Unveiling the structural and functional perspectives of a bifunctional α-l-arabinofuranosidase/endo-β-1,4-xylanase (BoGH43_35) from Bacteroides ovatus

Abstract

Arabinoxylan, a complex hemicellulose, can be degraded to its constituent sugars by concerted action of hemicellulases like α-l-arabinofuranosidase, endo-β-1,4-xylanase and xylosidase. In this study, a novel bifunctional α-l-arabinofuranosidase/endo-β-1,4-xylanase (BoGH43_35) of glycoside hydrolase family 43 subfamily 35 from Bacteroides ovatus was characterized by computational and experimental approaches. Sequence analysis identified Asp34 and Glu251 as the conserved catalytic residues. Structure analysis of BoGH43_35 disclosed 5-bladed β-propeller fold adopted by the N-terminal GH43 catalytic module followed by two independently folded carbohydrate-binding modules family 6 (CBM6A and CBM6B), displaying jellyroll type β-sandwich fold. Molecular Dynamics simulation of BoGH43_35 for 200 ns showed RMSD 0.35 nm, confirming structural stability and compactness of modeled structure. Molecular docking of BoGH43_35 with arabino-xylooligosaccharides and xylooligosaccharides by using AutoDock 4.2.7 demonstrated most favourable binding with arabinose (−5.01 kcal/mol) followed by arabinoxylobiose (−4.35 kcal/mol), xylotriose (−4.65 kcal/mol), xylotetraose (−4.18 kcal/mol) and xylobiose (−3.66 kcal/mol) showing affinity with both types of oligosaccharides. RMSD value of BoGH43_35-arabinose complex decreased to 0.28 nm upon MD simulation from 0.35 nm for only BoGH43_35, indicating stability of enzyme-substrate complex throughout the trajectory. The binding analysis of BoGH43_35 with wheat arabinoxylan by fluorescence spectroscopy gave K_a, 3.1 × 10² M⁻¹, ΔG -14.2 kJ mole⁻¹ and number of binding sites 2.2. Dynamic light scattering of BoGH43_35 showed hydrodynamic radius (R_h) of 4.0 nm, slightly higher than the radius of gyration (2.69 nm) from MD simulation. Additionally, zeta potential measurements (−9.3 mV at 0.5 mg/mL and −9.4 mV at 1.0 mg/mL) denoted its fair resistance towards aggregation in aqueous solution.