Small angle X-ray scattering and based structure and function analysis of a novel xylobiohydrolase (GH30A) from .

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, GH30A, from . Secondary structure analysis of GH30A in an aqueous environment using Circular Dichroism and modeling revealed an α/β/α sandwich structure with a central β-barrel comprising eight β-strands. Superposition of the homology-modelled structure of GH30A 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. GH30A displayed a high binding affinity with xylobiose with an association constant () of 7.83 × 10 M, as determined by isothermal titration calorimetry. Molecular dynamics (MD) simulations of GH30A and GH30A-xylobiose complex in solution showed reduced RMSD, 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 GH30A 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 () of GH30A, determined by DLS, was 3.7 nm. This study significantly contributed valuable insights into the structure and functional aspects of GH30A.