The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
Takuya Koseki - One of the best experts on this subject based on the ideXlab platform.
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characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate binding module
Applied Microbiology and Biotechnology, 2010Co-Authors: Takuya Koseki, Keiji Mochizuki, Hiroe Kisara, Tetsuya Murayama, Akimasa Miyanaga, Shinya Fushinobu, Yoshihito ShionoAbstract:We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (Mr) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.
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the family 42 carbohydrate binding module of family 54 α l arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose
Biochemical Journal, 2006Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Sachiko Nakamura, Atsushi Kuno, Jun Hirabayashi, Hirofumi ShounAbstract:α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.
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mutational analysis of n glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α l arabinofuranosidase 54
Biochimica et Biophysica Acta, 2006Co-Authors: Takuya Koseki, Akimasa Miyanaga, Shinya Fushinobu, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Hirofumi Shoun, Katsumi HashizumeAbstract:Abstract A role for N -linked oligosaccharides on the biochemical properties of recombinant α- l -arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N -linked glycosylation motifs (Asn 83 –Thr–Thr and Asn 202 –Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N -linked glycosylation sites are located in the catalytic domain. Asn 83 , Asn 202 , and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn 202 may contribute to thermostability and catalysis.
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crystal structure of a family 54 α l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystal structure of a family 54 alpha l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
Hiroshi Matsuzawa - One of the best experts on this subject based on the ideXlab platform.
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the family 42 carbohydrate binding module of family 54 α l arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose
Biochemical Journal, 2006Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Sachiko Nakamura, Atsushi Kuno, Jun Hirabayashi, Hirofumi ShounAbstract:α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.
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mutational analysis of n glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α l arabinofuranosidase 54
Biochimica et Biophysica Acta, 2006Co-Authors: Takuya Koseki, Akimasa Miyanaga, Shinya Fushinobu, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Hirofumi Shoun, Katsumi HashizumeAbstract:Abstract A role for N -linked oligosaccharides on the biochemical properties of recombinant α- l -arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N -linked glycosylation motifs (Asn 83 –Thr–Thr and Asn 202 –Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N -linked glycosylation sites are located in the catalytic domain. Asn 83 , Asn 202 , and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn 202 may contribute to thermostability and catalysis.
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crystal structure of a family 54 α l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystal structure of a family 54 alpha l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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role of two α l arabinofuranosidases in arabinoxylan degradation and characteristics of the encoding genes from shochu koji molds Aspergillus kawachii and Aspergillus awamori
Journal of Bioscience and Bioengineering, 2003Co-Authors: Takuya Koseki, Masaki Okuda, Shigetoshi Sudoh, Yasuzo Kizaki, Kimio Iwano, Isao Aramaki, Hiroshi MatsuzawaAbstract:Abstract Two different α- l -arabinofuranosidases from Aspergillus kawachii were purified and characterized. The two enzymes acted synergically with xylanase in the degradation of arabinoxylan and resulted in an increase in the amount of ferulic acid release by feruloyl esterase. Both enzymes were acidophilic and acid stable enzymes which had an optimum pH of 4.0 and were stable at pH 3.0–7.0. The general properties of the enzymes including pH optima and pH stability were similar to those of Aspergillus awamori. These results suggest that the α- l -arabinofuranosidases contribute to an increase in cereal utilization and formation of aroma in shochu brewing. Two different genes encoding α- l -arabinofuranosidases from A. kawachii, designated as AkabfA and AkabjB, and those from A. awamori, designated as AwabfA and AwabjB, were also cloned and characterized. The difference between the sequences of AkabfA and AwabfA was only one nucleotide, resulting in an amino acid difference in the sequence, and the enzymes were assigned to family 51 of glycoside hydrolases. On the other hand, the differences between the sequences of AkabjB and AwabjB and between their encoding proteins were two nucleotides and one amino acid residue, respectively, and the enzymes were assigned to family 54 of glycoside hydrolases. On comparison of the abfA and abjB genes among A. kawachii, A. awamori, and A. niger, the relationship between the two genes for A. kawachii and A. awamori was much closer than those between A. niger and the others. Northern analyses showed that transcription of AkabfB was greater than that of AkabfA in the presence of l -arabitol and l -arabinose, and that transcriptions of both genes were not induced in the presence of sucrose and glucose.
Akimasa Miyanaga - One of the best experts on this subject based on the ideXlab platform.
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characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate binding module
Applied Microbiology and Biotechnology, 2010Co-Authors: Takuya Koseki, Keiji Mochizuki, Hiroe Kisara, Tetsuya Murayama, Akimasa Miyanaga, Shinya Fushinobu, Yoshihito ShionoAbstract:We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (Mr) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.
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the family 42 carbohydrate binding module of family 54 α l arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose
Biochemical Journal, 2006Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Sachiko Nakamura, Atsushi Kuno, Jun Hirabayashi, Hirofumi ShounAbstract:α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.
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mutational analysis of n glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α l arabinofuranosidase 54
Biochimica et Biophysica Acta, 2006Co-Authors: Takuya Koseki, Akimasa Miyanaga, Shinya Fushinobu, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Hirofumi Shoun, Katsumi HashizumeAbstract:Abstract A role for N -linked oligosaccharides on the biochemical properties of recombinant α- l -arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N -linked glycosylation motifs (Asn 83 –Thr–Thr and Asn 202 –Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N -linked glycosylation sites are located in the catalytic domain. Asn 83 , Asn 202 , and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn 202 may contribute to thermostability and catalysis.
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crystal structure of a family 54 α l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystal structure of a family 54 alpha l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
Shinya Fushinobu - One of the best experts on this subject based on the ideXlab platform.
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characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate binding module
Applied Microbiology and Biotechnology, 2010Co-Authors: Takuya Koseki, Keiji Mochizuki, Hiroe Kisara, Tetsuya Murayama, Akimasa Miyanaga, Shinya Fushinobu, Yoshihito ShionoAbstract:We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (Mr) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.
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mutational analysis of n glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α l arabinofuranosidase 54
Biochimica et Biophysica Acta, 2006Co-Authors: Takuya Koseki, Akimasa Miyanaga, Shinya Fushinobu, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Hirofumi Shoun, Katsumi HashizumeAbstract:Abstract A role for N -linked oligosaccharides on the biochemical properties of recombinant α- l -arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N -linked glycosylation motifs (Asn 83 –Thr–Thr and Asn 202 –Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N -linked glycosylation sites are located in the catalytic domain. Asn 83 , Asn 202 , and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn 202 may contribute to thermostability and catalysis.
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crystal structure of a family 54 α l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystal structure of a family 54 alpha l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystallographic and mutational analyses of an extremely acidophilic and acid stable xylanase biased distribution of acidic residues and importance of asp37 for catalysis at low ph
Protein Engineering, 1998Co-Authors: Shinya Fushinobu, Kiyoshi Ito, Michiko Konno, Takayoshi Wakagi, Hiroshi MatsuzawaAbstract:Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0. The crystal structure of xylanase C was determined at 2.0 A resolution (R-factor = 19.4%). The overall structure was similar to those of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 A), as in other xylanases with low pH optima. Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis. Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH. In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wild-type enzyme. On structural comparison with xylanases with higher pH optima, another striking feature of the xylanase C structure was found; the enzyme has numerous acidic residues concentrated on the surface (so-called 'Ser/Thr surface' in most family 11 xylanases). The relationship of the stability against extreme pH conditions and high salt concentrations with the spatially biased distribution of charged residues on the proteins is discussed.
Hirofumi Shoun - One of the best experts on this subject based on the ideXlab platform.
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the family 42 carbohydrate binding module of family 54 α l arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose
Biochemical Journal, 2006Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Sachiko Nakamura, Atsushi Kuno, Jun Hirabayashi, Hirofumi ShounAbstract:α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.
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mutational analysis of n glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α l arabinofuranosidase 54
Biochimica et Biophysica Acta, 2006Co-Authors: Takuya Koseki, Akimasa Miyanaga, Shinya Fushinobu, Takayoshi Wakagi, Hiroshi Matsuzawa, Yozo Miwa, Yuichiro Mese, Hirofumi Shoun, Katsumi HashizumeAbstract:Abstract A role for N -linked oligosaccharides on the biochemical properties of recombinant α- l -arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N -linked glycosylation motifs (Asn 83 –Thr–Thr and Asn 202 –Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N -linked glycosylation sites are located in the catalytic domain. Asn 83 , Asn 202 , and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn 202 may contribute to thermostability and catalysis.
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crystal structure of a family 54 α l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
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crystal structure of a family 54 alpha l arabinofuranosidase reveals a novel carbohydrate binding module that can bind arabinose
Journal of Biological Chemistry, 2004Co-Authors: Akimasa Miyanaga, Takuya Koseki, Takayoshi Wakagi, Hiroshi Matsuzawa, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
