Deciphering the structural and biochemical aspects of xylosidase from Pseudopedobacter saltans

Xylose, a key constituent of the heterogeneous hemicellulose polymer, occurs in lignocellulosic biomass and forms xylan polymers through β-1,4 glycosidic linkages. The β-1,4-xylosidase enzyme was isolated from Pseudopedobacter saltans (PsGH43) to find an effective enzyme with enhanced activity to depolymerize xylo-oligosaccharides. β-1,4-xylosidase belongs to the GH43¹ family as classified in the Carbohydrate-Active Enzyme Database (CAZy). PsGH43² was found to be active only on xylose-based substrate, 4NPX³, with maximum activity occurring at a pH 7 and 30 °C (K_m 1.96 ± 0.2 mM and V_max 0.43 mM/min). The study also confirms the influence of Ca²⁺ ions on enzymatic activity and thermal stability. Subsequently, native PsGH43 was crystallized at optimum conditions and the structure was determined at 2.5 Å resolution. Crystallographic analysis revealed an asymmetric unit containing eight monomers and 16 calcium ions wherein a tetramer constituted the functional unit. Each monomer exhibits a characteristic GH43 N-terminal β-propeller fold that serves as a catalytic domain accommodating one calcium ion in the centre, while the C-terminal β-sandwich fold associated with the CBM6⁴ family preserves another calcium ion. Our study reveals a novel tetrameric arrangement of β-1,4-xylosidase which unravels its functional indispensability. This study opens newer avenues to engineer a potential enzyme for biofuel and bioethanol industry.