Competitive inhibition of family GH11 Aspergillus fumigatus endo-xylanase A by Oryza sativa xylanase inhibitor protein: Investigating key interface residues and non-covalent interactions

•Beechwood xylan hydrolysates released by reAfXylA11 exhibited significant antioxidant activity.•Interaction between reAfXylA11 and rePOsXIP resulted in static fluorescence quenching.•rePOsXIP competitively inhibited reAfXylA11 with a ki value of 120.98 nM.•Residues R153 in OsXIP and E197 in AfXylA11 were critical for their non-covalent interactions evidenced by MD simulations. Glycoside hydrolase (GH) family 11 Aspergillus fumigatus endo-xylanase A (AfXylA11) was recombinantly expressed in Pichia pastoris X33 and secreted into culture medium. Hydrolysis of beechwood xylan by recombinant AfXylA11 (reAfXylA11) predominantly generated xylooligosaccharides (XOs), chiefly comprising xylotriose (X2) and xylotetraose (X3). These hydrolysates demonstrated significant antioxidant capabilities. Competitive inhibition of reAfXylA11 was observed in the presence of the Oryza sativa xylanase inhibitor protein (OsXIP), with an inhibition constant (Ki) of 120.98 nM. Molecular dynamics (MD) simulations and conformational analyses of the AfXylA11-OsXIP complex revealed that the extended loop (Lα4β5, K150-V158) of OsXIP penetrated the active groove of AfXylA11, thereby obstructing xylan from interacting with catalytic sites. The R153 at the bottom of the U-shaped loop, was found to form durable hydrogen bonds with E197 (acid/base catalyst) and Y97 of AfXylA11. Additionally, OsXIP established stable interactions with the “cord” and “thumb” regions of AfXylA11 during MD simulations. The binding free energy of the AfXylA11-OsXIP complex was determined to be -74.3 ± 7.6 kcal/mol, with electrostatic interactions and van der Waals forces being the predominant contributors to enzyme-inhibitor interaction. Further MD simulations demonstrated that the mutation of Y116A or S120A in the “cord” region of AfXylA11 significantly reduced its binding affinity to OsXIP by weakening interface residue interactions. Delving into the conformational dynamics of the AfXylA11-OsXIP complex yields insights into the molecular mechanisms dictating their competitive inhibition. This investigation offers useful basis for engineering mutant xylanases that resist to the inhibitory protein yet retain high catalytic efficacy. [Display omitted]