Abstract
β-
d-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. β-
d-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 β-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (β/α)
8 barrel fold. Here, we report the crystal structure of the GH39
Geobacillus stearothermophilus β-
d-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-β-
d-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.