Differential inhibition of GH family 11 endo-xylanase by rice xylanase inhibitor and verification by a modified yeast two-hybrid system.

Rice xylanase inhibitor (RIXI) is a XIP-type xylanase inhibitor protein that protects rice cells from pathogenic organisms. RIXI inhibits most microbial xylanases and thus decreases their practical application. The recombinant RIXI (rePRIXI) showed evident inhibitory activities against several family 11 endo-xylanases. After interaction with rePRIXI at 50 °C for 40 min, the residual activities of reBaxA50, reBaxA, TfxA_CD214, and TfxA_CD were 55.6%, 30.3%, 30.09%, and 11.20%, respectively. Intrinsic fluorescence of reBaxA50 and TfxA_CD214 was statically quenched after interaction with rePRIXI. rePRIXI decreased hydrolysis of beechwood xylan by reBaxA50 and TfxA_CD214. Molecular dynamics simulations revealed the long loop (residues 144-153) of RIXI inserts into the catalytic cleft of family 11 xylanases. Native PAGE results revealed the formation of RIXI-xylanase complex after their interaction in the test tube. Interactions were also observed between RIXI and xylanases in living yeast cells. The results of inhibitory activity assay and modified yeast two-hybrid revealed that the inhibitory activity of RIXI on family 11 xylanase improved with the interaction strength of the RIXI-xylanase complex, indicating their positive correlation. The modified yeast two-hybrid system is relatively simple and has low cost, and its use may be extended to other studies on protein-protein interactions.