�-Xylosidases (EC 3.2.1.37) are glycosyl hydrolases with an important role in xylan biodegradation, liberating xylose from the nonreducing ends of xylobiose and xylooligo-saccharides. The fungus Penicillium purpurogenum secretes a wide variety of xylanolytic enzymes to the medium, including two with �-D-xylosidase activity. This work focuses on a biochemical and structural analysis of these enzymes: 1) arabinofuranosidase 3, a bifunctional a-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-a-L-arabinofuranoside and p-nitrophenyl-�-D-xylopyranoside with a KM of 0.65 mM and 12 mM, respectively, and 2) �-D-xylosidase 1. The �-D-xylosidase 1 gene was expressed in Pichia pastoris GS115 including a polyhistidine tag. The recombinant enzyme was purified to homogeneity with a Ni-NTA column and characterized; it was active on p-nitrophenyl-�-D-xylopyranoside with a KM of 0.55 mM, but showed no activity with p-nitrophenyl-a-L-arabinofuranoside. Based on sequence similarities both can be classified in family 43 of the glycosyl hydrolases. Structural models for each protein were built using homology modeling tools, based on the crystal structure of family 43 arabinoxylan arabinofurano hydrolase from Bacillus subtilis (PDB:3C7E). The conformational interactions between the proteins and both ligands were explored by molecular docking and molecular dynamics simulations underpinning the atomic interactions involved. Structural analysis corroborates that the most important residues in the interactions between the proteins and ligands are Asp-15, Asp-135 and Glu-240 for �-D-xylosidase 1 and Asp-12, Asp-127 and Glu-187 for arabinofuranosidase 3. Docking results show no significant affinity energy differences for the binding of both substrates to arabinofuranosidase 3, consistent with the enzyme bifunctionality. For �-D-xylosidase 1, a significant difference (4 Kcal/mole) in affinity energy was found for both substrates, being lower for p-nitrophenyl-�-D-xylopyranoside, which exhibits higher affinity as expected from the biochemical data. Grant support: FONDECYT 1100084, 1130180 (J.E) and 1131003 (FGN); Universidad Andr�s Bello (DI 03�10/R and 61-12/R). FONDECYT Post-Doctoral Fellowship (3120032, C.R.).

Two glycosyl hydrolases from family 43 with �-xylosidase activity differ in substrate specificity: Biochemical and structural analysis of these differences

246th National Meeting (2013)

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