Dataset Information

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 ?-1,4-mannanase from Amphibacillus xylanus.

ABSTRACT: ?-1,4-Mannanase degrades ?-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 ?-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 ?-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 ?-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.

SUBMITTER: You X

PROVIDER: S-EPMC6066303 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus.

You Xin X Qin Zhen Z Yan Qiaojuan Q Yang Shaoqing S Li Yanxiao Y Jiang Zhengqiang Z

The Journal of biological chemistry 20180605 30

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 comp ...[more]

PMID: 29871927

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Project description:As classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identified Arcticibacterium luteifluviistationis carboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase. AlCMCase originated from an Arctic marine bacterium, Arcticibacterium luteifluviistationis SM1504T It shows low identity (<35%) with other GH10 xylanases. The gene encoding AlCMCase was overexpressed in Escherichia coli Biochemical characterization showed that recombinant AlCMCase is a cold-adapted and salt-tolerant enzyme. AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity of AlCMCase is negligible. Correspondingly, AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides. AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity as AlCMCase. These results suggest that AlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters of AlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCE Cellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase, AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover, AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.

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Dataset Information

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 ?-1,4-mannanase from Amphibacillus xylanus.

Publications

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from <i>Amphibacillus xylanus</i>.

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