Penicillium purpurogenum produces two GH family 43 enzymes with ß-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.

ß-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with ß-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-ß-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, ß-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3-pNPXyl, ABF3-pNPAra and XYL1-pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1-pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.