Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein

Abstract

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn²⁺ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (K_i) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was -30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239_OsXIP stably interacted with the catalytic site E140_AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104_AfXylA10/V99_AfXylA10-Q5_OsXIP, R256_AfXylA10-E235_OsXIP, D155_AfXylA10-Y243_OsXIP and D145_AfXylA10-R194_OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.

Keywords: Aspergillus fumigatus xylanase A (AfXylA10); Competitive inhibition; Molecular dynamics (MD) simulations.