The Experts below are selected from a list of 10962 Experts worldwide ranked by ideXlab platform
Zhengqiang Jiang - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of a novel exo-oligoxylanase from Paenibacillus barengoltzii suitable for monosaccharification from corncobs
Biotechnology for biofuels, 2019Co-Authors: Xueqiang Liu, Shaoqing Yang, Zhengqiang Jiang, Xin You, Yu Liu, Qiaojuan YanAbstract:Xylan is the major component of hemicelluloses, which are the second most abundant polysaccharides in nature, accounting for approximately one-third of all renewable organic carbon resources on earth. Efficient degradation of xylan is the prerequisite for biofuel production. Enzymatic degradation has been demonstrated to be more attractive due to low energy consumption and environmental friendliness, when compared with chemical degradation. Exo-xylanases, as a rate-limiting factor, play an important role in the xylose production. It is of great value to identify novel exo-xylanases for efficient bioconversion of xylan in biorefinery industry. A novel glycoside hydrolase (GH) Family 8 reducing-end xylose-releasing exo-oligoxylanase (Rex)-encoding gene (PbRex8) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The deduced amino acid sequence of PbRex8 shared the hiGHest identity of 74% with a Rex from Bacillus halodurans. The recombinant enzyme (PbRex8) was purified and biochemically characterized. The optimal pH and temperature of PbRex8 were 5.5 and 55 °C, respectively. PbRex8 showed prominent activity on xylooligosaccharides (XOSs), and trace activity on xylan. It also exhibited β-1,3-1,4-glucanase and xylobiase activities. The enzyme efficiently converted corncob xylan to xylose coupled with a GH Family 10 endo-xylanase, with a xylose yield of 83%. The crystal structure of PbRex8 was resolved at 1.88 A. Structural comparison suggests that Arg67 can hydrogen-bond to xylose moieties in the -1 subsite, and Asn122 and Arg253 are close to xylose moieties in the -3 subsite, the hypotheses of which were further verified by mutation analysis. In addition, Trp205, Trp132, Tyr372, Tyr277 and Tyr369 in the grove of PbRex8 were found to involve in glucooligosaccharides interactions. This is the first report on a GH Family 8 Rex from P. barengoltzii. A novel reducing-end xylose-releasing exo-oligoxylanase suitable for xylose production from corncobs was identified, biochemically characterized and structurally elucidated. The properties of PbRex8 may make it an excellent candidate in biorefinery industries.
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Structural insiGHts into the catalytic mechanism of a novel glycoside hydrolase Family 113 beta-1,4-mannanase from Amphibacillus xylanus
The Journal of biological chemistry, 2018Co-Authors: Xin You, Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Li Yanxiao, Zhengqiang JiangAbstract:β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) Family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme–manno-oligosaccharides from this Family has been reported. Here, crystal structures of a GH Family 113 β-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had hiGHer affinity for −1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (−4 to +2) exist in the groove, but mannose units preferentially occupied subsites −4 to −1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH Family 113 β-1,4-mannanase–manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH Family 113 members, and provide novel insiGHt into protein engineering of GHs to improve their hydrolysis activity.
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A first glycoside hydrolase Family 50 endo-β-1,3-d-glucanase from Pseudomonas aeruginosa.
Enzyme and microbial technology, 2017Co-Authors: Qiaojuan Yan, Zhengqiang Jiang, Ling WangAbstract:A novel β-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the hiGHest identity of 34% with the β-agarase belonging to glycoside hydrolase (GH) Family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The Km and Vmax of PaBglu50A for curdlan and laminarin were 94.4mgml-1 and 23.4μmolmin-1mg-1, 3.65mgml-1 and 8.89μmolmin-1mg-1, respectively. All characterized members of GH Family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo-β-1,3-glucanase, making a significant difference from the reported enzymes of GH Family 50. These results suggested that PaBglu50A is the first endo-type β-1,3-glucanase (EC 3.2.1.39) in GH Family 50.
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A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase Family 3 β-N-acetylglucosaminidase.
Scientific reports, 2015Co-Authors: Zhen Qin, Shaoqing Yang, Yibei Xiao, Xinbin Yang, Jeroen R. Mesters, Zhengqiang JiangAbstract:Glycoside hydrolase (GH) Family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH Family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 A resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH Family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH Family 3 proteins.
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Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering
Applied microbiology and biotechnology, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Zhengqiang JiangAbstract:A glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH Family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited hiGH hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold hiGHer than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insiGHt into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.
Zhen Qin - One of the best experts on this subject based on the ideXlab platform.
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Structure-function analysis of Gynuella sunshinyii chitosanase uncovers the mechanism of substrate binding in GH Family 46 members.
International journal of biological macromolecules, 2020Co-Authors: Yani Wang, Zhen Qin, Liqiang Fan, Liming ZhaoAbstract:Chitooligosaccharides (COS) is a kind of functional carbohydrates with great application potential as its various biological functions in food, cosmetics, and pharmaceutical fields. Exploring the relationship between structure and function of chitosanase is essential for the controllable preparation of chitooligosaccharides with the specific degree of polymerization (DP). GsCsn46A is a cold-adapted glycosyl hydrolase (GH) Family 46 chitosanase with application potential for the controllable preparation of chitooligosaccharides. Here, we present two complex structures with substrate chitopentaose and chitotetraose of GsCsn46A, respectively. The overall structure of GsCsn46A contains nine α-helices and two β-strands that folds into two globular domains with the substrate between them. The unique binding positions of both chitopentaose and chitotetraose revealed two novel sugar residues in the negatively-numbered subsites of GH Family 46 chitosanases. The structure-function analysis of GsCsn46A uncovers the substrate binding and catalysis mechanism of GH Family 46 chitosanases. Structural basis mutagenesis in GsCsn46A indicated that altering interactions near +3 subsite would help produce hydrolysis products with hiGHer DP. Specifically, the mutant N21W of GsCsn46A nearly eliminated the ability of hydrolyzing chitotetraose after long-time degradation.
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efficient immobilization of bacterial GH Family 46 chitosanase by carbohydrate binding module fusion for the controllable preparation of chitooligosaccharides
Journal of Agricultural and Food Chemistry, 2019Co-Authors: Si Lin, Zhen Qin, Liqiang Fan, Qiming Chen, Jiachun Zhou, Liming ZhaoAbstract:Chitooligosaccharide has been reported to possess diverse bioactivities. The development of novel strategies for obtaining optimum degree of polymerization (DP) chitooligosaccharides has become increasingly important. In this study, two glycoside hydrolase Family 46 chitosanases were studied for immobilization on curdlan (insoluble β-1,3-glucan) using a novel carbohydrate binding module (CBM) Family 56 domain from a β-1,3-glucanase. The CBM56 domain provided a spontaneous and specific sorption of the fusion proteins onto a curdlan carrier, and two fusion enzymes showed increased enzyme stability in comparison with native enzymes. Furthermore, a continuous packed-bed reactor was constructed with chitosanase immobilized on a curdlan carrier to control the enzymatic hydrolysis of chitosan. Three chitooligosaccharide products with different molecular weiGHts were prepared in optimized reaction conditions. This study provides a novel CBM tag for the stabilization and immobilization of enzymes. The controllable hydrolysis strategy offers potential for the industrial-scale preparation of chitooligosaccharides with different desired DPs.
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Structural insiGHts into the catalytic mechanism of a novel glycoside hydrolase Family 113 beta-1,4-mannanase from Amphibacillus xylanus
The Journal of biological chemistry, 2018Co-Authors: Xin You, Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Li Yanxiao, Zhengqiang JiangAbstract:β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) Family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme–manno-oligosaccharides from this Family has been reported. Here, crystal structures of a GH Family 113 β-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had hiGHer affinity for −1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (−4 to +2) exist in the groove, but mannose units preferentially occupied subsites −4 to −1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH Family 113 β-1,4-mannanase–manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH Family 113 members, and provide novel insiGHt into protein engineering of GHs to improve their hydrolysis activity.
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A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase Family 3 β-N-acetylglucosaminidase.
Scientific reports, 2015Co-Authors: Zhen Qin, Shaoqing Yang, Yibei Xiao, Xinbin Yang, Jeroen R. Mesters, Zhengqiang JiangAbstract:Glycoside hydrolase (GH) Family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH Family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 A resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH Family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH Family 3 proteins.
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Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering
Applied microbiology and biotechnology, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Zhengqiang JiangAbstract:A glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH Family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited hiGH hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold hiGHer than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insiGHt into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.
Qiaojuan Yan - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of a novel exo-oligoxylanase from Paenibacillus barengoltzii suitable for monosaccharification from corncobs
Biotechnology for biofuels, 2019Co-Authors: Xueqiang Liu, Shaoqing Yang, Zhengqiang Jiang, Xin You, Yu Liu, Qiaojuan YanAbstract:Xylan is the major component of hemicelluloses, which are the second most abundant polysaccharides in nature, accounting for approximately one-third of all renewable organic carbon resources on earth. Efficient degradation of xylan is the prerequisite for biofuel production. Enzymatic degradation has been demonstrated to be more attractive due to low energy consumption and environmental friendliness, when compared with chemical degradation. Exo-xylanases, as a rate-limiting factor, play an important role in the xylose production. It is of great value to identify novel exo-xylanases for efficient bioconversion of xylan in biorefinery industry. A novel glycoside hydrolase (GH) Family 8 reducing-end xylose-releasing exo-oligoxylanase (Rex)-encoding gene (PbRex8) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The deduced amino acid sequence of PbRex8 shared the hiGHest identity of 74% with a Rex from Bacillus halodurans. The recombinant enzyme (PbRex8) was purified and biochemically characterized. The optimal pH and temperature of PbRex8 were 5.5 and 55 °C, respectively. PbRex8 showed prominent activity on xylooligosaccharides (XOSs), and trace activity on xylan. It also exhibited β-1,3-1,4-glucanase and xylobiase activities. The enzyme efficiently converted corncob xylan to xylose coupled with a GH Family 10 endo-xylanase, with a xylose yield of 83%. The crystal structure of PbRex8 was resolved at 1.88 A. Structural comparison suggests that Arg67 can hydrogen-bond to xylose moieties in the -1 subsite, and Asn122 and Arg253 are close to xylose moieties in the -3 subsite, the hypotheses of which were further verified by mutation analysis. In addition, Trp205, Trp132, Tyr372, Tyr277 and Tyr369 in the grove of PbRex8 were found to involve in glucooligosaccharides interactions. This is the first report on a GH Family 8 Rex from P. barengoltzii. A novel reducing-end xylose-releasing exo-oligoxylanase suitable for xylose production from corncobs was identified, biochemically characterized and structurally elucidated. The properties of PbRex8 may make it an excellent candidate in biorefinery industries.
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Structural insiGHts into the catalytic mechanism of a novel glycoside hydrolase Family 113 beta-1,4-mannanase from Amphibacillus xylanus
The Journal of biological chemistry, 2018Co-Authors: Xin You, Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Li Yanxiao, Zhengqiang JiangAbstract:β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) Family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme–manno-oligosaccharides from this Family has been reported. Here, crystal structures of a GH Family 113 β-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had hiGHer affinity for −1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (−4 to +2) exist in the groove, but mannose units preferentially occupied subsites −4 to −1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH Family 113 β-1,4-mannanase–manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH Family 113 members, and provide novel insiGHt into protein engineering of GHs to improve their hydrolysis activity.
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A first glycoside hydrolase Family 50 endo-β-1,3-d-glucanase from Pseudomonas aeruginosa.
Enzyme and microbial technology, 2017Co-Authors: Qiaojuan Yan, Zhengqiang Jiang, Ling WangAbstract:A novel β-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the hiGHest identity of 34% with the β-agarase belonging to glycoside hydrolase (GH) Family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The Km and Vmax of PaBglu50A for curdlan and laminarin were 94.4mgml-1 and 23.4μmolmin-1mg-1, 3.65mgml-1 and 8.89μmolmin-1mg-1, respectively. All characterized members of GH Family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo-β-1,3-glucanase, making a significant difference from the reported enzymes of GH Family 50. These results suggested that PaBglu50A is the first endo-type β-1,3-glucanase (EC 3.2.1.39) in GH Family 50.
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Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering
Applied microbiology and biotechnology, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Zhengqiang JiangAbstract:A glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH Family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited hiGH hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold hiGHer than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insiGHt into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.
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The first crystal structure of a glycoside hydrolase Family 17 β-1,3-glucanosyltransferase displays a unique catalytic cleft.
Acta crystallographica. Section D Biological crystallography, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Jian Lei, Shaoqing Yang, Zhengqiang JiangAbstract:β-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (β/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH Family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH Family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH Family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear β-1,3-glucan and the remaining glucan is transferred to the end of another β-1,3-glucan acceptor. The first crystal structure of a GH Family 17 β-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH Family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.
Shaoqing Yang - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of a novel exo-oligoxylanase from Paenibacillus barengoltzii suitable for monosaccharification from corncobs
Biotechnology for biofuels, 2019Co-Authors: Xueqiang Liu, Shaoqing Yang, Zhengqiang Jiang, Xin You, Yu Liu, Qiaojuan YanAbstract:Xylan is the major component of hemicelluloses, which are the second most abundant polysaccharides in nature, accounting for approximately one-third of all renewable organic carbon resources on earth. Efficient degradation of xylan is the prerequisite for biofuel production. Enzymatic degradation has been demonstrated to be more attractive due to low energy consumption and environmental friendliness, when compared with chemical degradation. Exo-xylanases, as a rate-limiting factor, play an important role in the xylose production. It is of great value to identify novel exo-xylanases for efficient bioconversion of xylan in biorefinery industry. A novel glycoside hydrolase (GH) Family 8 reducing-end xylose-releasing exo-oligoxylanase (Rex)-encoding gene (PbRex8) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The deduced amino acid sequence of PbRex8 shared the hiGHest identity of 74% with a Rex from Bacillus halodurans. The recombinant enzyme (PbRex8) was purified and biochemically characterized. The optimal pH and temperature of PbRex8 were 5.5 and 55 °C, respectively. PbRex8 showed prominent activity on xylooligosaccharides (XOSs), and trace activity on xylan. It also exhibited β-1,3-1,4-glucanase and xylobiase activities. The enzyme efficiently converted corncob xylan to xylose coupled with a GH Family 10 endo-xylanase, with a xylose yield of 83%. The crystal structure of PbRex8 was resolved at 1.88 A. Structural comparison suggests that Arg67 can hydrogen-bond to xylose moieties in the -1 subsite, and Asn122 and Arg253 are close to xylose moieties in the -3 subsite, the hypotheses of which were further verified by mutation analysis. In addition, Trp205, Trp132, Tyr372, Tyr277 and Tyr369 in the grove of PbRex8 were found to involve in glucooligosaccharides interactions. This is the first report on a GH Family 8 Rex from P. barengoltzii. A novel reducing-end xylose-releasing exo-oligoxylanase suitable for xylose production from corncobs was identified, biochemically characterized and structurally elucidated. The properties of PbRex8 may make it an excellent candidate in biorefinery industries.
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Structural insiGHts into the catalytic mechanism of a novel glycoside hydrolase Family 113 beta-1,4-mannanase from Amphibacillus xylanus
The Journal of biological chemistry, 2018Co-Authors: Xin You, Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Li Yanxiao, Zhengqiang JiangAbstract:β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) Family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme–manno-oligosaccharides from this Family has been reported. Here, crystal structures of a GH Family 113 β-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had hiGHer affinity for −1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (−4 to +2) exist in the groove, but mannose units preferentially occupied subsites −4 to −1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH Family 113 β-1,4-mannanase–manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH Family 113 members, and provide novel insiGHt into protein engineering of GHs to improve their hydrolysis activity.
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A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase Family 3 β-N-acetylglucosaminidase.
Scientific reports, 2015Co-Authors: Zhen Qin, Shaoqing Yang, Yibei Xiao, Xinbin Yang, Jeroen R. Mesters, Zhengqiang JiangAbstract:Glycoside hydrolase (GH) Family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH Family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 A resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH Family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH Family 3 proteins.
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Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering
Applied microbiology and biotechnology, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Zhengqiang JiangAbstract:A glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH Family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited hiGH hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold hiGHer than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insiGHt into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.
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The first crystal structure of a glycoside hydrolase Family 17 β-1,3-glucanosyltransferase displays a unique catalytic cleft.
Acta crystallographica. Section D Biological crystallography, 2015Co-Authors: Zhen Qin, Qiaojuan Yan, Jian Lei, Shaoqing Yang, Zhengqiang JiangAbstract:β-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) Family 17 β-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (β/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH Family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH Family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH Family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear β-1,3-glucan and the remaining glucan is transferred to the end of another β-1,3-glucan acceptor. The first crystal structure of a GH Family 17 β-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH Family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.
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Characterization and diversity of the complete set of GH Family 3 enzymes from Rhodothermus marinus DSM 4253.
Scientific reports, 2020Co-Authors: Kazi Zubaida Gulshan Ara, Anna Månberger, Marek Gabriško, Javier A. Linares-pastén, Andrius Jasilionis, Ólafur H. Friðjónsson, Guðmundur Óli Hreggviðsson, Štefan Janeček, Eva Nordberg KarlssonAbstract:The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH Family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.