Sensitivity of family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the T33I mutant to Oryza sativa xylanase inhibitor protein (OsXIP): An experimental and computational study.

Glycoside hydrolase (GH) family 10 and 11 xylanases are inhibited by many xylanase inhibitor proteins (XIPs). We recombinantly expressed the Oryza sativa xylanase inhibitor protein (OsXIP) in Pichia pastoris GS115, with a molecular mass of 47.0 kDa. Family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the mutant T33I (DS199) were inhibited by the recombinant OsXIP (rePOsXIP) through competive inhibition, with corresponding inhibition constants (K) of 54.09 and 12.16 nM. After incubation with rePOsXIP (70 nM) at 40 °C for 40 min, inhibitory rates of reBaxA and DS199 (0.2 U) were 23.7% and 76.7%, respectively. Xylooligosaccharides with low concentration were released from beechwood xylan by reBaxA and DS199 in the presence of reOsXIP. Intrinsic fluorescences of reBaxA and DS199 were statically quenched by rePOsXIP in a concentration-dependent manner. Molecular dynamics (MD) simulations and conformational analysis of OsXIP-BaxA and OsXIP-DS199 revealed that the long loop (Lαβ) of OsXIP inserted into the catalytic grooves of BaxA and DS199. The DS199 enhanced the binding affinity to OsXIP, causing conformational alterations on protein-protein interface residues, thereby forming more hydrogen bonds and van der Waals forces. MM/GBSA analysis revealed that the binding free energy (∆G) of OsXIP-DS199 was enhanced by 2.08 kcal/mol compared to that of OsXIP-BaxA. The OsXIP binding induced a conformational changes among residues in the cord and thumb regions of BaxA and DS199. In particular, the TRYNAP residues in the thumb region of DS199 was maintained close to OsXIP by specific bonds. Additional MD simulations revealed that Y113A or T93A mutation of BaxA suppressed the binding affinity by diminishing interface associations of OsXIP-BaxA. This study partially elucidats the molecular basis of inhibitory mechanism and structure-function relationships of GH11 xylanases. Our findings inform rational designs of mutant xylanases with higher resistance to inhibitor proteins.