-3002 2013/01 [Refereed][Not invited]
  • Kyung-Mo Song, Masayuki Okuyama, Kazuyuki Kobayashi, Haruhide Mori, Atsuo Kimura
    Bioscience, Biotechnology and Biochemistry 77 (10) 2117 - 2124 0916-8451 2013 [Refereed][Not invited]
     
    For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.
  • Hitoshi Iwaya, Jae-Sung Lee, Shinya Yamagishi, Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Shiho Suzuki, Shinichi Kitamura, Hiroshi Hara, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka
    PLOS ONE 7 (11) e50658  1932-6203 2012/11 [Refereed][Not invited]
     
    Background: Isomaltosyloligosaccharides (IMO) and dextran (Dex) are hardly digestible in the small intestine and thus influence the luminal environment and affect the maintenance of health. There is wide variation in the degree of polymerization (DP) in Dex and IMO (short-sized IMO, S-IMO; long-sized IMO, L-IMO), and the physiological influence of these compounds may be dependent on their DP. Methodology/Principal Findings: Five-week-old male Wistar rats were given a semi-purified diet with or without 30 g/kg diet of the S-IMO (DP = 3.3), L-IMO (DP = 8.4), or Dex (DP = 1230) for two weeks. Dextran sulfate sodium (DSS) was administered to the rats for one week to induce experimental colitis. We evaluated the clinical symptoms during the DSS treatment period by scoring the body weight loss, stool consistency, and rectal bleeding. The development of colitis induced by DSS was delayed in the rats fed S-IMO and Dex diets. The DSS treatment promoted an accumulation of neutrophils in the colonic mucosa in the rats fed the control, S-IMO, and L-IMO diets, as assessed by a measurement of myeloperoxidase (MPO) activity. In contrast, no increase in MPO activity was observed in the Dex-diet-fed rats even with DSS treatment. Immune cell populations in peripheral blood were also modified by the DP of ingested saccharides. Dietary S-IMO increased the concentration of n-butyric acid in the cecal contents and the levels of glucagon-like peptide-2 in the colonic mucosa. Conclusion/Significance: Our study provided evidence that the physiological effects of alpha-glucosaccharides on colitis depend on their DP, linkage type, and digestibility. Citation: Iwaya H, Lee J-S, Yamagishi S, Shinoki A, Lang W, et al. (2012) The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization. PLoS ONE 7(11): e50658. doi: 10.1371/journal.pone.0050658
  • Lukana Ngiwsara, Gaku Iwai, Takayoshi Tagami, Natsuko Sato, Hiroyuki Nakai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (10) 1967 - 1974 0916-8451 2012/10 [Refereed][Not invited]
     
    Honeybees, Apis mellifera, possess three alpha-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-M preferred sucrose to maltose as substrate and formed only alpha-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the alpha-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) alpha-glucosidases and related enzymes.
  • Young-Min Kim, Eiji Yamamoto, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Masayuki Okuyama, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura
    FEBS JOURNAL 279 (17) 3185 - 3191 1742-464X 2012/09 [Refereed][Not invited]
     
    Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.
  • Nobuhiro Suzuki, Young-Min Kim, Zui Fujimoto, Mitsuru Momma, Masayuki Okuyama, Haruhide Mori, Kazumi Funane, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 (24) 19916 - 19926 0021-9258 2012/06 [Refereed][Not invited]
     
    Dextranase is an enzyme that hydrolyzes dextran alpha-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln(100) to Ile(732) of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 angstrom resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like beta-sandwich fold; domain A contained the enzyme's catalytic module, comprising a (beta/alpha)(8)-barrel; and domain C formed a beta-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, -4 to -1, in the catalytic cleft. The complexed structure with 4',5'-epoxypentyl-alpha-D-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp(385) side chain. These structures collectively indicated that Asp(385) was the catalytic nucleophile and that Glu(453) was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
  • Young-Min Kim, Yoshiaki Kiso, Tomoe Muraki, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Weeranuch Lang, Hee-Kwon Kang, Masayuki Okuyama, Haruhide Mori, Ryuichiro Suzuki, Kazumi Funane, Nobuhiro Suzuki, Mitsuru Momma, Zui Fujimoto, Tetsuya Oguma, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 (24) 19927 - 19935 0021-9258 2012/06 [Refereed][Not invited]
     
    A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1,500 to 1/40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using alpha-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a beta- or alpha-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from alpha-isomaltotetraosyl fluoride in the presence of NaN3.
  • Young-Min Kim, Wataru Saburi, Shukun Yu, Hiroyuki Nakai, Janjira Maneesan, Min-Sun Kang, Seiya Chiba, Doman Kim, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 (27) 22441 - 22444 0021-9258 2012/06 [Refereed][Not invited]
     
    alpha-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form alpha-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for alpha-glucosidases. alpha-Glucosidase-catalyzed hydration produced alpha-glucose from AF, which plateaued with time. Combined reaction with alpha-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger alpha-glucosidase (31AG), which is stable in organic solvent, produced ethyl alpha-glucoside from AF in 80% ethanol. The findings indicate that alpha-glucosidases catalyze trans-addition. This is the first report of alpha-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.
  • Atip Chantarudee, Preecha Phuwapraisirisan, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao
    BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 45  1472-6882 2012/04 [Refereed][Not invited]
     
    Background: Bee pollen is composed of floral pollen mixed with nectar and bee secretion that is collected by foraging honey (Apis sp.) and stingless bees. It is rich in nutrients, such as sugars, proteins, lipids, vitamins and flavonoids, and has been ascribed antiproliferative, anti-allergenic, anti-angiogenic and free radical scavenging activities. This research aimed at a preliminary investigation of the chemical constituents and free radical scavenging activity in A. mellifera bee pollen. Methods: Bee pollen was directly collected from A. mellifera colonies in Nan province, Thailand, in June, 2010, whilst floral corn (Zea mays L.) pollen was collected from the nearby corn fields. The pollen was then sequentially extracted with methanol, dichloromethane (DCM) and hexane, and each crude extract was tested for free radical scavenging activity using the DPPH assay, evaluating the percentage scavenging activity and the effective concentration at 50% (EC50). The most active crude fraction from the bee pollen was then further enriched for bioactive components by silica gel 60 quick and adsorption or Sephadex LH-20 size exclusion chromatography. The purity of all fractions in each step was observed by thin layer chromatography and the bioactivity assessed by the DPPH assay. The chemical structures of the most active fractions were analyzed by nuclear magnetic resonance. Results: The crude DCM extract of both the bee corn pollen and floral corn pollen provided the highest active free radical scavenging activity of the three solvent extracts, but it was significantly (over 28-fold) higher in the bee corn pollen (EC50 = 7.42 +/- 0.12 mu g/ml), than the floral corn pollen (EC50 = 212 +/- 13.6% mu g/ml). After fractionation to homogeneity, the phenolic hydroquinone and the flavone 7-O-R-apigenin were found as the minor and major bioactive compounds, respectively. Bee corn pollen contained a reasonably diverse array of nutritional components, including biotin (56.7 mu g/100 g), invert sugar (19.9 g/100 g), vitamin A and beta carotene (1.53 mg/100 g). Conclusions: Bee pollen derived from corn (Z. mays), a non-toxic or edible plant, provided a better free radical scavenging activity than floral corn pollen.
  • Dungporn Teerasripreecha, Preecha Phuwapraisirisan, Songchan Puthong, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao
    BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 27  1472-6882 2012/03 [Refereed][Not invited]
     
    Background: Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity. Methods: Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G(2)), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy. Results: The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC50 values across the five cancer cell lines ranging from 41.3 to 52.4 mu g/ml and from 43.8 to 53.5 mu g/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC50 values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 mu g/ml for the cardanol and < 3.13 to 5.97 mu g/ml (6.82 - 13.0 mu M) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts. Conclusion: This is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.
  • Degree of polymerization in dietary α-1,6-gluco¬sac¬cha¬rides modulates symptom of experimental colitis in rats.
    Iwaya H, Lee JS, Yamagishi S, Shinoki A, Lang W, Kang HK, Okuyama M, Mori H, Hara H, Kimura A, Ishizuka S
    Plos One 7 (11) e50658 - e50658 2012 [Refereed][Not invited]
  • Characterization of some enzymatic properties of recombinant α-glucosidase III from the Thai honeybee, Apis cerana indica Fabricus.
    Kaewmuangmoon J, Yoshiyama M, Kimura K, Okuyama M, Mori H, Kimura A, Chanchao C
    Afr J Biotechnol 11 (96) 16220 - 16232 2012 [Refereed][Not invited]
  • Masayuki Okuyama
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (12) 2269 - 2277 0916-8451 2011/12 [Refereed][Not invited]
     
    A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on alpha-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli a-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe alpha-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining alpha-glycosidases, including GH family 97 retaining alpha-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.
  • Nobuhiro Suzuki, Young-Min Kim, Zui Fujimoto, Mitsuru Momma, Hee-Kwon Kang, Kazumi Funane, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 67 (Pt 12) 1542 - 1544 1744-3091 2011/12 [Refereed][Not invited]
     
    Streptococcus mutans dextranase hydrolyzes the internal a-1,6-linkages of dextran and belongs to glycoside hydrolase family 66. An N- and C-terminal deletion mutant of S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 angstrom and belonged to space group P21, with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 angstrom, beta = 102.3 degrees. Assuming that the asymmetric unit of the crystal contained one molecule, the Matthews coefficient was calculated to be 4.07 angstrom 3 Da-1; assuming the presence of two molecules in the asymmetric unit it was calculated to be 2.03 angstrom 3 Da-1.
  • Momoko Kobayashi, Hironori Hondoh, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (8) 1557 - 1563 0916-8451 2011/08 [Refereed][Not invited]
     
    Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl2 increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl2 also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca2+, were considered to stabilize SmDG. On the other hand, CaCl2 had no significant effect on catalytic reaction. The enhanced stability by Ca2+ was probably related to calcium binding in the beta -> alpha loop 1 of the (beta/alpha)(8) barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.
  • Young-Min Kim, Ryoko Shimizu, Hiroyuki Nakai, Haruhide Mori, Masayuki Okuyama, Min-Sun Kang, Zui Fujimoto, Kazumi Funane, Doman Kim, Atsuo Kimura
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 91 (2) 329 - 339 0175-7598 2011/07 [Refereed][Not invited]
     
    Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.
  • Min-Sun Kang, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    BIOCHIMIE 91 (11-12) 1434 - 1442 0300-9084 2009/11 [Refereed][Not invited]
     
    Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES-GroEL chaperones. Recombinant LJAG31 was an alpha-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k(cat)/K-m) toward nigerose, maltulose, and kojibiose than other natural substrates having an alpha-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial alpha-1,3-glucosidase to be characterized with a high k(cat)/K-m value for nigerose [alpha-D-Glcp-(1 -> 3)-D-Glcp]. Transglucosylation of 4-nitrophenyl M-D-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl alpha-nigeroside and 4-nitrophenyl alpha-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) alpha-1,3-glucosidase of glycoside hydrolase family 31. (C) 2009 Elsevier Masson SAS. All rights reserved.
  • Haruhide Mori, Jin-Ha Lee, Masayuki Okuyama, Mamoru Nishimoto, Masao Ohguchi, Doman Kim, Atsuo Kimura, Seiya Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 (11) 2466 - 2473 0916-8451 2009/11 [Refereed][Not invited]
     
    Trehalase, an anomer-inverting glycosidase, hydrolyzes only alpha,alpha-trehalose in natural substrates to release equimolecular beta-glucose and alpha-glucose. Since the hydrolytic reaction is reversible, alpha,alpha-[1,1'-H-2]trehalose is capable of synthesis from [1-H-2]glucose through the reverse reaction of trehalase. alpha-Secondary deuterium kinetic isotope effects (alpha-SDKIEs) for the hydrolysis of synthesized alpha,alpha-[1,1'-H-2]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high k(H)/k(D) value of 1.53 for alpha-SDKIEs was observed. The data imply that the catalytic reaction of the trehalase occurs by the oxocarbenium ion intermediate mechanism. In addition, the hydrolytic reaction of glycosidase is discussed from the viewpoint of chemical reactivity for the hydrolysis of acetal in organic chemistry. As to the hydrolytic reaction mechanism of glycosidases, oxocarbenium ion intermediate and nucleophilic displacement mechanisms have been widely recognized, but it is pointed out for the first time that the former mechanism is rational and valid and generally the latter mechanism is unlikely to occur in the hydrolytic reaction of glycosidases.
  • Masayuki Okuyama, Momoyo Kitamura, Hironori Hondoh, Min-Sun Kang, Haruhide Mori, Atsuo Kimura, Isao Tanaka, Min Yao
    JOURNAL OF MOLECULAR BIOLOGY 392 (5) 1232 - 1241 0022-2836 2009/10 [Refereed][Not invited]
     
    Glycoside hydrolase family 97 (GH 97) is a unique glycoside family that contains inverting and retaining glycosidases. Of these, BtGH97a (SusB) and BtGH97b (UniProtKB/TrEMBL entry QM6L0), derived from Bacteroides thetaiotaomicron, have been characterized as an inverting a-glucoside hydrolase and a retaining a-galactosidase, respectively. Previous studies on the three-dimensional structures of BtGH97a and site-directed mutagenesis 508 indicated that Glu532 acts as an acid catalyst and that G function as the catalytic base in the inverting mechanism. However, BtGH97b lacks base catalysts but possesses a putative catalytic nucleophilic residue, Asp415. Here, we report that Asp415 in BtGH97b is the nucleophilic catalyst based on the results of crystal structure analysis and site-directed mutagenesis study. Structural comparison between BtGH97b and BtGH97a indicated that OD1 of Asp415 in BtGH97b is located at a position spatially identical with the catalytic water molecule of BtGH97a, which attacks on the anomeric carbon from the beta-face (i.e., Asp415 is poised for nucleophilic attack on the anomeric carbon). Site-directed mutagenesis of Asp415 leads to inactivation of the enzyme, and the activity is rescued by an external nucleophilic azide ion. That is, Asp415 functions as a nucleophilic catalyst. The multiple amino acid sequence alignment of GH 97 members indicated that almost half of the GH 97 enzymes possess base catalyst residues at the end of beta-strands 3 and 5, while the other half of the fan-Lily show a conserved nucleophilic residue at the end of beta-strand 4. The different positions of functional groups on the beta-face of the substrate, which seem to be due to "hopping of the functional group" during evolution, have led to divergence of catalytic mechanism within the same family. (C) 2009 Elsevier Ltd. All rights reserved.
  • Wataru Saburi, Hironori Hondoh, Young-Min Kim, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    BIOLOGIA 63 (6) 1000 - 1005 0006-3088 2008/12 [Not refereed][Not invited]
     
    Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an alpha-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short- and long-chain substrates, while O16G acts on only short- chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short beta -> alpha loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short beta -> alpha loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.
  • Momoyo Kitamura, Masayuki Okuyama, Fumiko Tanzawa, Haruhide Mori, Yu Kitago, Nobuhisa Watanabe, Atsuo Kimura, Isao Tanaka, Min Yao
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 (52) 36328 - 36337 0021-9258 2008/12 [Refereed][Not invited]
     
    SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6 angstrom resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu(532) is positioned to provide protonic assistance to leaving group departure, with Glu(439) and Glu(508) both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.
  • Masayuki Okuyama, Haruhide Mori, Hironori Hondoh, Hiroyuki Nakai, Wataru Saburi, Min Sung Kang, Young Min Kim, Mamoru Nishimoto, Jintanart Wongchawalit, Takeshi Yamamoto, Mee Son, Jin Ha Lee, San San Mar, Kenji Fukuda, Seiya Chiba, Atsuo Kimura
    Carbohydrate-Active Enzymes: Structure, Function and Applications 64 - 76 2008/09 [Refereed][Not invited]
     
    α-Glucosidase (EC 3.2.1.20), an exo-glycosylase to hydrolyze α-glucosidic linkage, is characterized by the variety of substrate specificity. Enzyme also catalyzes the transglucosylation, on which industrial interests focus due to the production of valuable glucooligosaccharides. α-Glucosidase is a physiologically important enzyme in most of organisms (microorganisms, insects, plants and animals including human). Therefore, there are many types of α-glucosidases to display unique functions, in which we are interested. This report describes the recently analyzed unique functions of α-glucosidases by mainly focusing on honeybee α-glucosidase isoenzymes, dextran glucosidase, multiple forms of rice α-glucosidases, and Escherichia coli α-xylosidase. © 2008 Woodhead Publishing Limited. All rights reserved.
  • Hironori Hondoh, Wataru Saburi, Haruhide Mori, Masayuki Okuyama, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura
    JOURNAL OF MOLECULAR BIOLOGY 378 (4) 913 - 922 0022-2836 2008/05 [Refereed][Not invited]
     
    We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the alpha-1,6-glucosidic linkage of isomaltooli-gosaccharides from their non-reducing ends to produce alpha-glucose. By using the mutant of catalytic acid Glu236 -> Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 angstrom resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (beta/alpha)(8)-barrel in domain A, which is common to the alpha-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites -1 to +2. The environment of the glucose residue at subsite -1 is similar to the environment of this residue in the alpha-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite -1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile alpha-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile alpha-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the alpha-amylase structure from Bacillus subtilis. The comparison with the alpha-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of alpha-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes. (c) 2008 Elsevier Ltd. All rights reserved.
  • M-S. Kang, M. Okuyama, K. Yaoi, Y. Mitsuishi, Y-M. Kim, H. Mori, A. Kimura
    BIOCATALYSIS AND BIOTRANSFORMATION 26 (1-2) 96 - 103 1024-2422 2008 [Not refereed][Not invited]
     
    Escherichia coli YicI is a retaining alpha-xylosidase, which strictly recognizes the alpha-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli -xylosidase (YicI). Detection of distinguishing features between alpha-xylosidases and alpha-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an alpha-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, alpha-D-Xylp-(1 -> 6)-D-Manp, alpha-D-Xylp-(1 -> 6)-D-Fruf, and alpha-D-Xylp-(1 -> 3)-D-Frup, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal alpha-glucosidases.
  • Hiroyuki Nakai, Shigeki Tanizawa, Tatsuya Ito, Koutaro Kamiya, Young-Min Kim, Takeshi Yamamoto, Kazuki Matsubara, Makoto Sakai, Hiroyuki Sato, Tokio Imbe, Masayuki Okuyama, Haruhide Mori, Seiya Chiba, Yoshio Sano, Atsuo Kimura
    Biocatalysis and Biotransformation 26 (1-2) 104 - 110 1024-2422 2008 [Refereed][Not invited]
     
    Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized α-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) α-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by α-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the α-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.
  • Min-Sun Kang, Masayuki Okuyama, Katsuro Yaoi, Yasushi Mitsuishi, Young-Min Kim, Haruhide Mori, Doman Kim, Atsuo Kimura
    FEBS JOURNAL 274 (23) 6074 - 6084 1742-464X 2007/12 [Refereed][Not invited]
     
    The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1 -> 6)-D-Manp, alpha-D-Xylp-(1 -> 6)-D-Fruf, and alpha-D-Xylp-(1 -> 3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-D-Xylp-(1 -> 6)-D-Manp and alpha-D-Xylp(1 -> 6)-D-Fruf inhibited intestinal alpha-glucosidases.
  • Hiroyuki Nakai, Shigeki Tanizawa, Tatsuya Ito, Koutarou Kamiya, Young-Min Kim, Takeshi Yamamoto, Kazuki Matsubara, Makoto Sakai, Hiroyuki Sato, Tokio Imbe, Masayuki Okuyama, Haruhide Mori, Yoshio Sano, Seiya Chiba, Atsuo Kimura
    JOURNAL OF BIOCHEMISTRY 142 (4) 491 - 500 0021-924X 2007/10 [Refereed][Not invited]
     
    In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.
  • Wataru Saburi, Hironori Hondoh, Hideaki Unno, Masayuki Okuyama, Haruhide Mori, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 63 (Pt 9) 774 - 776 1744-3091 2007/09 [Refereed][Not invited]
     
    Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal alpha-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 angstrom. A native data set was collected to 2.2 angstrom resolution from a single crystal.
  • Jin-Ha Lee, Saori Saito, Haruhide Mori, Mamoru Nishimoto, Masayuki Okuyama, Doman Kim, Jintanart Wongchawalit, Atsuo Kimura, Seiya Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (9) 2256 - 2265 0916-8451 2007/09 [Refereed][Not invited]
     
    cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5 ' untranslated region (UTR) of 869 bp, and the 3 ' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M-r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.
  • Mamoru Nishimoto, Haruhide Mori, Tsuneharu Moteki, Yukiko Takamura, Gaku Iwai, Yu Miyaguchi, Masayuki Okuyama, Jintanart Wongchawalit, Rudee Surarit, Jisnuson Svasti, Atsuo Kimura, Seiya Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (7) 1703 - 1716 0916-8451 2007/07 [Refereed][Not invited]
     
    cDNAs encoding three alpha-glucosidases (HBGases I, II, and 111) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 by in length, and included ORFs of 1,767, 1,743, and 1,704 by encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases 1, 11, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (3844% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.
  • Hiroyuki Nakai, Tatsuya Ito, Masatoshi Hayashi, Koutarou Kamiya, Takeshi Yamamoto, Kazuki Matsubara, Young-Min Kim, Wongchawalit Jintanart, Masayuki Okuyama, Haruhide Mori, Seiya Chiba, Yoshio Sano, Atsuo Kimura
    BIOCHIMIE 89 (1) 49 - 62 0300-9084 2007/01 [Refereed][Not invited]
     
    Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice. (c) 2006 Elsevier Masson SAS. All rights reserved.
  • Jintanart Wongchawalit, Takeshi Yamamoto, Hiroyuki Nakai, Young-Min Kim, Natsuko Sato, Mamoru Nishimoto, Masayuki Okuyama, Haruhide Mori, Osamu Saji, Chanpen Chanchao, Siriwat Wongsiri, Rudee Surarit, Jisnuson Svasti, Seiya Chiba, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 (12) 2889 - 2898 0916-8451 2006/12 [Refereed][Not invited]
     
    a-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.
  • NAKAI Hiroyuki, ITO Tatsuya, TANIZAWA Shigeki, MATSUBARA Kazuki, YAMAMOTO Takeshi, OKUYAMA Masayuki, MORI Haruhide, CHIBA Seiya, SANO Yoshio, KIMURA Atsuo
    Journal of Applied Glycoscience The Japanese Society of Applied Glycoscience 53 (2) 137 - 142 1344-7882 2006/07 [Refereed][Not invited]
     
    In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by de novo synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.
  • M Okuyama, A Kaneko, H Mori, S Chiba, A Kimura
    FEBS LETTERS 580 (11) 2707 - 2711 0014-5793 2006/05 [Refereed][Not invited]
     
    Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta -> alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C3071/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Wataru Saburi, Haruhide Mori, Saori Saito, Masayuki Okuyama, Atsuo Kimura
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1764 (4) 688 - 698 1570-9639 2006/04 [Refereed][Not invited]
     
    Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta -> alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar k(cat)/k(m) value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta -> alpha loop 4 of B. subtilis oligo- 1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta ->alpha loop 4 were important for high activity to long-chain substrates. (c) 2006 Elsevier B.V. All rights reserved.
  • Saburi W, Mori H, Saito S, Okuyama M, Kimura A
    Biochimica et biophysica acta 1764 (4) 688 - 698 0006-3002 2006/04 [Refereed][Not invited]
  • F Sato, M Okuyama, H Nakai, H Mori, A Kimura, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 (10) 1905 - 1913 0916-8451 2005/10 [Refereed][Not invited]
     
    A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K-m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.
  • M Kitamura, T Ose, M Okuyama, H Watanabe, M Yao, H Mori, A Kimura, Tanaka, I
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 (Pt 2) 178 - 179 1744-3091 2005/02 [Refereed][Not invited]
  • YM Kim, M Okuyama, H Mori, H Nakai, W Saburi, S Chiba, A Kimura
    TETRAHEDRON-ASYMMETRY 16 (2) 403 - 409 0957-4166 2005/01 [Refereed][Not invited]
     
    Aspergillus niger alpha-glucosidase (ANGase) was used for an efficient syntheses of alkyl alpha-D-2-deoxyglucosides (A2DGs) and for regioselectivity studies of alkoxy-hydro additions Of D-glucal in the presence of alkyl alcohols. ANGase showed a high stability with respect to the high concentration of alkyl alcohols. The reaction conditions were optimized for pH, temperature, alkyl alcohol concentration, and D-glucal concentration. On the basis of MS and NMR analyses, A2DGs were confirmed to have only an alpha-2deoxyglucosidic bond and the two-dimensional NMR (HMBC) spectra showed to be made up of 2-deoxyglucosyl and alkyl moieties. (C) 2004 Elsevier Ltd. All rights reserved.
  • Nakai H, Okuyama M, Kim YM, Saburi W, Wongchawalit J, Mori H, Chiba S, Kimura A
    Biologia, Bratislava 60 131 - 135 2005 [Refereed][Not invited]
  • Nakai H., Okuyama M., Kim YM., Saburi W., Wongchawalit J., Mori H., Chiba S. and Kimura A. "Molecular analysis of alpha-glucosidase belonging to GH-family 31" Biologia 60: 131-135(2005)*
    2005 [Not refereed][Not invited]
  • Okuyama M., Tanimoto Y., Ito T., Anzai A., Mori H., Kimura A., Matsui H. and Chiba S. "Purification and characterization of the hyper-glycosylated extracellular alpha-glucosidase from Schizosaccharomyces pombe" Enzyme and Microbial Technol., 37:472-48・・・
    2005 [Not refereed][Not invited]
     
    Okuyama M., Tanimoto Y., Ito T., Anzai A., Mori H., Kimura A., Matsui H. and Chiba S. "Purification and characterization of the hyper-glycosylated extracellular alpha-glucosidase from Schizosaccharomyces pombe"
    Enzyme and Microbial Technol., 37:472-480(2005)*
  • KIM YM., Okuyama M., Mori H., Chiba S. and Kimura A.: "Enzymatic synthesis of alkyl alpha-2-deoxyglucosides by alkyl alcohol resistant alpha-glucosidase from Aspergillus niger", Tetrahedron-asymmetr. 16 403-409 (2005)*
    2005 [Not refereed][Not invited]
  • M Kubota, M Tsuji, M Nishimoto, J Wongchawalit, M Okuyama, H Mori, H Matsui, R Surarit, J Svasti, A Kimura, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (11) 2346 - 2352 0916-8451 2004/11 [Refereed][Not invited]
     
    Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an et-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey a-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.
  • M Okuyama, H Mori, S Chiba, A Kimura
    PROTEIN EXPRESSION AND PURIFICATION 37 (1) 170 - 179 1046-5928 2004/09 [Refereed][Not invited]
     
    The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E coli were purified and characterized. YicI has the activity of Otxylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24 h at 4 degreesC and also is stable up to 47 degreesC with incubation for 15 min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6-O-alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E coli. (C) 2004 Elsevier Inc. All rights reserved.
  • T Yamamoto, T Unno, Y Watanabe, M Yamamoto, M Okuyama, H Mori, S Chiba, A Kimura
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1700 (2) 189 - 198 1570-9639 2004/08 [Refereed][Not invited]
     
    alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 5 1,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(o)/K-m value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)- O-alpha-maltosyl-maltose were novel saccharides. (C) 2004 Elsevier B.V All rights reserved.
  • Yamamoto T, Unno T, Watanabe Y, Yamamoto M, Okuyama M, Mori H, Chiba S, Kimura A
    Biochimica et biophysica acta 1700 (2) 189 - 198 0006-3002 2004/08 [Refereed][Not invited]
  • SS Mar, H Mori, JH Lee, K Fukuda, W Saburi, A Fukuhara, M Okuyama, S Chiba, A Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (5) 1080 - 1093 0916-8451 2003/05 [Refereed][Not invited]
     
    Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): k(cat), 71.8 and 55.5 s(-1), K-m, 0.113 and 0.097 mg /ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: k(cat), 62.4 and 85.3 s(-1), K-m, 0.22 and 0.37 mm, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on /near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.
  • Son Mee, Mori Haruhide, Okuyama Masayuki, Kimura Atsuo, Chiba Seiya
    Journal of Applied Glycoscience The Japanese Society of Applied Glycoscience 50 (1) 41 - 44 1344-7882 2003 [Not refereed][Not invited]
     
    The hydrolytic reaction of carbohydrate-hydrolase is essentially accompanied by a reverse reaction (the condensation reaction), meaning that only the substrate capable of being hydrolyzed is produced by the reverse reaction. Honeybee α-glucosidase I can't hydrolyze isomaltose, but is capable of hydrolyzing maltose, kojibiose and slightly nigerose. Nevertheless, the enzyme catalyzes the formation and accumulation of isomaltose from glucose together with α-glucobioses such as maltose, kojibiose and nigerose. This finding is in conflict with the data that the enzyme has no hydrolytic activity toward isomaltose. However, the conflict for the peculiar phenomenon on the reaction was rationally explained by the evidence that isomaltose might be formed by the intramolecular transglucosylation via other α-glucobioses that are easily produced from glucose by the condensation reaction. It is suggested that the usual transglycosylation of carbohydrate-hydrolase may be accompanied by an intramolecular transfer reaction.
  • Son, M., Mori, H., Okuyama, M., Kimura A. and Chiba, S.:" Evidence of Intramolecular Transglucosylation Catalyzed by an alpha-Glucosidase", Journal of Applied Glycoscience, 50: 41-44(2003)*
    2003 [Not refereed][Not invited]
  • K Fukuda, H Mori, M Okuyama, A Kimura, H Ozaki, M Yoneyama, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 (10) 2060 - 2067 0916-8451 2002/10 [Refereed][Not invited]
     
    Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and K. values on pH, when the substrate concentration was sufficiently lower than K-m, gave the values of 4.1 and 7.2 for the ionization constants, pK(e1) and pKe(2) of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe(1) and pKe(2), values shifted to higher, to + 0.60 and + 0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities A(i)'s in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive A(i) values (A(1) was 1.10kcal/mol and A(2) was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, kthe subsites 3 and 4 showed negative A(i) values (A(3) was - 0.21 kcal /mol and A(4) was - 2.8 kcal /mol).
  • FUKUDA Kenji, SHIRAKAWA Kou, MORI Haruhide, OKUYAMA Masayuki, KIMURA Atsuo, OZAKI Hachiro, YONEYAMA Michio, CHIBA Seiya
    Journal of applied glycoscience 日本応用糖質科学会 49 (3) 265 - 272 1340-3494 2002/07/18 [Not refereed][Not invited]
     
    The substrate specificity and the active site of β-D-glucosidase F-1 (Mr, 50, 000; optimum pH, 5.5) purified from a Streptomyces sp. were kinetically investigated. The β-D-glucosidase showed a broad substrate specificity for synthetic glycosides and disaccharides having β-glycosidic linkage, but the former was more favorable substrate than the latter. The enzyme was characterized by the ability to hydrolyze rapidly not only ρ-nitrophenyl β-glucoside (Km, 0.72 mM; k0, 63 s-1) but also ρ-nitrophenyl β-fucoside (Km, 0.19 mM; k0, 44 s-1) and laminaribiose (Km, 1.6 mM; k0, 70 s-1). The kinetic study was made as to whether the hydrolyses of these synthetic glycosides were catalyzed at a single active site or at dual active sites in the β-D-glucosidase. In the experiments with the mixed substrates of p-nitrophenyl β-glucoside (PNPG) and ρ-nitrophenyl β-fucoside (PNPF) or ρ-nitrophenyl β-galactoside (PNPGal), the kinetic features, the linearity of Lineweaver-Burk plots and the dependence of the apparent maximal veloities and Km value on the mole fraction (f) of PNPG in the mixed substrate, f = [PNPG/([PNPG] + [PNPF or PNPGal]) agreed very closely with those theoretically predicted for a single catalytic site mechanism. The findings strongly support the notion that the β-D-glucosidase attacks the synthetic glycosides at a common active site.
  • M Okuyama, H Mori, K Watanabe, A Kimura, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 (4) 928 - 933 0916-8451 2002/04 [Refereed][Not invited]
     
    Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively. PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not. No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside. This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.
  • FUKUDA K, SHIRAKAWA K, MORI H, OKUYAMA M, KIMURA A, OZAKI H, YONEYAMA M, CHIBA S
    J. Appl. Glycosci. 49 (3) 265 - 272 1344-7882 2002 [Not refereed][Not invited]
  • Fukuda, K., Shirakawa, K., Mori, H., Okuyama, M., Kimura, A., Ozaki H., Yoneyama, M. and Chiba, S. "Kinetic Studies on Substrate Specificity and Active site of β-D-Glucosidase F1 from Streptomyces sp.", Journal of Applied Glycoscience, 49, 265-272(2002)
    2002 [Not refereed][Not invited]
  • Jin-Ha Lee, Masahisa Tsuji, Mitsuru Nakamura, Mamoru Nishimoto, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Hirokazu Matsui, Seiya Chiba
    Bioscience, Biotechnology and Biochemistry 65 (12) 2657 - 2665 0916-8451 2001/12 [Refereed][Not invited]
     
    Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl β-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2s-1. The enzyme was an "inverting" type which produced β-glucose from α, α-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of +0.2 and +0.5 pH unit, respectively. The ionization heat (ΔH) of ionizable group 1 was estimated to be +1.8 kcal/mol, and the ΔH value of group 2 was +1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO-, ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe2 value is high.
  • M Okuyama, A Okuno, N Shimizu, H Mori, A Kimura, S Chiba
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (8) 2270 - 2280 0014-2956 2001/04 [Refereed][Not invited]
     
    cDNA encoding Schizosaccharomyces pombe alpha -glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M-r 106 138. The deduced amino-acid sequence showed a high homology to those of alpha -glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha -glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha -glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.
  • SAEKI Takeshi, OKUYAMA Masayuki, MORI Haruhide, KIMURA Atsuo, CHIBA Seiya
    Journal of applied glycoscience 日本応用糖質科学会 45 (3) 281 - 283 1340-3494 1998/08/31 [Not refereed][Not invited]

MISC

Presentations

  • Discovery of α-L-glucosidase in nature  [Not invited]
    Masayuki Okuyama, Hye-jin Kang, Rikako Shishiuchi, Takayoshi Tagami
    3rd Japan-Switzerland-Germany Workshop on Biocatalysis and Bioprocess Development  2023/10
  • 糖質加水分解酵素ファミリー97における触媒機構と多様性  [Invited]
    奥山 正幸
    日本農芸化学会2015年度岡山大会  2015/03

Association Memberships

  • 日本農芸化学会   日本応用糖質科学会   

Works

  • 「Glycoside hydrolase family 31酵素の構造と機能」、日本農芸化学会平成17年度大会シンポジウム－糖質酵素の最先端研究を担う若きサイエンティストの視点－
    2005
  • 「glycosidase変異酵素が触媒するglycosynthase反応」, 『第29回糖質科学懇話会』

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
    Date (from‐to) : 2019/10 -2023/03 
    Author : 木村 淳夫, 橋床 泰之, 崎浜 靖子, 奥山 正幸, 田上 貴祥
     
    我々は世界で初めてメガロ糖（MS）の生産に成功した。性質を調べると、BCS IIに属す化合物（難水溶性・高膜透過性の薬剤や食品素材など）を可溶化する画期的な機能が発見された。またMSは「BCS II化合物を溶質とする糖質水溶化剤」と捉えることもできた。一方、難溶性ベンジル系アゾ色素もBCS IIに属し、かつ「東南アジア諸国における名高い環境汚染物質」である点に注目し、MSとアゾ分解酵素を組合せることで、実験室レベルではあるが、色素の可溶化と酵素分解に成功した。以上は初めて生産したMS、すなわち従来型MSの知見である。極最近に従来型MSより高機能な新奇MS（新型MS）を発見した。本申請では、新型MSによるアゾ色素の酵素分解を目的とし、現地試験をタイで実施する。最終的な到達目標はアゾ色素の汚染解消（すなわち環境問題解決への貢献）である。 本年度は、新型コロナウイルス感染症の流行および相手国の政情不安から渡航が困難となり、現地調査に大きな支障が生じ、計画の遂行に予想外の大きな遅れが発生した。本状況下で得られた成果を述べる。多糖選抜および新型MS調製の項目に関し、昨年度に行った研究計画上の工夫（調査が不可な期間は、日タイ共通植物種を対象に研究を先行。渡タイ可能時に現地で結果検証）により研究を進めた。共通植物種から取得したMSが示すBCS II化合物の可溶化能を測定し、優れた能力を有する新奇MSを見出した。さらに調査対象の植物種を増やし、多糖調製・MS分離・構造と機能の解析を継続している。なお、現地で実施が必要なMS高機能化および色素汚染解消に関する計画に大幅な遅延が生じているが、渡航開始後に打開を目指したい。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2017/04 -2021/03 
    Author : KIMURA Atsuo
     
    We succeeded in production of megalosaccharides and found their valuable function to solubilize water-insoluble compounds. However, the period of our research is very short, so that we have many problems that must be solved. This project challenges a resolution of three important problems, from which we will obtain the fundamental knowledge about megalosaccharides. Furthermore, it also contributes to the development of application research on megalosaccharides. The purposes of this program are 1) analysis of molecular mechanism of polysaccharide-forming enzyme to produce megalosaccharide, 2) synthesis of new megalosaccharide with high functionality, and 3) improvement of azo-dye pollution using megalosaccharide.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
    Date (from‐to) : 2018/06 -2020/03 
    Author : OKUYAMA MASAYUKI
     
    This study was conducted on the basis that most of the natural carbohydrates are composed of the D-series, but I believe that even if L-series monosaccharides and their oligosaccharides are naturally occuring, they are not theoretically strange. We discovered a new specificity for L-glycoside in an existing enzyme, and succeeded in obtaining an enzyme with a novel L-glycoside specificity from a sequence database. In addition, these L-glycosidases were used to synthesize oligosaccharides consisting of L-series carbohydrates and a mixture of D- and L-series oligosaccharides.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2014/04 -2017/03 
    Author : KIMURA, Atsuo
     
    This project aims at identification and application of structural elements that regulate and improve the carbohydrase-catalyzed transfer activities by elucidating four promising phenomena, which we newly found. Results obtained are as follows: 1) we identify the structural elements to control the α-1,3-transfer reaction displayed by α-1,3-glucoside-transfer enzyme; 2) we reveal the reaction mechanism of the catalytic residue-mutated glycosidase that shows the high transfer yield; 3) a pore canal at the active site of glycosidase plays a role that supplies the catalytic water by study at the molecular level. 4) we analyze the polysaccharide-producing enzyme and identify its structural elements to contribute to the synthesis of oligosaccharides.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014 -2016 
    Author : Okuyama Masayuki
     
    The purpose of this study is to make donor substrates for transglycosylation, which glycosidases catalyze. Using β-fructofranosidase and levansucrase, I succeeded in transferring a fructosyl group of sucrose to each saccharide, such as xylose, galactose, mannose, isomaltooligosaccharides, maltooligosaccharides, and glucuronic acid. The reaction product can be a donor substrate of the transglycosylation by glycosidases. Furthermore, I investigated the transglycosylation properties of several glycosidases and modified their specificity through molecular analyses. In addition, nucleotide sugar was successfully synthesized by the reaction of sucrose synthase with fructosyl saccharide as substrate.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))
    Date (from‐to) : 2010 -2011 
    Author : Masayuki OKUYAMA
     
    The purpose of this project was to clarify molecular evolution ofα-glucosidases distributed largely in organisms. An.-glucosidase, which hydrolyzesα-1, 3-glucosidic linkage in endoplasmic reticulum, also showed specificity forα-1, 4-glucosidic linkage. Structural studies of an.-glucosidase indicated that the loop structure, which covers the active pocket, was involved in substrate recognition. Trp residue in the active pocket of an yeast.-glucosidase had important role for substrate specificity. Novel.-glucosidase, which preferredα-1, 3-glucosidic linkage, was discovered from bacteria. It seems that an ancestor enzyme originally hadα-1, 3 specificity and its derivatives have acquired various specificity during the molecular evolution. Moreover, it appears that protein with a new function remained in the related family
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2008 -2010 
    Author : KIMURA Atsuo, OKUYAMA Masayuki
     
    Glycosylases are enzymes, which are currently utilized for the production of oligosaccharides. Interestingly, production-ability is dependent on the kinds of glycosylases, meaning that their structural elements contribute to the reaction specificity. Our research aims at elucidating structural elements of glycosylase-catalyzed transglycosylation. Our research achieved i) elucidation of structural elements contributing to the oligosaccharide production ; ii) establishment of the theoretical background of oligosaccharide production ; iii) finding the ways to regulate the oligosaccharide production.
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    Date (from‐to) : 2008 -2009 
    Author : 木村 淳夫, 森 春英, 奥山 正幸
     
    1残基のアミノ酸置換で「多糖の加水分解酵素(デキストラナーゼ)を合成酵素に転換できる現象」を見出した。この反応機構を分子解析することが、本申請の目的である。このような合成反応は例がなく、世界で初めての現象である。また、産業利用への発展にも期待したい。この残基は触媒アミノ酸と考えられる。本現象は試験管内の観察であるが、このような点突然変異した酵素が実際に生物で機能している可能性を得た。進化の過程においてアミノ酸置換は容易に生じ、1つのアミノ酸を変異させることで酵素分子を「加水分解→合成」にする戦略は、進化的に効率が良い。この戦略の検証も本申請の目的である。本年度は次の結果を得た。多糖合成の分子解析(デキストラナーゼ):1)酵素の結晶化と立体構造解析:昨年度に大量精製した親酵素とGly置換体を用いて結晶化条件を検討した。良好な結晶化条件を確定でき、X線構造解析を進行中である。2)他のアミノ酸による変異酵素:Asp→Gly置換体が合成反応を示したが、より効率の良いアミノ酸置換も想定されたため、他の残基への点変異を試みた。その結果、Gly置換体が最も高い反応効率を与えた。Glyは最もサイズの小さな残基であり、Asp→Gly置換で生じた大きな空間が重要と考えられた。すなわち、このサイズの大きい空間に陰イオンが侵入し合成反応が進行したと考えられた。3)陰イオンの解析:アザイドイオンが最も反応効率の良い陰イオンであった。従って本イオンのサイズ・強度が合成反応に最適であることが分かった。反応の至適pHを確定でき、pK_a値に大きな変化がないと予想できた。4)生成物の構造解析:生成多糖はデキストラン様の構造であった。触媒残基の変異酵素の解析(ウニ酵素):5)遺伝子の発現:酵素遺伝子の異種宿主発現を行った。酵素蛋白質は封入体を形成せず発現しているが、塩存在下であっても酵素活性が極めて低かった。
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))
    Date (from‐to) : 2008 -2009 
    Author : Masayuki OKUYAMA
     
    I discovered an unique glycoside hydrolases family which contains inverting- and retaining-catalytic enzymes. This divergence was due to the simple difference of the position of catalytic residues. We attempted to convert the catalytic mechanism of these enzyme each other. However, the attempt was failed. These results indicated that two similar enzymes diverged from a common ancestor in early stage of evolution and each enzyme evolved in different ways.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2005 -2007 
    Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki
     
    This project is about the enzymes having the structure similar to α-glucosidase; i.e. α-xylosidase, glucan lyase, cyclic-tetrasaccharide-forming enzyme, and α-glucosidase, each of which catalyzes the different reaction. Three-dimensional structure available recently allows us to analyze the molecular mechanism of reactions exhibited by four enzymes. The purposes of research are 1) to elucidate the relationship between substrate and amino acid residue(s) in the catalytic site; 2) to analyze the function of catalytic residues; 3) to elucidate the structural element(s) to display the above-described different reactions; 4) to synthesize the useful enzyme. Results are as follows. (1) We analyzed the amino acid residues in the catalytic site of α-xylosidase to recognize α-xyloside-structure, and succeeded in conversion of α-xylosidase into α-glucosidase by the mutagenesis of its structural elements. (2) The catalytic residues were identified and their functions were investigated. (3) Amino acid replacement of α-glucosidase (a hydrolyzing enzyme) lost its hydrolytic activity and enhanced the transglucosidation ability, meaning the conversion of hydrolyzing enzyme into transferring enzyme. (4) We have succeed in change of transferring products.
  • 文部科学省：科学研究費補助金(若手研究(B))
    Date (from‐to) : 2005 -2006 
    Author : 奥山 正幸
     
    構造プロテオミクス、プロテオーム解析など網羅的なタンパク質の機能解析の一方で、各論的な分子機能解析も今後の重要な課題となる。本研究の目的は分子機能解析の一環としての自殺基質を用いた糖質関連酵素の活性中心の一般的な探索法の開発である。自殺基質(EAG)は、酵素が基質を認識する機構を考慮した糖分子と反応基にはカルボキシ基と反応性の高いエポキシ環を用い、これら二つの分子群の距離をアルキル基鎖長を増減することで調節し、より特異性の高い自殺基質を設計した。酵素失活の分子機構解析に関しては失活が自殺基質的であることを反応動力学的に実証した。応用例も開発した。1.自殺基質の失活反応の解析;自殺基質の効果を解析するモデル酵素としてisomalto-dextranaseを用いた。アルキル基の長さ(炭素数3-6)が異なる自殺基質(E3G〜E6G)を混合し阻害効果を調べた。各自殺基質(EAG)濃度で失活の擬一次速度定数を求め、EAG濃度に対して擬一次速度定数をプロットすると両者の関係は直線ではなく飽和曲線となりMichaelis-Menten型の失活反応を示すことがわかった。すなわち失活はメカニカルベースであることがわかった。またE5Gの二次速度定数は他のEAGの6倍から10倍高くなっており、アルキル基の鎖長が自殺基質としての能力に重要なファクターであることがわかった。2.EAGを用いたα-amylase活性の特異的測定法;EAG利用の応用として生物試料破砕液からα-amylase活性を特異的に測定する方法の開発を試みた。β-amylase阻害剤E4Gとα-glucosidase阻害剤CBEをα-amylase:β-amylase:α-glucosidase混液と混合し後者二酵素を特異的に失活させ、α-amylaseのみの活性測定に成功した。このことは酵素を精製することなく粗酵素液中などで特定の酵素を失活させることができること示している。またこの結果は標識した自殺基質を用いて修飾後、ペプチドマスフィンガープリントなどにより、粗酵素液でも特定の酵素の活性中心のアミノ酸配列を知ることができるツールに成り得る可能性を示している。3.自殺基質との複合体立体構造の解析については期間内に解析を完了することはできなかった。現在も引き続き進行中である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2004 -2005 
    Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki
     
    We have analyzed three sucrose-degrading isozymes (α-glucosidase I,II, and III) in European honeybees, since these enzymes exhibited the interesting activity to sucrose of high concentration. α-Glucosidase I was a unique allosteric enzyme to display negative cooperativity in extremely high sucrose concentration. α-Glucosidase II also exhibited the allosteric property of positive cooperativity. α-Glucosidase III was a Michaelis-Menten-type enzyme to be secreted to nectar (bees gathered from flower) and to contribute to the formation of honey. Purpose of this project is i)existence of allosteric α-glucosidase in Asian honeybees, ii)investigation of honey-forming mechanism, and iii)regulation in expression of three α-glucosidase isozymes. Four kinds of honeybee species were investigated in this research. From typical Asian honeybees (living in Thailand, Korea and Japan), three isozymes were isolated by chromatographic method. One of them was an allosteric enzyme having similar properties to European honeybee α-glucosidase I, allowing to molecular analysis of negative cooperativity. The second enzyme was α-glucosidase II-type isozyme. Enzyme in honey was also investigated, and showed similar to one of enzymes (α-glucosidase III-type isozyme). cDNAs of these three enzyme were cloned. It was found that the expression of three isozymes was regulated. In small honeybees living in Thailand, three α-glucosidases were also found, in which two α-glucosidases were isolated. Their properties investigated were almost identical to those of α-glucosidases I and II. Enzyme in honey was compared with adult bee enzymes. All honeybee species studied in this project contained allosteric enzyme. Asian honeybees were divided into two types : i)having three α-glucosidase isozymes and ii)having two α-glucosidase isozymes. Recently, we found that bumblebees also had two α-glucosidase isozymes. Further research will elucidate the interesting molecular mechanism of sucrose-degrading enzymes in flower bees.
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    Date (from‐to) : 2004 -2005 
    Author : 木村 淳夫, 森 春英, 奥山 正幸
     
    糖質の加水分解酵素は、2つの酸性アミノ酸(AspやGlu)を触媒残基とし、それぞれが-COO^-と-COOHの荷電状態を形成し、協奏的に加水分解を触媒する。応用性の高い糖転移作用も示すが、分解と転移は2つの酸性アミノ酸でなされ分割できない。最近、α-グルコシダーゼにある-COO^-型の触媒基であるAspをCysに置換した。本酵素(Asp→Cys)には活性はないが、温和な酸化で活性を発揮した。Cysの-SHが酸化され-SOOHとなり、活性中心内で-SOO^-に解離し-COO^-の代わりを行うと考えている。この酵素は分解能を失い、糖転移能が上昇し95%の収率を与えた。本研究の目的は、-SOO^-酵素に見出された「非分解・高転移」の現象を解析することである。具体的には、1)酸化したCys残基の構造決定、2)糖転移反応の解析、3)他の酵素を合成酵素にする先駆けとして、触媒基を-SOO^-にした糖質酵素の構築と機帯解析、である。計画は順調に進行し、1)と2)が完了した。本年度は、この現象の応用を図るために3)の課題を中心に研究を進行させた。レバン合成酵素とキチン分解酵素を取り上げ、触媒残基をCysに置換し、酸化処理を行った。両酵素のCys変異体には活性がなかったが、穏やかな酸化により活性が回復した。導入したSH基が-SOOHに変化したことを確認した。Cys酸化酵素は、親酵素と異なる性質を示した(レバン合成酵素:至適pHや転移作用の変化、キチン分解酵素:至適pHや協同性の変化)。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2002 -2004 
    Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki
     
    Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.

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  • デキストラン生成酵素遺伝子、デキストラン生成酵素およびその製造方法、デキストランの製造方法
    特許公開２００７－１８１４５２

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    Date (from-to) : 2010-Today
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