Competitive Inhibition of Bacillus amyloliquefaciens Pectate Lyase B by Oryza sativa Xylanase Inhibitor Protein Explored through Experimental and Molecular Simulation Studies

Pectate lyases facilitate pathogen invasion by degrading pectic polysaccharides in the plant cell wall. Although xylanase inhibitor proteins (XIPs) are recognized as plant defense proteins against xylanases, their ability to inhibit pectate lyases remained unknown. Here, recombinant Bacillus amyloliquefaciens pectate lyase B (rePelB) displayed optimal activity at 60 °C and pH 5.0 and was enhanced more than 2-fold by 5 mM Ca2+. Recombinant Oryza sativa XIP (reOsXIP) competitively inhibited rePelB with a Ki of 98.68 nM. Fluorescence spectroscopy indicated static quenching upon rePelB-reOsXIP interaction. Molecular dynamics simulations revealed that the Lα4β5 loop of OsXIP inserts into the PelB catalytic groove, with a calculated binding free energy of -87.6 ± 7.4 kcal/mol. Alanine substitutions of PelB residues K99, S337, and V338 weakened binding and reduced inhibition by reOsXIP. These results provide the first evidence that XIP competitively inhibits a pectate lyase, highlighting a broader defensive role against plant cell wall-degrading enzymes.