Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared to other members of the same family

Arabinoxylan arabinofuranohydrolases (AXH) are α-L-arabinofuranosidases that specifically hydrolyze the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues from arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-monosubstituted xylose residues: BsAXH-m2,3. Crystallographic analysis reveals a two domain structure for this enzyme: a catalytic domain displaying a 5-bladed β-propeller fold characteristic to glycoside hydrolase family (GH) 43 and a carbohydrate binding module (CBM) with a β-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions which probably allow the positional flexibility needed to hydrolyze both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, β-xylosidase and α-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.