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Crystal structure of ?-L-arabinobiosidase belonging to glycoside hydrolase family 121.

ABSTRACT: Enzymes acting on ?-L-arabinofuranosides have been extensively studied; however, the structures and functions of ?-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of ?-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular ?-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing ?-1,2-linked arabinofuranose disaccharide (?-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (?/?)6 barrel domain and two flanking (N- and C-terminal) ?-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the ?-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 ?-glycosidase, GH94 chitobiose phosphorylase, GH142 ?-L-arabinofuranosidase, GH78 ?-L-rhamnosidase, and GH37 ?,?-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. ?-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the ?-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

SUBMITTER: Saito K

PROVIDER: S-EPMC7263609 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121.

Saito Keita K Viborg Alexander Holm AH Sakamoto Shiho S Arakawa Takatoshi T Yamada Chihaya C Fujita Kiyotaka K Fushinobu Shinya S

PloS one 20200601 6

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degr ...[more]

PMID: 32479540

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Project description:Backgroundβ-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects.ResultsA β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively.ConclusionsA new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Dataset Information

Crystal structure of ?-L-arabinobiosidase belonging to glycoside hydrolase family 121.

Publications

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121.

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