Small angle X-ray scattering and in silico based structure and function analysis of a novel xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus

Xylobiohydrolase plays a crucial role in the hydrolysis of xylan, a complex polysaccharide present in the cell walls of plants. This study focuses on the solution structure and substrate binding analysis of a novel xylobiohydrolase, AcGH30A, from Acetivibrio clariflavus. Secondary structure analysis of AcGH30A in an aqueous environment using Circular Dichroism and in silico modeling revealed an alpha/beta/alpha sandwich structure with a central beta-barrel comprising eight beta-strands. Superposition of the homology-modelled structure of AcGH30A with its closest homolog showed that the active-site contains Glu175 and Glu268 as the catalytic residues. Molecular docking confirmed xylobiose as the preferred ligand, showcasing polar interactions with the catalytic amino acids, indicating its xylobiohydrolase activity. AcGH30A displayed a high binding affinity with xylobiose with an association constant (Ka) of 7.83 x 105 M-1, as determined by isothermal titration calorimetry. Molecular dynamics (MD) simulations of AcGH30A and AcGH30A-xylobiose complex in solution showed reduced RMSD, Rg and SASA values, confirming the stability and compactness of the complex. MD simulations further highlighted the crucial role of Glu175 in hydrogen bonding with the ligand, which acts as an acid or base. Small-angle X-ray scattering (SAXS) analysis of AcGH30A showed its molecular shape as an earbud with a globular structure existing in a monodispersed state, which was corroborated by dynamic light scattering (DLS). The hydrodynamic radius (Rh) of AcGH30A, determined by DLS, was 3.7 nm. This study significantly contributed valuable insights into the structure and functional aspects of AcGH30A.