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Naoto Shibuya - One of the best experts on this subject based on the ideXlab platform.
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differences in the recognition of glucan elicitor signals between rice and soybean beta glucan fragments from the rice blast disease fungus pyricularia oryzae that elicit phytoalexin biosynthesis in suspension cultured rice cells
The Plant Cell, 2000Co-Authors: Takeshi Yamaguchi, Akira Yamada, Namgi Hong, Tomoya Ogawa, Tadashi Ishii, Naoto ShibuyaAbstract:Partial acid/enzymatic hydrolysis of the β-(1→3, 1→6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-β-(1→3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcβ(1→3)Glcβ(1→3)(Glcβ(1→6)) Glcβ(1→3)Glucitol (most active fragment); (2) Glcβ(1→3)(Glcβ(1→6))Glcβ(1→3)Glcβ(1→3)Glucitol; and (3) Glcβ(1→6) Glcβ(1→3)Glcβ(1→3)Glcβ(1→3)Glucitol. However, a synthetic hexa-β-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the β-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.
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differences in the recognition of glucan elicitor signals between rice and soybean β glucan fragments from the rice blast disease fungus pyricularia oryzae that elicit phytoalexin biosynthesis in suspension cultured rice cells
The Plant Cell, 2000Co-Authors: Takeshi Yamaguchi, Akira Yamada, Namgi Hong, Tomoya Ogawa, Tadashi Ishii, Naoto ShibuyaAbstract:Partial acid/enzymatic hydrolysis of the β-(1→3, 1→6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-β-(1→3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcβ(1→3)Glcβ(1→3)(Glcβ(1→6)) Glcβ(1→3)Glucitol (most active fragment); (2) Glcβ(1→3)(Glcβ(1→6))Glcβ(1→3)Glcβ(1→3)Glucitol; and (3) Glcβ(1→6) Glcβ(1→3)Glcβ(1→3)Glcβ(1→3)Glucitol. However, a synthetic hexa-β-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the β-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.
Hayao Taguchi - One of the best experts on this subject based on the ideXlab platform.
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colorimetric determination of β 1 2 Glucooligosaccharides for an enzymatic assay using 3 methyl 2 benzothiazolinonehydrazone
Analytical Biochemistry, 2018Co-Authors: Kaito Kobayashi, Hiroyuki Nakai, Masahiro Nakajima, Hiroki Aramasa, Hayao TaguchiAbstract:A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of β-1,2-glucanases, which produce β-1,2-Glucooligosaccharides from β-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of β-1,2-Glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other β-1,2-Glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for β-1,2-Glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a β-1,2-glucanase using the modified MBTH method.
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characterization and structural analysis of a novel exo type enzyme acting on β 1 2 Glucooligosaccharides from parabacteroides distasonis
Biochemistry, 2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Yuta Takahashi, Nobukiyo Tanaka, Kaito Kobayashi, Naohisa Sugimoto, Masahiro Nakajima, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-Glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superi...
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characterization and structural analysis of a novel exo type enzyme acting on β 1 2 Glucooligosaccharides from parabacteroides distasonis
Biochemistry, 2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Nobukiyo Tanaka, Kaito Kobayashi, Masahiro Nakajima, Y Takahashi, N Sugimoto, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) ...
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Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β‑1,2-Glucooligosaccharides from Parabacteroides distasonis
2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Yuta Takahashi, Nobukiyo Tanaka, Kaito Kobayashi, Naohisa Sugimoto, Masahiro Nakajima, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-Glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite −3 in CpSGL. Docking analysis involving a β-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-β-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064
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functional and structural analysis of a β glucosidase involved in β 1 2 glucan metabolism in listeria innocua
PLOS ONE, 2016Co-Authors: Masahiro Nakajima, Hiroyuki Nakai, Akimasa Miyanaga, Ryuta Yoshida, Koichi Abe, Y Takahashi, N Sugimoto, Hiroyuki Toyoizumi, Motomitsu Kitaoka, Hayao TaguchiAbstract:Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-Glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.
Takeshi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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differences in the recognition of glucan elicitor signals between rice and soybean beta glucan fragments from the rice blast disease fungus pyricularia oryzae that elicit phytoalexin biosynthesis in suspension cultured rice cells
The Plant Cell, 2000Co-Authors: Takeshi Yamaguchi, Akira Yamada, Namgi Hong, Tomoya Ogawa, Tadashi Ishii, Naoto ShibuyaAbstract:Partial acid/enzymatic hydrolysis of the β-(1→3, 1→6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-β-(1→3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcβ(1→3)Glcβ(1→3)(Glcβ(1→6)) Glcβ(1→3)Glucitol (most active fragment); (2) Glcβ(1→3)(Glcβ(1→6))Glcβ(1→3)Glcβ(1→3)Glucitol; and (3) Glcβ(1→6) Glcβ(1→3)Glcβ(1→3)Glcβ(1→3)Glucitol. However, a synthetic hexa-β-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the β-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.
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differences in the recognition of glucan elicitor signals between rice and soybean β glucan fragments from the rice blast disease fungus pyricularia oryzae that elicit phytoalexin biosynthesis in suspension cultured rice cells
The Plant Cell, 2000Co-Authors: Takeshi Yamaguchi, Akira Yamada, Namgi Hong, Tomoya Ogawa, Tadashi Ishii, Naoto ShibuyaAbstract:Partial acid/enzymatic hydrolysis of the β-(1→3, 1→6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-β-(1→3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcβ(1→3)Glcβ(1→3)(Glcβ(1→6)) Glcβ(1→3)Glucitol (most active fragment); (2) Glcβ(1→3)(Glcβ(1→6))Glcβ(1→3)Glcβ(1→3)Glucitol; and (3) Glcβ(1→6) Glcβ(1→3)Glcβ(1→3)Glcβ(1→3)Glucitol. However, a synthetic hexa-β-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the β-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.
Ying-chieh Tsai - One of the best experts on this subject based on the ideXlab platform.
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functional roles of the 6 s cysteinyl 8alpha n1 histidyl fad in glucooligosaccharide oxidase from acremonium strictum
Journal of Biological Chemistry, 2008Co-Authors: Chun-hsiang Huang, Peter Macheroux, Ying-chieh Tsai, Andreas Winkler, Chialin Chen, Shwu-huey LiawAbstract:Abstract The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8α-positions of the flavin isoalloxazine ring cross-linked to Cys130 and His70, respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of ∼+69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with kcat values reduced to ∼2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.
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functional roles of the 6 s cysteinyl 8alpha n1 histidyl fad in glucooligosaccharide oxidase from acremonium strictum
Journal of Biological Chemistry, 2008Co-Authors: Chun-hsiang Huang, Peter Macheroux, Ying-chieh Tsai, Andreas Winkler, Chialin Chen, Wenlin Lai, Shwu-huey LiawAbstract:The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8alpha-positions of the flavin isoalloxazine ring cross-linked to Cys(130) and His(70), respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of approximately +69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with k(cat) values reduced to approximately 2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.
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structural characterization of glucooligosaccharide oxidase from acremonium strictum
Applied and Environmental Microbiology, 2005Co-Authors: Shwu-huey Liaw, Ying-chieh TsaiAbstract:Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.
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crystal structure of glucooligosaccharide oxidase from acremonium strictum a novel flavinylation of 6 s cysteinyl 8alpha n1 histidyl fad
Journal of Biological Chemistry, 2005Co-Authors: Chun-hsiang Huang, Andrea Vasella, Chun-jung Chen, Ying-chieh Tsai, Shwu-huey LiawAbstract:Abstract Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-A resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8α-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8α-methyl group of the isoalloxazine ring with Cys130 and His70, respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a β-d-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin N5. Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.
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immobilization of glucooligosaccharide oxidase of acremonium strictum for oligosaccharic acid production
Biotechnology Techniques, 1996Co-Authors: Yinglong Hwang, Ying-chieh TsaiAbstract:The glucooligosaccharide oxidase was covalently immobilized to chitosan with polyethyleneimine and glutaraldehyde. Immobilization improved thermal stability. When used for conversion of starch hydrolysate to oligosaccharic acids, the immobilized enzyme maintained 75% initial activity after 60 days of continuous operation. Strong substrate inhibition was seen at high concentrations of cellobiose and lactose for free enzyme but not for immobilized enzyme.
Hiroyuki Nakai - One of the best experts on this subject based on the ideXlab platform.
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colorimetric determination of β 1 2 Glucooligosaccharides for an enzymatic assay using 3 methyl 2 benzothiazolinonehydrazone
Analytical Biochemistry, 2018Co-Authors: Kaito Kobayashi, Hiroyuki Nakai, Masahiro Nakajima, Hiroki Aramasa, Hayao TaguchiAbstract:A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of β-1,2-glucanases, which produce β-1,2-Glucooligosaccharides from β-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of β-1,2-Glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other β-1,2-Glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for β-1,2-Glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a β-1,2-glucanase using the modified MBTH method.
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characterization and structural analysis of a novel exo type enzyme acting on β 1 2 Glucooligosaccharides from parabacteroides distasonis
Biochemistry, 2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Yuta Takahashi, Nobukiyo Tanaka, Kaito Kobayashi, Naohisa Sugimoto, Masahiro Nakajima, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-Glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superi...
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characterization and structural analysis of a novel exo type enzyme acting on β 1 2 Glucooligosaccharides from parabacteroides distasonis
Biochemistry, 2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Nobukiyo Tanaka, Kaito Kobayashi, Masahiro Nakajima, Y Takahashi, N Sugimoto, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) ...
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Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β‑1,2-Glucooligosaccharides from Parabacteroides distasonis
2018Co-Authors: Hisaka Shimizu, Hiroyuki Nakai, Akimasa Miyanaga, Yuta Takahashi, Nobukiyo Tanaka, Kaito Kobayashi, Naohisa Sugimoto, Masahiro Nakajima, Hayao TaguchiAbstract:β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-Glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite −3 in CpSGL. Docking analysis involving a β-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-β-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064
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functional and structural analysis of a β glucosidase involved in β 1 2 glucan metabolism in listeria innocua
PLOS ONE, 2016Co-Authors: Masahiro Nakajima, Hiroyuki Nakai, Akimasa Miyanaga, Ryuta Yoshida, Koichi Abe, Y Takahashi, N Sugimoto, Hiroyuki Toyoizumi, Motomitsu Kitaoka, Hayao TaguchiAbstract:Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-Glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.