Structural basis for inhibition of TcrXyn10A xylanase by nonspecific xylose binding at subsite − 2

Xylanases play a critical role in the degradation of lignocellulose-based biomass for the production of biofuels, value-added chemicals, and various industrial applications. Xylanase activity is often inhibited by reaction products such as xylose, which reduces the efficiency of industrial applications involving xylan degradation. Xylanase TcrXyn10A is an industrially promising enzyme that efficiently hydrolyzes agro-food byproducts, but its catalytic activity decreases in the presence of xylose, and the mechanism underlying this inhibition remains unclear. To understand the product inhibition mechanism, the crystal structure and molecular dynamics simulations of xylose-bound TcrXyn10A were analyzed. The xylose molecule binds to the − 2 subsite within the substrate-binding cleft of one of the two molecules in the asymmetric unit, indicating that xylose acts as a competitive inhibitor with low binding affinity. The bound xylose is stabilized by hydrogen bonds and a π–π interaction with five conserved residues located at the subsite − 2. Xylose binding induces subtle movements of interacting residues, resulting in a narrowing of the substrate-binding cleft around subsites − 2 and − 1. Structural comparison with other xylobiose-bound GH10 xylanases revealed that the binding mode of xylose at the − 2 subsite differed depending on the number of xylose units occupying the binding cleft. These findings provide insights into the molecular basis of xylose-induced inhibition in TcrXyn10A and contribute to a broader understanding of product inhibition mechanisms across the GH10 family.