Site-directed mutagenesis of family GH10 Aspergillus fumigatus xylanase A and the interaction with Oryza sativa xylanase inhibitor protein

Abstract

In this study three alanine mutants (V99A, D145A and V99A/D145A) of family GH10 Aspergillus fumigatus xylanase A (AfXylA10) were engineered and characterized. These variants exhibited molecular mass of approximately 52.0 kDa and optimum temperature at 53 °C. The optimum pH of V99A/D145A was pH 4.0, and that of V99A and D145A was 5.0. Three variants were competitively inhibited by Oryza sativa xylanase inhibitor protein, rePOsXIP in a typical dose-dependent manner, with Ki of 214.8, 170.3 and 126.2 nM, respectively. Mutant V99A showed lower sensitivity to rePOsXIP and lower activity compared to reAfXylA10. However, D145A obtained higher activity and similar sensitivity to rePOsXIP. The underlying interaction mechanism was delved by molecular dynamics (MD) simulations and computational analysis. Binding free energy of V99A-OsXIP, D145A-OsXIP and V99A/D145A-OsXIP systems, calculated using the MM/PBSA method, were −19.7 ± 9.4, −26.9 ± 6.0, and −45.4 ± 9.8 kcal/mol, respectively. Representational snapshot of these systems from MD simulations showed that the α7-helix of OsXIP entered the active tunnel of mutant xylanases, which would hinder xylan from entering the catalytic groove. Particularly, residue K239 in OsXIP's α7-helix formed stable hydrogen bonding with the acid-base catalytic site (E140) of these mutants. Laplacian of electron density values of E140xylanase- K239OsXIP in V99A-OsXIP, D145A-OsXIP, and V99A/D145A-OsXIP systems were 0.07545, 0.09155 and 0.09764 au., respectively. The study provided preliminary insight into how V99AfXylA10 and D145AfXylA10 residue influence enzymatic properties and the interaction with OsXIP. These findings might inform design of mutant xylanase with enhanced catalytic activity and reduced sensitivity to xylanase inhibitory proteins (XIs).