大沼 貴之(オオヌマ タカユキ)

農学部 生物機能科学科教授

Last Updated :2024/06/18

■教員コメント

コメント

様々な生物に存在する酵素やタンパク質の構造や機能、その利用に関して。キチンやキトサンを中心とする糖質関連。

■研究者基本情報

学位

  • 博士(農学)(2002年03月 九州大学)

研究キーワード

  • キチン質分解酵素   植物ー微生物間相互作用   生体分子間相互作用   アレルゲン   

現在の研究分野(キーワード)

様々な生物に存在する酵素やタンパク質の構造や機能、その利用に関して。キチンやキトサンを中心とする糖質関連。

研究分野

  • ライフサイエンス / 応用生物化学 / 酵素学

■経歴

経歴

  • 2021年 - 現在  近畿大学農学部 生物機能科学科教授
  • 2015年 - 2021年  近畿大学農学部 バイオサイエンス学科准教授
  • 2012年 - 2015年  近畿大学農学部 バイオサイエンス学科講師
  • 2008年 - 2012年  近畿大学農学部 バイオサイエンス学科助教
  • 2006年 - 2008年  独立行政法人農業生物資源研究所植物・微生物間相互作用研究ユニット特別研究員
  • 2003年 - 2006年  カリフォルニア大学バークレー校/米国農務省Department of Plant and Microbial Biology/Plant Gene Expression Center博士研究員
  • 2002年 - 2003年  イリノイ大学アーバナ-シャンペーン校獣医学部博士研究員

学歴

  •         - 2002年03月   九州大学   大学院 生物資源環境科学研究科
  •         - 1999年03月   名城大学   大学院 農学研究科
  •         - 1997年03月   名城大学   農学部   農芸化学科

委員歴

  • 2021年09月 - 現在   日本応用糖質科学会   「応用糖質科学」副編集委員長
  • 2017年 - 現在   日本応用糖質科学会   評議員
  • 2014年 - 現在   日本キチン・キトサン学会   編集委員
  • 2013年 - 現在   日本応用糖質科学会   和文誌編集員
  • 2014年 - 2016年   日本キチン・キトサン学会   理事

■研究活動情報

受賞

  • 2019年09月 日本応用糖質科学会 技術開発賞
     放線菌糖質関連酵素の食品分野への展開
  • 2017年08月 日本キチン・キトサン学会 奨励賞
     キチン分解酵素の構造と機能および糖鎖合成への応用 
    受賞者: 大沼貴之
  • 2015年09月 日本応用糖質科学会 奨励賞
     キチン質分解酵素の構造と機能および利用に関する研究 
    受賞者: 大沼貴之

論文

  • Md Azadur RAHMAN; Shuji TAKAHASHI; Norihiko SASAKI; Toshiyuki ITOH; Takayuki OHNUMA; Toshiki NOKAMI
    Electrochemistry 91 11 112013 - 112013 2023年11月
  • Takayuki Ohnuma; Jun Tsujii; Chikara Kataoka; Teruki Yoshimoto; Daijiro Takeshita; Outi Lampela; André H Juffer; Wipa Suginta; Tamo Fukamizo
    Scientific reports 13 1 20558 - 20558 2023年11月 
    Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc)n translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc)n from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)3-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)3-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.
  • Tomonari Tanaka; Yoshiaki Habuchi; Rika Okuno; Shota Nishimura; Sotaro Tsuji; Yuji Aso; Takayuki Ohnuma
    Carbohydrate research 523 108740 - 108740 2023年01月 
    O-Glycosylated N-acetyl-β-d-glucosamine-selective N-acetyl-β-d-glucosaminidase (O-GlcNAcase), belonging to glycoside hydrolase family 84 (GH84), is known as a retaining glycosidase with the possibility of enzymatic transglycosylation. However, no enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase has been reported. Here, enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase was first reported. The enzymatic transglycosylation catalyzed by the GH84 O-GlcNAcase from Bacteroides thetaiotaomicron (BtGH84 O-GlcNAcase) was attained using 1,2-oxazoline derivative of N-acetyl-d-glucosamine (GlcNAc oxazoline) as a glycosyl donor substrate. The β-linked N-acetyl-d-glucosamine (GlcNAc) derivative was enzymatically synthesized using N-(2-hydroxyethyl)acrylamide as an acceptor substrate. Interestingly, the β1,6-linked disaccharide derivative of GlcNAc was also obtained in the case of using the GlcNAc derivative with a triazole-linked acrylamide group as an acceptor substrate. Additionally, a one-pot chemo-enzymatic transglycosylation starting from unprotected GlcNAc through GlcNAc oxazoline successfully showed through the combination with the direct synthesis of GlcNAc oxazoline in water and the enzymatic transglycosylation.
  • Jun Tanaka; Tomoya Takashima; Naojiro Abe; Tamo Fukamizo; Tomoyuki Numata; Takayuki Ohnuma
    Plant science : an international journal of experimental plant biology 111524 - 111524 2022年10月 
    Two rice GH18 chitinases, Oschib1 and Oschib2, belonging to family 8 of plant pathogenesis-related proteins (PR proteins) were expressed, purified, and characterized. These enzymes, which have the structural features of class IIIb chitinases, preferentially cleaved the second glycosidic linkage from the non-reducing end of substrate chitin oligosaccharides as opposed to rice class IIIa enzymes, OsChib3a and OsChib3b, which mainly cleaved the fourth linkage from the non-reducing end of chitin hexasaccharide [ (GlcNAc)6]. Oschib1 and Oschiab2 inhibited the growth of Fusarium solani, but showed only a weak or no antifungal activity against Aspergillus niger and Trichoderma viride on the agar plates. Structural analysis of Oschib1 and Oschib2 revealed that these enzymes have two large loops extruded from the (β/α)8 TIM-barrel fold, which are absent in the structures of class IIIa chitinases. The differences in the cleavage site preferences toward chitin oligosaccharides between plant class IIIa and IIIb chitinases are likely attributed to the additional loop structures found in the IIIb enzymes. The class IIIb chitinases, Oschib1 and Oschib2, seem to play important roles for the effective hydrolysis of chitin oligosaccharides released from the cell wall of the pathogenic fungi by the cooperative actions with the extracellular chitinases in rice.
  • Naoyuki Umemoto; Natsuki Saito; Masato Noguchi; Shin-Ichiro Shoda; Takayuki Ohnuma; Takeshi Watanabe; Shohei Sakuda; Tamo Fukamizo
    Journal of agricultural and food chemistry 70 40 12897 - 12906 2022年10月 
    Sugar oxazolines, (GlcNAc)n-oxa (n = 2, 3, 4, and 5), were synthesized from a mixture of chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, and 5), and utilized for synthesis of (GlcNAc)7 with higher elicitor activity using plant chitinase mutants as the catalysts. From isothermal titration calorimetry, the binding affinity of (GlcNAc)2-oxa toward an inactive mutant obtained from Arabidopsis thaliana GH18 chitinase was found to be higher than those of the other (GlcNAc)n-oxa (n = 3, 4, and 5). To synthesize (GlcNAc)7, the donor/acceptor substrates with different size combinations, (GlcNAc)2-oxa/(GlcNAc)5 (1), (GlcNAc)3-oxa/(GlcNAc)4 (2), (GlcNAc)4-oxa/(GlcNAc)3 (3), and (GlcNAc)5-oxa/(GlcNAc)2 (4), were incubated with hypertransglycosylating mutants of GH18 chitinases from A. thaliana and Cycas revoluta. The synthetic activities of these plant chitinase mutants were lower than that of a mutant of Bacillus circulans chitinase A1. Nevertheless, in the plant chitinase mutants, the synthetic efficiency of combination (1) was higher than those of the other combinations (2), (3), and (4), suggesting that the synthetic reaction is mostly dominated by the binding affinities of (GlcNAc)n-oxa. In contrast, the Bacillus enzyme mutant with a different subsite arrangement synthesized (GlcNAc)7 from combination (1) in the lowest efficiency. Donor/acceptor-size dependency of the enzymatic synthesis appeared to be strongly related to the subsite arrangement of the enzyme used as the catalyst. The A. thaliana chitinase mutant was found to be useful when combination (1) is employed for the substrates.
  • Yoshihito Kitaoku; Toki Taira; Tomoyuki Numata; Takayuki Ohnuma; Tamo Fukamizo
    Plant science : an international journal of experimental plant biology 321 111310 - 111310 2022年08月 
    A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.
  • Daiki Kawamoto; Tomoya Takashima; Tamo Fukamizo; Tomoyuki Numata; Takayuki Ohnuma
    Glycobiology 32 4 356 - 364 2021年11月 
    Plant GH19 chitinases have several loop structures, which may define their enzymatic properties. Among these loops, the longest loop, Loop-III, is most frequently conserved in GH19 enzymes. A GH19 chitinase from the moss Bryum coronatum (BcChi-A) has only one loop structure, Loop-III, which is connected to the catalytically important β-sheet region. Here, we produced and characterized a Loop-III-deleted mutant of BcChi-A (BcChi-A-ΔIII) and found that its stability and chitinase activity were strongly reduced. The deletion of Loop-III also moderately affected the chitooligosaccharide binding ability as well as the binding mode to the substrate-binding groove. The crystal structure of an inactive mutant of BcChi-A-ΔIII was successfully solved, revealing that the remaining polypeptide chain has an almost identical fold to that of the original protein. Loop-III is not necessarily essential for the folding of the enzyme protein. However, closer examination of the crystal structure revealed that the deletion of Loop-III altered the arrangement of the catalytic triad, Glu61, Glu70 and Ser102, and the orientation of the Trp103 side chain, which is important for sugar residue binding. We concluded that Loop-III is not directly involved in the enzymatic activity but assists the enzyme function by stabilizing the conformation of the β-sheet region and the adjacent substrate-binding platform from behind the core-functional regions.
  • Makoto Ogata; Tamo Fukamizo; Takayuki Ohnuma
    Frontiers in Molecular Biosciences 8 654706 - 654706 2021年06月 
    4-O-β-tri-N-acetylchitotriosyl moranoline (GN3M) is a transition-state analogue for hen egg white lysozyme (HEWL) and identified as the most potent inhibitor till date. Isothermal titration calorimetry experiments provided the thermodynamic parameters for binding of GN3M to HEWL and revealed that the binding is driven by a favorable enthalpy change (ΔH° = −11.0 kcal/mol) with an entropic penalty (−TΔS° = 2.6 kcal/mol), resulting in a free energy change (ΔG°) of −8.4 kcal/mol [Ogata et al. (2013) 288, 6,072–6,082]. Dissection of the entropic term showed that a favorable solvation entropy change (−TΔSsolv° = −9.2 kcal/mol) is its sole contributor. The change in heat capacity (ΔCp°) for the binding of GN3M was determined to be −120.2 cal/K·mol. These results indicate that the bound water molecules play a crucial role in the tight interaction between GN3M and HEWL.
  • Takayuki Ohnuma; Tomoki Taku; Takeshi Nagatani; Atsushi Horii; Shun Imaoka; Tomonari Tanaka
    Bioscience, biotechnology, and biochemistry 85 7 1716 - 1719 2021年04月 
    Chemo-enzymatic synthesis of lacto-N-biose I (LNB) catalyzed by β-1,3 galactosidase from Bacillus circulans (BgaC) has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl β-galactopyranoside [DMT-β-Gal] and GlcNAc as the donor and acceptor substrates, respectively. BgaC transferred the Gal moiety to the acceptor, giving rise to LNB. The maximum yield of LNB was obtained at the acceptor: donor substrate ratio of 1:30.
  • Takuya Nagata; Shoko Shinya; Takayuki Ohnuma; Tamo Fukamizo
    Scientific reports 11 1 2494 - 2494 2021年01月 [査読有り]
     
    GH19 and GH22 glycoside hydrolases belonging to the lysozyme superfamily have a related structure/function. A highly conserved tryptophan residue, Trp103, located in the binding groove of a GH19 chitinase from moss Bryum coronatum (BcChi-A) appears to have a function similar to that of well-known Trp62 in GH22 lysozymes. Here, we found that mutation of Trp103 to phenylalanine (W103F) or alanine (W103A) strongly reduced the enzymatic activity of BcChi-A. NMR experiments and the X-ray crystal structure suggested a hydrogen bond between the Trp103 side chain and the -2 sugar. Chitooligosaccharide binding experiments using NMR indicated that the W103F mutation reduced the sugar-binding abilities of nearby amino acid residues (Tyr105/Asn106) in addition to Trp103. This appeared to be derived from enhanced aromatic stacking of Phe103 with Tyr105 induced by disruption of the Trp103 hydrogen bond with the -2 sugar. Since the stacking with Tyr105 was unlikely in W103A, Tyr105/Asn106 of W103A was not so affected as in W103F. However, the W103A mutation appeared to reduce the catalytic potency, resulting in the lowest enzymatic activity in W103A. We concluded that Trp103 does not only interact with the sugar, but also controls other amino acids responsible for substrate binding and catalysis. Trp103 (GH19) and Trp62 (GH22) with such a multi-functionality may be advantageous for enzyme action and conserved in the divergent evolution in the lysozyme superfamily.
  • Yusuke Morimoto; Shuji Takahashi; Yuta Isoda; Toshiki Nokami; Tamo Fukamizo; Wipa Suginta; Takayuki Ohnuma
    Carbohydrate research 499 108201 - 108201 2020年11月 [査読有り]
     
    We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-β-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-β-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 μM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 μM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = -2.5 ± 0.1 kcal/mol and -TΔS° = -5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of -8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔSsolv° = -16 ± 2 kcal/mol) is the main contributor to the entropic term.
  • Takashima T; Taku T; Yamanaka T; Fukamizo T; Numata T; Ohnuma T
    Molecular immunology 116 199 - 207 2019年11月 [査読有り]
     
    A 38 kDa β-1,3-glucanase allergen from Cryptomeria japonica pollen (CJP38) was recombinantly produced in E. coli and purified to homogeneity with the use of Ni-affinity resin. CJP38 hydrolyzed β-1,3-glucans such as CM-curdlan and laminarioligosaccharides in an endo-splitting manner. The optimum pH and temperature for β-1,3-glucanase activity were approximately 4.5 and 50 °C, respectively. The enzyme was stable at 30-60 °C and pH 4.0-10.5. Furthermore, CJP38 catalyzed a transglycosylation reaction to yield reaction products with a molecular weight higher than those of the starting laminarioligosaccharide substrates. The three-dimensional structure of CJP38 was determined using X-ray crystallography at 1.5 Å resolution. CJP38 exhibited the typical (β/α)8 TIM-barrel motif, similar to allergenic β-1,3-glucanases from banana (Mus a 5) and rubber tree latex (Hev b 2). Amino acid sequence alignment of these proteins indicated that the two-consensus IgE epitopes identified on the molecular surfaces of Mus a 5 and Hev b 2 were highly conserved in CJP38. Their conformations and surface locations were quite similar for these proteins. Sequence and structural conservation of these regions suggest that CJP38 is a candidate allergen responsible for the pollen-latex-fruit syndrome relating to Japanese cedar pollinosis.
  • Kitaoku Y; Nishimura S; Hirono T; Suginta W; Ohnuma T; Fukamizo T
    Glycobiology 29 7 565 - 575 2019年07月 [査読有り]
     
    Two N-terminal lysin motifs (LysMs) found in a chitinase from the green alga Volvox carteri (VcLysM1 and VcLysM2) were produced, and their structures and chitin-binding properties were characterized. The binding affinities of VcLysM1 toward chitin oligomers determined by isothermal titration calorimetry (ITC) were higher than those of VcLysM2 by 0.8-1.1 kcal/mol of ΔG°. Based on the NMR solution structures of the two LysMs, the differences in binding affinities were found to result from amino acid substitutions at the binding site. The NMR spectrum of a two-domain protein (VcLysM1+2), in which VcLysM1 and VcLysM2 are linked in tandem through a flexible linker, suggested that the individual domains of VcLysM1+2 independently fold and do not interact with each other. ITC analysis of chitin-oligomer binding revealed two different binding sites in VcLysM1+2, showing no cooperativity. The binding affinities of the VcLysM1 domain in VcLysM1+2 were lower than those of VcLysM1 alone, probably due to the flexible linker destabilizing the interaction between the chito-oligosaccahrides and VcLysM1 domain. Overall, two LysMs attached to the chitinase from the primitive plant species, V. carteri, were found to resemble bacterial LysMs reported thus far.
  • Shirasaka N; Harazono K; Nakahigashi R; Mitsui K; Tanaka J; Tanazawa S; Mitsutomi M; Ohnuma T
    Journal of Applied Glycoscience 66 3 83 - 88 2019年 [査読有り]
     
    We characterized SaHEX, which is a glycoside hydrolase (GH) family 20 exo-β-N-acetylhexosaminidase found in Streptomyces avermitilis. SaHEX exolytically hydrolyzed chitin oligosaccharides from their non-reducing ends, and yielded N-acetylglucosamine (GlcNAc) as the end product. According to the initial rate of substrate hydrolysis, the rates of (GlcNAc)3 and (GlcNAc)5 hydrolysis were greater than the rates for the other oligosaccharides. The enzyme exhibited antifungal activity against Aspergillus niger, which was probably due to hydrolytic activity with regard to chitin in the hyphal tips. Therefore, SaHEX has potential for use in GlcNAc production and food preservation.
  • Ohnuma T; Tanaka T; Urasaki A; Dozen S; Fukamizo T
    Journal of biochemistry 165 6 497 - 503 2018年12月 [査読有り]
     
    A novel method for the chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by mutants of BcChi-A, an inverting family GH19 chitinase from Bryum coronatum, has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside [DMT-α-(GlcNAc)2)] as a donor substrate. Based on the glycosynthase derived from BcChi-A, Glu70, which acts as a catalytic base, and Ser102, which fixes a nucleophilic water molecule, were changed to generate several single and double mutants of BcChi-A, which were employed in synthetic reactions. Among the double mutants tested, E70G/S102G, E70G/S102C and E70G/S102A were found to successfully synthesize chitotetraose [ (GlcNAc)4] from DMT-α-(GlcNAc)2 and (GlcNAc)2; however, the single mutants, E70G, S102G, S102C and S102A, did not. Among the mutants, E70G/S102A showed the highest synthetic activity. This is the first report of a glycosynthase that employs a dimethoxytriazine-type glycoside as a donor substrate.
  • Feng Y; Kitaoku Y; Tanaka J; Taira T; Ohnuma T; Aachmann FL; Fukamizo T
    Plant molecular biology 97 6 553 - 564 2018年08月 [査読有り]
     
    KEY MESSAGE: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.
  • Iinuma C; Saito A; Ohnuma T; Tenconi E; Rosu A; Colson S; Mizutani Y; Liu F; Świątek-Połatyńska M; Wezel GV; Rigali S; Fujii T; Miyashita K
    Microbes and environments 33 3 272 - 281 2018年08月 [査読有り]
     
    In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N'- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005-7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcESco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 μM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcESco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcESco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcESco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFGSco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcESco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.
  • Tomoya Takashima; Tomoyuki Numata; Toki Taira; Tamo Fukamizo; Takayuki Ohnuma
    Journal of Agricultural and Food Chemistry 66 22 5699 - 5706 2018年06月 [査読有り]
     
    CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc)n, polysaccharide substrates, glycol chitin, and β-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc)n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward β-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.
  • Taira T; Gushiken C; Sugata K; Ohnuma T; Fukamizo T
    Bioscience, biotechnology, and biochemistry 82 7 1 - 11 2018年04月 [査読有り]
     
    A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [ (GlcNAc)4-6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)2 and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)2 or GlcNAc, which are easily taken up by the cells.
  • Tomoya Takashima; Takayuki Ohnuma; Tamo Fukamizo
    Carbohydrate Research 458-459 52 - 59 2018年03月 [査読有り]
     
    CJP-4 is a two-domain chitinase from Japanese cedar (Cryptomeria japonica) pollen, consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. The substrate binding to an inactive mutant protein of full-length CJP-4, in which the catalytic acid Glu108 was mutated to glutamine, CJP-4(E108Q), was analyzed by NMR spectroscopy. Based on the chemical shift perturbations of 1H-15N HSQC signals of Gly26 (CBM18 domain) and Trp185 (GH19 domain), the association constants for individual domains of CJP-4(E108Q) toward soluble chitin hexamer (GlcNAc)6 were determined to be 2300 and 3500 M−1, respectively. Isothermal titration calorimetry provided a similar association constant for (GlcNAc)6 (1980 M−1) with the one-site binding model. One (GlcNAc)6 molecule appeared to bind to a single binding site of CJP-4(E108Q), spanning from CBM18 to GH19 domains. When chitin nanofibers, insoluble chitinase substrate, were added to the CJP-4(E108Q) solution, strong line-broadening was observed for the majority of the backbone resonances in CBM18 domain but not in GH19 domain, indicating a binding preference of CBM18 domain to the insoluble chitin. We here demonstrated importance of CBM18 domain in insoluble chitin recognition based on the NMR binding data obtained for full-length CJP-4. Chitin nanofibers were found to be useful for spectroscopic observation of insoluble chitin binding to proteins.
  • Takayuki Ohnuma; Toki Taira; Naoyuki Umemoto; Yoshihito Kitaoku; Morten Sorlie; Tomoyuki Numata; Tamo Fukamizo
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 494 3-4 736 - 741 2017年12月 [査読有り]
     
    We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 angstrom. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (Delta G(r)degrees) for binding of (GIcNAc)(6), (G1cNAc)(5), and (GIcNAc)(4) to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol(-1), respectively, by ITC. Thermodynamic dissection of the binding energetics of (GIcNAc)(6) revealed that the driving force is the enthalpy change (Delta H-r degrees = -11.7 +/- 0.2 kcal/mol) and the solvation entropy change (-T Delta S-solv degrees = -5.9 +/- 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module. (C) 2017 Elsevier Inc. All rights reserved.
  • Jun Tanaka; Tamo Fukamizo; Takayuki Ohnuma
    GLYCOBIOLOGY 27 5 477 - 485 2017年05月 [査読有り]
     
    The catalytic domains of family GH19 chitinases have been found to consist of a conserved, alpha-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important beta-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)(n) (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)(n) (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)(n), but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sorlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the beta-stranded region is important for (GlcNAc)(n) binding, but is not essential for antifungal activity.
  • Tomoya Takashima; Takayuki Ohnuma; Tamo Fukamizo
    BIOMOLECULAR NMR ASSIGNMENTS 11 1 85 - 90 2017年04月 [査読有り]
     
    A two-domain family GH19 chitinase from Japanese cedar (Cryptomeria japonica) pollen, CJP-4, which consists of an N-terminal CBM18 domain and a GH19 catalytic domain, is known to be an important allergen, that causes pollinosis. We report here the resonance assignments of the NMR spectrum of CJP-4. The backbone resonances were almost completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of a chitin dimer to the enzyme solution perturbed the chemical shifts of the resonances of amino acid residues forming a long extended binding site spanning from the CBM18 domain to the GH19 catalytic domain.
  • Shoko Takenaka; Takayuki Ohnuma; Tamo Fukamizo
    Journal of Applied Glycoscience 64 2 39 - 42 2017年01月 [査読有り]
     
    Chitinases belonging to the GH19 family have diverse loop structure arrangements. A GH19 chitinase from rye seeds (RSC-c) has a full set of (six) loop structures that form an extended binding cleft from -4 to +4 ("loopful"), while that from moss (BcChi-A) lacks several loops and forms a shortened binding cleft from -2 to +2 ("loopless"). We herein inserted a loop involved in sugar residue binding at subsites +3 and +4 of RSC-c (Loop-II) into BcChi-A (BcChi-A+L-II), and the thermal stability and enzymatic activity of BcChi-A+L-II were then characterized and compared with those of BcChi-A. The transition temperature of thermal unfolding decreased from 77.2 ˚C (BcChi-A) to 63.3 ˚C (BcChi-A+L-II) by insertion of Loop-II. Enzymatic activities toward the chitin tetramer (GlcNAc)4 and the polymeric substrate glycol chitin were also suppressed by the Loop-II insertion to 12 and 9 %, respectively. The Loop-II inserted into BcChi-A was found to be markedly flexible and disadvantageous for protein stability and enzymatic activity.
  • Yoshihito Kitaoku; Tamo Fukamizo; Tomoyuki Numata; Takayuki Ohnuma
    PLANT MOLECULAR BIOLOGY 93 1-2 97 - 108 2017年01月 [査読有り]
     
    The chitinase-mediated defense system in higher plants has been intensively studied from physiological and structural viewpoints. However, the defense system in the most primitive plant species, such as green algae, has not yet been elucidated in details. In this study, we solved the crystal structure of a family CBM-50 LysM module attached to the N-terminus of chitinase from Volvox carteri, and successfully analyzed its chitin-binding ability by NMR spectroscopy and isothermal titration calorimetry. Trp96 of the LysM module appeared to make a CH-pi stacking interaction with the reducing end sugar residue of the ligand. We believe the data included in this manuscript provide novel insights into the molecular basis of chitinase-mediated defense system in green algae. A chitinase from the multicellular green alga, Volvox carteri, contains two N-terminal lysin motifs (VcLysM1 and VcLysM2), that belong to the CBM-50 family, in addition to a catalytic domain. We produced a recombinant protein of VcLysM2 in order to examine its structure and function. The X-ray crystal structure of VcLysM2 was successfully solved at a resolution of 1.2 , and revealed that the protein adopts the beta alpha alpha beta fold typical of members belonging to the CBM-50 family. NMR spectra of C-13- and N-15-labeled proteins were analyzed in order to completely assign the main chain resonances of the H-1,N-15-HSQC spectrum in a sequential manner. NMR-based titration experiments of chitin oligosaccharides, (GlcNAc)(n) (n = 3-6), revealed the ligand-binding site of VcLysM2, in which the Trp96 side chain appeared to interact with the terminal GlcNAc residue of the ligand. We then mutated Trp96 to alanine (VcLysM2-W96A), and the mutant protein was characterized. Based on isothermal titration calorimetry, the affinity of (GlcNAc)(6) toward VcLysM2 (-6.9 kcal/mol) was found to be markedly higher than that of (GlcNAc)(3) (-4.1 kcal/mol), whereas the difference in affinities between (GlcNAc)(6) and (GlcNAc)(3) in VcLysM2-W96A (-5.1 and -4.0 kcal/mol, respectively) was only moderate. This suggests that the Trp96 side chain of VcLysM2 interacts with the sugar residue of (GlcNAc)(6) not with (GlcNAc)(3). VcLysM2 appears to preferentially bind (GlcNAc)(n) with longer chains and plays a major role in the degradation of the chitinous components of enzyme targets.
  • Wipa Suginta; Paknisa Sirimontree; Natchanok Sritho; Takayuki Ohnuma; Tamo Fukamizo
    INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 93 Pt A 1111 - 1117 2016年12月 [査読有り]
     
    Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (alpha/beta)(8) TIM barrel catalytic domain; and iii) the alpha + beta insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD(VhChiA). The recombinant ChBD(VhChiA) was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD(VhChiA) contained beta-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD(VhChiA) bound to both alpha- and beta-chitins, with the greatest affinity for beta-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions. (C) 2016 Elsevier B.V. All rights reserved.
  • Takayuki Ohnuma; Satoshi Dozen; Yuji Honda; Motomitsu Kitaoka; Tamo Fukamizo
    JOURNAL OF BIOCHEMISTRY 160 2 93 - 100 2016年08月 [査読有り]
     
    We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, + 1, + 2, + 3 and + 4. When wild-type RSC-c was incubated with alpha-(G1cNAc)(3)-F [alpha-(GleNAc)(3) fluoride], (G1cNAc)(3) and hydrogen fluoride were produced through the Hehre resynthesis-hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (G1cNAc)(7) from alpha-(GlcNAc)(3)-F in the presence of (G1cNAc)(4). S120A, with the highest F- -releasing activity, produced a larger amount of (G1cNAc)(7), a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F--releasing activity was only moderately affected, produced the largest amount of (G1eNAc)(7) from alpha-(GleNAc)(3)-F and (G1cNAc)(4) without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.
  • Shoko Shinya; Shigenori Nishimura; Yoshihito Kitaoku; Tomoyuki Numata; Hisashi Kimoto; Hideo Kusaoke; Takayuki Ohnuma; Tamo Fukamizo
    BIOCHEMICAL JOURNAL 473 8 1085 - 1095 2016年04月 [査読有り]
     
    An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a beta-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in H-1-N-15-HSQC resonances, the chitosan tetramer (GlcN)(4) was found to bind to the loop region extruded from the core beta-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)(3), in which the bound (GlcN)(3) stood upright on its non-reducing end at the binding site. Glu(14) of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36) and Glu(61) formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36) of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36) to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.
  • Yoshihito Kitaoku; Naoyuki Umemoto; Takayuki Ohnuma; Tomoyuki Numata; Toki Taira; Shohei Sakuda; Tamo Fukamizo
    PLANTA 242 4 895 - 907 2015年10月 [査読有り]
     
    We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (alpha/beta)(8) fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) (n) (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)(3) and (GlcNAc)(4) toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.
  • Saki Inamine; Shoko Onaga; Takayuki Ohnuma; Tamo Fukamizo; Toki Taira
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 8 1296 - 1304 2015年08月 [査読有り]
     
    Chitinase-A (EaChiA), molecular mass 36kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.
  • Naoyuki Umemoto; Takayuki Ohnuma; Takuo Osawa; Tomoyuki Numata; Tamo Fukamizo
    FEBS LETTERS 589 18 2327 - 2333 2015年08月 [査読有り]
     
    Transglycosylation (TG) activity of a family GH18 chitinase from the cycad, Cycas revoluta, (CrChiA) was modulated by removing or introducing a tryptophan side chain. The removal from subsite +3 through mutation of Trp168 to alanine suppressed TG activity, while introduction into subsite +1 through mutation of Gly77 to tryptophan (CrChiA-G77W) enhanced TG activity. The crystal structures of an inactive double mutant of CrChiA (CrChiA-G77W/E119Q) with one or two N-acetylglucosamine residues occupying subsites +1 or +1/+2, respectively, revealed that the Trp77 side chain was oriented toward +1 GlcNAc to be stacked with it face-to-face, but rotated away from subsite +1 in the absence of GlcNAc at the subsite. Aromatic residues in the aglycon-binding site are key determinants of TG activity of GH18 chitinases. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Naoyuki Umemoto; Yuka Kanda; Takayuki Ohnuma; Takuo Osawa; Tomoyuki Numata; Shohei Sakuda; Toki Taira; Tamo Fukamizo
    PLANT JOURNAL 82 1 54 - 66 2015年04月 [査読有り]
     
    A classV (glycoside hydrolase family18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of classV chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (/)(8) barrel fold with an (+) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8kcal mol(-1). However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinaseA from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant classV chitinases.
  • Paknisa Sirimontree; Wipa Suginta; Natchanok Sritho; Yuka Kanda; Shoko Shinya; Takayuki Ohnuma; Tamo Fukamizo
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 12 2014 - 2021 2014年12月 [査読有り]
     
    Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc(n), with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc(n). On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.
  • Tomoyo Nishihira; Asami Miyano; Takayuki Ohnuma; Takeshi Gotoh; Saori Takahashi; Kazue Narihiro; Kazuhiko Yamashita; Tamo Fukamizo
    Journal of Applied Glycoscience 61 4 113 - 116 2014年11月 [査読有り]
  • Shoko Shinya; Atsushi Urasaki; Takayuki Ohnuma; Toki Taira; Akari Suzuki; Makoto Ogata; Taichi Usui; Outi Lampela; Andre H. Juffer; Tamo Fukamizo
    GLYCOBIOLOGY 24 10 945 - 955 2014年10月 [査読有り]
     
    Tri-N-acetylchitotriosyl moranoline, (GlcNAc)(3)-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-beta-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288: 6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)(2)-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (T-m) was elevated by 4.3 and 5.8 degrees C, respectively, upon the addition of (GlcNAc)(2)-M, while the chitin dimer, (GlcNAc)(2), elevated T-m only by 1.0 and 1.4 degrees C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc) 3 and (GlcNAc)(2)-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)(2) was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using 15N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)(2) and (GlcNAc)(2)-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)(2)-M is markedly stronger than that of (GlcNAc)(2) for both enzymes. However, (GlcNAc)(2)-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 mu M). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)(2)-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)(2)-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite + 1. This situation may result in the moderate inhibition toward the BcChi19Acatalyzed hydrolysis.
  • Ohnuma T; Umemoto N; Nagata T; Shinya S; Numata T; Taira T; Fukamizo T
    Biochimica et biophysica acta 1844 4 793 - 802 4 2014年04月 [査読有り]
     
    DESCRIPTIONS: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)4 revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the Oε1 atom of the catalytic base (Glu70), and the Oγ atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis.
  • Maria Mahata; Shoko Shinya; Eiko Masaki; Takashi Yamamoto; Takayuki Ohnuma; Ryszard Brzezinski; Tapan K. Mazumder; Kazuhiko Yamashita; Kazue Narihiro; Tamo Fukamizo
    CARBOHYDRATE RESEARCH 383 27 - 33 2014年01月 [査読有り]
     
    The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS C-13 NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25 m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)(2)-GlcNAc, and (GlcN)(2) were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact. (C) 2013 Elsevier Ltd. All rights reserved.
  • Takayuki Ohnuma; Naoyuki Umemoto; Toki Taira; Tamo Fukamizo; Tomoyuki Numata
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 69 Pt 12 1360 - 1362 2013年12月 [査読有り]
     
    The catalytic mechanism of family GH19 chitinases is not well understood owing to insufficient information regarding the three-dimensional structures of enzyme-substrate complexes. Here, the crystallization and preliminary X-ray diffraction analysis of a selenomethionine-labelled active-site mutant of 'loopless' family GH19 chitinase from the moss Bryum coronatum in complex with chitotetraose, (GlcNAc) 4, are reported. The crystals were grown using the vapour-diffusion method. They diffracted to 1.58 angstrom resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 74.5, b = 58.4, c = 48.1 angstrom, beta = 115.6 degrees. The asymmetric unit of the crystals is expected to contain one protein molecule, with a Matthews coefficient of 2.08 angstrom(3) Da(-1) and a solvent content of 41%.
  • Shoko Shinya; Takayuki Ohnuma; Reina Yamashiro; Hisashi Kimoto; Hideo Kusaoke; Padmanabhan Anbazhagan; Andre H. Juffer; Tamo Fukamizo
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 42 30042 - 30053 2013年10月 [査読有り]
     
    Background: Carbohydrate binding modules (CBMs) specific to chitosan have yet to be identified. Results: Two CBMs located at the C terminus of a chitosanase from Paenibacillus sp. IK-5 specifically bound chitosan oligosaccharides. Conclusion: Individual CBMs can accommodate at least two glucosamine units at loops extruded from the core -sandwich. Significance: The synergistic action of the two CBMs appears to facilitate chitosan hydrolysis. Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T-m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)(6). The T-m elevation (T-m) in DD1 was the highest (10.3 degrees C) upon the addition of (GlcN)(6) and was markedly higher than that in DD2 (1.0 degrees C). A synergistic effect was observed (T-m = 13.6 degrees C), when (GlcN)(6) was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)(n); G(r)degrees values were -7.8 (n = 6), -7.6 (n = 5), -7.6 (n = 4), -7.6 (n = 3), and -7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)(n) with the lower affinities (G(r)degrees = -5.0 (n = 3) approximate to -5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)(6) for individual DD1 and DD2 sites (G(r)degrees = -8.6 and -6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)(n) (n = 2 approximate to 6) were found to bind to loops extruded from the core -sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.
  • Takayuki Ohnuma; Naoyuki Umemoto; Kaori Kondo; Tomoyuki Numata; Tamo Fukamizo
    FEBS LETTERS 587 16 2691 - 2697 2013年08月 [査読有り]
     
    Crystallographic analysis of a mutated form of "loopful" GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)(4) revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)(4) molecules. One (GlcNAc)(4) molecule bound to subsites -4 to -1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Ogata M; Umemoto N; Ohnuma T; Numata T; Suzuki A; Usui T; Fukamizo T
    The Journal of biological chemistry 288 9 6072 - 6082 9 2013年03月 [査読有り]
     
    4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri- Nacetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)4, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with Ki values of 2.01 × 10-5 and 1.84 × 10-6 M, respectively. From IT Canalysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔHr°), which is similar to those obtained for 2 and (GlcNAc)4. However, the entropy loss (-TΔSr°) for the binding of 3 was smaller than those of 2 and (GlcNAc)4. Thusthe binding of 3 was found to bemorefavorable than those of the others. Judging from the Kd value of 3 (760 nM), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWLin a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted 4C1 chair conformation almost overlapping with the -1 sugar covalentlyboundtoAsp-52ofHEWL(Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835-838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Wakinaka T; Kiyohara M; Kurihara S; Hirata A; Chaiwangsri T; Ohnuma T; Fukamizo T; Katayama T; Ashida H; Yamamoto K
    Glycobiology 23 2 232 - 240 2 2013年02月 [査読有り]
     
    Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22: 361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 alpha-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed alpha 1,3-linked Gal in branched blood group B antigen [Gal alpha 1-3(Fuca1-2)Gal beta 1-R], but not in a linear xenotransplantation antigen (Gal alpha 1-3Gal beta 1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.
  • Naoyuki Umemoto; Takayuki Ohnuma; Mamiko Mizuhara; Hirokazu Sato; Karen Skriver; Tamo Fukamizo
    GLYCOBIOLOGY 23 1 81 - 90 2013年01月 [査読有り]
     
    A tryptophan side chain was introduced into subsite +1 of family GH-18 (class V) chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC, respectively) by the mutation of a glycine residue to tryptophan (G74W-NtChiV and G75W-AtChiC). The specific activity toward glycol chitin of the two mutant enzymes was 70-71% of that of the wild type. Using chitin oligosaccharides, (GlcNAc)(n) (n = 4, 5 and 6), as the substrates, we found the transglycosylation reaction to be significantly enhanced in G74W-NtChiV and G75W-AtChiC when compared with the corresponding wild-type enzymes. The introduced tryptophan side chain might protect the oxazolinium ion intermediate from attack by a nucleophilic water molecule. The enhancement of transglycosylation activity was much more distinct in G75W-AtChiC than in G74W-NtChiV. Nuclear magnetic resonance titration experiments using the inactive double mutants, E115Q/G74W-NtChiV and E116Q/G75W-AtChiC revealed that the association constant of (GlcNAc)(5) was considerably larger for the latter. Amino acid substitutions at the acceptor binding site might have resulted in the larger association constant for G75W-AtChiC, giving rise to the higher transglycosylation activity of G75W-AtChiC.
  • Marie-Ève Lacombe-Harvey; Mélanie Fortin; Takayuki Ohnuma; Tamo Fukamizo; Thomas Letzel; Ryszard Brzezinski
    BMC Biochemistry 14 1 23 - 23 2013年 [査読有り]
     
    Background: Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. Results: Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. Conclusions: Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan. © 2013 Lacombe-Harvey et al. licensee BioMed Central Ltd.
  • Takayuki Ohnuma; Tomoyuki Numata; Takuo Osawa; Hideko Inanaga; Yoko Okazaki; Shoko Shinya; Kaori Kondo; Tatsuya Fukuda; Tamo Fukamizo
    FEBS JOURNAL 279 19 3639 - 3651 2012年10月 [査読有り]
     
    The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, site-directed mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc)4, was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the 1H15N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc)4. Signal assignments revealed that most signals responsive to the addition of (GlcNAc)4 are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure. Database ?The atomic coordinates and structural factors have been deposited in the Protein Data Bank, under the accession codes 4DWX (unliganded form) and 4DYG ((GlcNAc)4 complex). Chitinase, EC 3.2.1.14. Backbone assignment data were deposited in the Biological Magnetic Resonance Data Bank ( http://www.bmrb.wisc.edu/bmrb/) with the code number 11467 Structured digital abstract RSC-c and RSC-c bind by x-ray crystallography (View interaction)
  • Shoko Shinya; Takuya Nagata; Takayuki Ohnuma; Toki Taira; Shigenori Nishimura; Tamo Fukamizo
    BIOMOLECULAR NMR ASSIGNMENTS 6 2 157 - 161 2012年10月 [査読有り]
     
    Family GH19 chitinases have been recognized as important in the plant defense against fungal pathogens. However, their substrate-recognition mechanism is still unknown. We report here the first resonance assignment of NMR spectrum of a GH19 chitinase from moss, Bryum coronatum (BcChi-A). The backbone signals were nearly completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of the chitin dimer to the enzyme solution perturbed the chemical shifts of HSQC resonances of the amino acid residues forming the putative substrate-binding cleft. Further NMR analysis of the ligand binding to BcChi-A will improve understanding of the substrate-recognition mechanism of GH-19 enzymes.
  • Takayuki Ohnuma; Tatsuya Fukuda; Satoshi Dozen; Yuji Honda; Motomitsu Kitaoka; Tanno Fukamizo
    BIOCHEMICAL JOURNAL 444 3 437 - 443 2012年06月 [査読有り]
     
    BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (G1cNAc)(n) [a beta-1,4-linked polysaccharide of G1cNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(G1cNAc)(2)-F [alpha-(G1cNAc)(2) fluoride] in the absence or presence of (G1cNAc)(2), (G1cNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [ (2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Seri(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, 5102D, S102G, S102H, S102T, E7OG and E70Q. all of the mutated enzymes, except S102T, hydrolytic activity towards (G1cNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S1020G and E70G were found to successfully synthesize (G1cNAc)4 as a major product from alpha-(G1eNAc)(2)-F in the presence of (G1cNAc)(2). The SIO2A mutant showed the greatest glycosynthase activity owing to its enhanced F-releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.
  • Naoyuki Umemoto; Takayuki Ohnuma; Henri Urpilainen; Takanori Yamamoto; Tomoyuki Numata; Tamo Fukamizo
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 4 778 - 784 2012年04月 [査読有り]
     
    Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5-7 degrees C, while the mutations of Trp190 lowered stability only by 2-4 degrees C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)(4), in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the beta-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.
  • Takayuki Ohnuma; Toki Taira; Tamo Fukamizo
    Journal of Applied Glycoscience 59 1 47 - 50 The Japanese Society of Applied Glycoscience 2012年04月 [査読有り]
     
    Recombinant class V chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC) were produced by the Escherichia coli expression system, and the antifungal activity of the enzymes was investigated using the hyphal extension inhibition assay on agar plates with Trichoderma viride as the test fungus. The activity of NtChiV was found to be much higher than that of AtChiC. The inactive mutants of both enzymes, in which the individual catalytic acids were mutated to glutamine, were also tested by the same assay system. The activity was impaired by the mutation, indicating that the hydrolytic activity contributes to the antifungal action of the enzymes. However, the activity of the enzymes toward glycol chitin substrate was not proportional to the antifungal activity, indicating that the hydrolytic activity does not exclusively contribute to the antifungal action. X-ray crystal structures of these enzymes revealed that the aglycon-binding region of NtChiV consists of a number of polar side chains but not in AtChiC. Polarity of the surface of substrate-binding cleft could be another factor controlling the antifungal action of class V chitinases.
  • Yasuyuki Arakane; Toki Taira; Takayuki Ohnuma; Tamo Fukamizo
    CURRENT DRUG TARGETS 13 4 442 - 470 2012年04月 [査読有り]
     
    Plants utilized for agricultural productions interact with insects, fungi, and bacteria under the field conditions, affecting thereby their productivity. Since chitin and its derivatives play important roles in the interactions between these organisms, chitin-related enzymes are effective tools or drug targets for controlling the interactions. Thus, the molecular biology, protein chemistry, and enzymology of the chitin-related enzymes have been intensively studied by many investigators. Identifications and classifications of the genes encoding chitin synthetases, chitinases, chitosanases, and chitin deacetylases in these organisms were conducted, and their physiological functions were defined by knockdown, knockout, or overexpression of the corresponding genes. Recombinant enzyme productions and mutation studies are also being conducted to understand their structure and function. All of these studies have opened the way to efficiently utilize these enzyme tools for enhancing the agricultural productions.
  • Tomonari Tanaka; Tomonori Wada; Masato Noguchi; Masaki Ishihara; Atsushi Kobayashi; Takayuki Ohnuma; Tamo Fukamizo; Ryszard Brzezinski; Shin-ichiro Shoda
    JOURNAL OF CARBOHYDRATE CHEMISTRY 31 8 634 - 646 2012年 [査読有り]
     
    A novel sugar adduct, 4,6-dimethoxy-1,3,5-triazin-2-yl beta-D-glucosaminide (GlcN-beta-DMT), has been prepared by the reaction of D-glucosamine (GlcN) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM). The adduct was recognized by exo-beta-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis and transferred the GlcN moiety, giving rise to the corresponding glucosaminides. This chemo-enzymatic process was successfully applied to D-galactosamine (GalN). GalN-beta-DMT prepared directly from GalN and DMT-MM behaved as an efficient glycosyl donor for transfer of the GalN moiety catalyzed by the same enzyme. The introduction of the 4,6-dimethoxy-1,3,5-triazin-2-yl leaving group to the anomeric center significantly enhanced transglycosylating ability, resulting in the efficient glycosidase-catalyzed synthesis of glycosaminides.
  • Takayuki Ohnuma; Morten Sorlie; Tatsuya Fukuda; Noriko Kawamoto; Toki Taira; Tamo Fukamizo
    FEBS JOURNAL 278 21 3991 - 4001 2011年11月 [査読有り]
     
    Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum (BcChi-A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions ('loop-less'), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). Chitin oligosaccharides [beta-1,4-linked oligosaccharides of N-acetylglucosamine with a polymerization degree of n, (GlcNAc)(n), n = 3-6] were hydrolyzed by BcChi-A at rates in the order (GlcNAc)(6) > (GlcNAc)(5) > (GlcNAc)(4) > > (GlcNAc)(3). From thermal unfolding experiments using the inactive BcChi-A mutant (BcChi-A-E61A), in which the catalytic residue Glu61 is mutated to Ala, we found that the transition temperature (T(m)) was elevated upon addition of (GlcNAc)(n) (n = 2-6) and that the elevation (Delta T(m)) was almost proportional to the degree of polymerization of (GlcNAc)(n). ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)(n) (n = 3-6) to BcChi-A-E61A, and revealed that the binding was driven by favorable enthalpy changes with unfavorable entropy changes. The change in heat capacity (Delta C(p)degrees) for (GlcNAc)(6) binding was found to be relatively small (-105 +/- 8 cal.K(-1) mol(-1)). The binding free energy changes for (GlcNAc)(6), (GlcNAc)(5), (GlcNAc)(4) and (GlcNAc)(3) were determined to be -8.5, -7.9, -6.6 and -5.0 kcal.mol(-1), respectively. Taken together, the substrate binding cleft of BcChi-A consists of at least six subsites, in contrast to the four-subsites binding cleft of the 'loopless' family 19 chitinase from Streptomyces coelicolor.
  • Shoko Shinya; Takayuki Ohnuma; Shunsuke Kawamura; Takao Torikata; Shigenori Nishimura; Etsuko Katoh; Tamo Fukamizo
    JOURNAL OF BIOCHEMISTRY 150 5 569 - 577 2011年11月 [査読有り]
     
    The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [ (GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 x 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 x 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (delta H = -6.1 kcal/mol and T delta S = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.
  • Tamo Fukamizo; Hirokazu Sato; Mamiko Mizuhara; Takayuki Ohnuma; Takeshi Gotoh; Kazuyuki Hiwatashi; Saori Takahashi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 9 1763 - 1769 2011年09月 [査読有り]
     
    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second beta-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [ (GlcNAc), n = 4, 5, and 6], producing the beta-anomer of (GlcNAc)(2). The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid beta-chitin, producing only (GlcNAc)2. The AcMNPV chitinase processively hydrolyzes solid beta-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.
  • Takayuki Ohnuma; Tomoyuki Numata; Takuo Osawa; Mamiko Mizuhara; Outi Lampela; Andre H. Juffer; Karen Skriver; Tamo Fukamizo
    PLANTA 234 1 123 - 137 2011年07月 [査読有り]
     
    Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 resolution. AtChiC was found to adopt an (beta/alpha)(8) fold with a small insertion domain composed of an alpha-helix and a five-stranded beta-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.
  • Toki Taira; Yoko Mahoe; Noriko Kawamoto; Shoko Onaga; Hironori Iwasaki; Takayuki Ohnuma; Tamo Fukamizo
    GLYCOBIOLOGY 21 5 644 - 654 2011年05月 [査読有り]
     
    Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.
  • Letzel T; Sahmel-Schneider E; Skriver K; Ohnuma T; Fukamizo T
    Carbohydrate research 346 6 863 - 866 2011年05月 [査読有り]
     
    4-Nitrophenyl penta-N-acetyl-beta-chitopentaoside [ (GlcNAc)(5)-pNP] was hydrolyzed by a family GH-19 class II barley chitinase, and the enzymatic reaction was monitored by real-time ESIMS. The wild-type enzyme hydrolyzed (GlcNAc)(5)-pNP producing predominantly (GlcNAc)(3)-pNP and a lesser amount of (GlcNAc)(2)-pNP, indicating that the (GlcNAc)(5) portion of the substrate binds predominantly to subsites -2 similar to +3 and less frequently to -3 similar to +2. However, (GlcNAc)(2)-pNP was mainly produced from (GlcNAc)(5)-pNP by mutated enzymes, in which Trp72 and Trp82 located at +3/+4 were substituted with alanine (W72A and W72A/W82A), indicating that the (GlcNAc)(5) portion of the substrate binds predominantly to subsites -3 similar to +2 of the mutants. The mutations of the tryptophan residues resulted in a significant shift of the substrate-binding mode to the glycon side, supporting the idea that the indole side chain of Trp72 interacts with the 4-nitrophenyl moiety of the substrate at subsite +4. (C) 2011 Elsevier Ltd. All rights reserved.
  • Takayuki Ohnuma; Tomoyuki Numata; Takuo Osawa; Mamiko Mizuhara; Kjell M. Varum; Tamo Fukamizo
    PLANT MOLECULAR BIOLOGY 75 3 291 - 304 2011年02月 [査読有り]
     
    A class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [ (NAG)(n)] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 resolution. The protein adopts a classical (beta/alpha)(8)-barrel fold (residues 1-233 and 303-348) with an insertion of a small (alpha + beta) domain (residues 234-302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG)(4) was also solved and exhibited a linear conformation of the bound oligosaccharide occupying -2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG)(4) binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to -1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme.
  • Takayuki Ohnuma; Takuo Osawa; Tamo Fukamizo; Tomoyuki Numata
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 66 12 1599 - 1601 2010年12月 [査読有り]
     
    The plant chitinases, which have been implicated in self-defence against pathogens, are divided into at least five classes (classes I, II, III, IV and V). Although the crystal structures of several plant chitinases have been solved, no crystal structure of a class V chitinase has been reported to date. Here, the crystallization of Nicotiana tabacum class V chitinase (NtChiV) using the vapour-diffusion method is reported. The NtChiV crystals diffracted to 1.2 A resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 62.4, b = 120.3, c = 51.9 A. The asymmetric unit of the crystals is expected to contain one molecule.
  • Toki Taira; Maho Fujiwara; Nicole Dennhart; Hiroko Hayashi; Shoko Onaga; Takayuki Ohnuma; Thomas Letzel; Shohei Sakuda; Tamo Fukamizo
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1804 4 668 - 675 2010年04月 [査読有り]
     
    Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from - 3 to - 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme. (C) 2009 Elsevier B.V. All rights reserved.
  • Taira T; Fujiwara M; Dennhart N; Hayashi H; Onaga S; Ohnuma T; Letzel T; Sakuda S; Fukamizo T
    Biochimica et biophysica acta 1804 4 668 - 675 2010年04月 [査読有り]
     
    Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.
  • Toki Taira; Hiroko Hayashi; Yoshiko Tajiri; Shoko Onaga; Gen-ichiro Uechi; Hironori Iwasaki; Takayuki Ohnuma; Tamo Fukamizo
    GLYCOBIOLOGY 19 12 1452 - 1461 2009年12月 [査読有り]
     
    Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.
  • Tamo Fukamizo; Ryoh Miyake; Atsushi Tamura; Takayuki Ohnuma; Karen Skriver; Niko V. Pursiainen; Andre H. Juffer
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1794 8 1159 - 1167 2009年08月 [査読有り]
     
    To examine the role of the loop structure consisting of residues 70-82 (70-82 loop) localized to +3/4 subsite of the substrate binding cleft of a family GH-19 endochitinase from barley seeds, Trp72 and Trp82 were mutated, and the mutated enzymes (W72A, W82A, and W72A/W82A) were characterized. Thermal stability and specific activities toward glycol chitin and chitin hexasaccharide were significantly affected by the individual mutations. When N-acetylglucosamine hexamer was hydrolyzed by the wild type, the beta-anomer of the substrate was preferentially hydrolyzed, producing the trimer predominantly and the dimer and tetramer in lesser amounts. When the mutated enzymes were used instead of the wild type, the enzyme cleavage sites in the hexamer substrate were clearly shifted, and the beta-anomer selectivity was eliminated. The mutation effects on the enzymatic activity and stability were much more substantial in W82A than in W72A, but surprisingly the effects of the W82A/W72A double mutation were intermediate between those of the two single mutations. A molecular dynamics simulation of the wild type and the Trp-mutated enzymes indicated that the 70-82 loop becomes more flexible upon mutation and the flexibility increases in the order of W72A, W72A/W82A and W82A. We conclude that Trp72 interacts with the sugar residue but Trp82 modulates the loop flexibility, which controls the protein stability and enzymatic properties. These tryptophan residues are likely to interact with each other, resulting in the non-additivity of mutational effects. (C) 2009 Elsevier B.V. All rights reserved.
  • Takayuki Ohnuma; Shoko Onaga; Katsuyoshi Murata; Toki Taira; Etsuko Katoh
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 8 5178 - 5187 2008年02月 [査読有り]
     
    The LysM domain probably binds peptidoglycans, but how it does so has yet to be described. For this report, we measured the thermal stabilities of recombinant LysM domains derived from Pteris ryukyuensis chitinase-A (PrChi-A) and monitored their binding to N-acetylglucosamine oligomers ((GlcNAc)(n)) using differential scanning calorimetry, isothermal titration calorimetry, and NMR spectroscopy. We thereby characterized certain of the domains' functional and structural features. We observed that the domains are very resistant to thermal denaturation and that this resistance depends on the presence of disulfide bonds. We also show that the stoichiometry of (GlcNAc)(n)/LysM domain binding is 1:1. (GlcNAc)(5) titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc)(5) binding. The binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments reiterate the critical involvement of Tyr(72) in (GlcNAc)(n)/LysM domain binding. Ours is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.
  • Masahide Sawano; Hitoshi Yamamoto; Kyoko Ogasahara; Shun-ichi Kidokoro; Shizue Katoh; Takayuki Ohnuma; Etsuko Katoh; Shigeyuki Yokoyama; Katsuhide Yutani
    BIOCHEMISTRY 47 2 721 - 730 2008年01月 [査読有り]
     
    In order to elucidate the stabilization mechanism of CutAl from Pyrococcus horikoshii (PhCutAl) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutAl proteins from Thermus thermophilus (TtCutAl) and Oryza sativa (OsCutAl) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutAl, TtCutAl and OsCutAl were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutAl and OsCutAl showed that the denaturation temperatures were remarkably high, 12.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutAl I showed higher stability to both heat and denaturant than PhCutAl Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutAl could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutAl basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutAl is superior to those of the other two CutA Is over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutAl contribute to the unusually high stability at neutral pH.
  • T Ohnuma; T Taira; T Yamagami; Y Aso; M Ishiguro
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 2 324 - 332 2004年02月 
    A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures. It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues. Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of Escherichia coli Origami(DE3) in a soluble form by inducing bacteria at a low temperature (20degreesC). Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against Trichoderma sp. in vitro. rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a. Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability. Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium. Complete abolishment of both activities was observed upon the mutation of Glu126 to Gin. The roles of these residues in both activities are discussed.
  • K Suzukawa; T Yamagami; T Ohnuma; H Hirakawa; S Kuhara; Y Aso; M Ishiguro
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 2 341 - 346 2003年02月 [査読有り]
     
    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.
  • T Taira; T Ohnuma; T Yamagami; Y Aso; M Ishiguro; M Ishihara
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 5 970 - 977 2002年05月 
    The antifungal activities of rye seed chitinase-a (RSC-a, class I) and -c (RSC-c, class II) were studied in detail using two different bioassays with Trichoderma sp. as well as binding and degradation experiments with the cell walls prepared from its mycelia. RSC-a inhibited more strongly the re-extension of the hyphae, containing mainly mature cells, than RSC-c did. Upon incubation of The fungus with fluorescent chitinases, FITC-labeled RSC-a was found to be located in the hyphal tips, lateral walls, and septa, while FITC-labeled RSC-c was only in the hyphal tip. RSC-a had a greater affinity for the cell walls than RSC-c. RSC-a liberated a larger amount of reducing sugar from the cell walls than RSC-c did. These results inferred that RSC-a first binds to the lateral walls and septa, consisting of the mature cell walls, and degrades mature chitin fiber, while RSC-c binds only to the hyphal tip followed by degradation of only nascent chitin. As a result, RSC-a inhibited fungal growth more effectively than RSC-c. Furthermore, it was suggested that the chitin-binding domain in RSC-a assists the antifungal action of RSC-a by binding to the fungal hypha.
  • T Ohnuma; M Yagi; T Yamagami; T Taira; Y Aso; M Ishiguro
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 2 277 - 284 2002年02月 
    We cloned a complete cDNA encoding rye seed chitinase-c, designated RSC-c, by rapid amplification of cDNA end and PCR procedures. The cDNA of RSC-c consists of 1,018 nucleotides and includes an open reading frame encoding a polypeptide of 266 amino acid residues. A recombinant RSC-c was produced by expression in Escherichia coli Origami(DE3) and purified. rRSC-c had almost the same chitinase activity toward glycolchitin and antifungal activity against Trichoderma sp. as the authentic RSC-c did. RSC-c mutants were subsequently constructed and characterized with respect to their chitinase and antifungal activities. Mutation of Glu67 to Gin completely abolished the chitinase activity and diminished the antifungal activity. Considerable decreases in both activities were observed in the mutations of Trp72 and Ser120 to Ala, and Glu89 to Gin. The roles of these residues in the catalytic event of RSC-c are discussed.
  • M Ishiguro; T Yamagami; M Tanigawa; K Tsutsumi; G Funatsu; T Ohnuma; Y Aso
    JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY 46 1 243 - 250 2001年10月 [査読有り]
     
    Chemical, modification of tulip bulb chitinase-1 (TBC-1) and pokeweed leaf chitinase (PLC-B) with N-bromosuccinimide (NBS) suggested the involvement of tryptophan residue (s) in the activity. In the case of TBC-1, at pH 4.0, about 1 mol of tryptophan residues out of 4 mol was oxidized with 5 mol of NBS per mol of TBC-1 and all 4 mol of tryptophan residues were oxidized with 15 mol of NBS. At pH 4.5, about 3 mol of tryptophan were finally oxidized although the oxidation rate of the first tryptophan residue was same as at pH 4.0. In both:cases, the oxidation of 1 mol of tryptophan residues caused 85% activity loss. Analysis of the oxytryptophan-containing peptides afforded to identify that Trp172 and Trp255 were those which reacted first with NBS at pH 4.0. When PLC-B was reacted with 16-folds molar of NBS at pH 4.0, all seven tryptophan residues were oxidized with a concomitant decrease of chitinase activity. Oxidized tryptophan residues were also determined as those at positions of 165 and 256 in PLC-B. From these results, it was inferred that the rapidly oxidized tryptophan residues in plant class III chhitinases were involved in their chitinase activity.

MISC

書籍等出版物

  • Lysozymes: Sources, Functions and Role in Diseas
    Shoko Shinya; Takayuki Ohnuma; Ryszard Brzezinski; Romy K. Scheerle; Johanna Grassmann; Thomas Letzel; Tamo Fukamizo (担当:分担執筆範囲:Lysozyme Superfamily: Progress in Functional Analysis using ESI-MS and NMR Spectroscopy)Nova Science Publishers, Inc. 2013年 ISBN: 9781622578429 127-154
  • Marine Medicinal Glycomics
    Naoyuki Umemoto; Takayuki Ohnuma; Tamo Fukamizo (担当:分担執筆範囲:Enzymatic Synthesis of Chitin Oligosaccharides with Longer Chains)Nova Science Publishers, Inc. 2013年 ISBN: 9781626186491 39-58
  • 糖質とタンパク質の相互作用解析、『機能性糖質素材の開発と食品への応用Ⅱ』
    大沼貴之; 深溝慶 (担当:分担執筆範囲:糖質とタンパク質の相互作用解析)シーエムシー出版 2013年 ISBN: 9784781308234 232-240
  • Thomas Letzel; Series Roger M Smith; Tamo Fukamizo; Takayuki Ohnuma; Seronei Chelulei Cheison; Christian Webhofer; Michael Schrader; Gabrielle Stohr; Andreas Tebbe; Boris Macek; Johanna Grassmann; Romy Scheerle; Michael Krappmann; Rene Wissiack; Friedrich Lottspeich; Johannes Hoos; Nicolas L Young; Benjamin A Garcia; Ulrich Kulozik; Wilfried M.A Niessen Royal Society of Chemistry 2011年08月 ISBN: 1849731829 194
  • キチン、キトサン、オリゴ糖およびその誘導体‐生物活性とその利用‐
    大沼 貴之; 深溝 慶; Department of Chemistry Director; Marine Bioprocess Research Center; Pukyong National; University, Busan; South Korea (担当:共著範囲:)CRC press 2010年07月

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2023年04月 -2026年03月 
    代表者 : 江口 陽子; 大沼 貴之
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2022年04月 -2025年03月 
    代表者 : 大沼 貴之
  • イネXIPによる病原菌抵抗機構の分子基盤の解明
    日本学術振興会:基盤研究(C)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 大沼貴之
  • 植物の細胞壁構築に関与するキチナーゼ様タンパク質CTLの構造と機能
    日本学術振興会:若手研究(B)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 大沼貴之
  • マメ科植物が生産するノッド因子加水分解酵素の構造と機能
    公益財団法人農芸化学研究奨励会:研究奨励金
    研究期間 : 2014年04月 -2015年03月 
    代表者 : 大沼貴之
  • 植物のキチンオリゴ糖エリシター受容体タンパク質の構造と機能
    日本学術振興会:若手研究(B)
    研究期間 : 2011年04月 -2013年03月 
    代表者 : 大沼貴之
  • Award of guest researcher scholarship
    Norwegian University of Life Sciences:Award of guest researcher scholarship
    研究期間 : 2011年 
    代表者 : 大沼貴之
  • 等温滴定型熱量計によるLysMドメインとキチンオリゴ糖の相互作用機構の解明
    日本学術振興会ーThe Research Council of Norway:特定国派遣研究者(ノルウェー生命科学大学)
    研究期間 : 2010年08月 -2010年09月 
    代表者 : 大沼貴之

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