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A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

ABSTRACT: Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the ?-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark ?8-?8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved ?8-?8 loop region of these enzymes influences xylan substrate specificity but not necessarily ?-1,4-xylanase function.

SUBMITTER: St John FJ

PROVIDER: S-EPMC4722856 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

St John Franz J FJ Dietrich Diane D Crooks Casey C Pozharski Edwin E González Javier M JM Bales Elizabeth E Smith Kennon K Hurlbert Jason C JC

Acta crystallographica. Section D, Biological crystallography 20141023 Pt 11

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the stru ...[more]

PMID: 25372685

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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.

Dataset Information

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

Publications

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

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