Structural insights into the catalytic mechanism of SsChiA from Sporisorium scitamineum.

Abstract

Sugarcane smut, caused by the biotrophic fungus Sporisorium scitamineum, represents a devastating global threat to sugarcane production. During fungal infection, secreted chitinases are involved not only in cell wall remodeling and nutrient acquisition, but also in the suppression of host immune recognition, making chitinases attractive targets for antifungal drug development. Despite their importance, the catalytic and inhibitory mechanisms underlying chitinases from S. scitamineum remain poorly understood. Here, we report the crystal structure of the S. scitamineum chitinase SsChiA at a resolution of 1.84 Å. SsChiA adopts a canonical (β/α)8 TIM-barrel catalytic domain coupled to a chitinase insertion domain (CID) characteristic of CID-containing GH18 chitinases, together forming a pronounced substrate-binding groove. Structural analysis revealed that the N-terminal His-tag peptide of a symmetry-related molecule specifically bound within this groove, which might suggest a competitive inhibitory mechanism mediated by occupancy of the catalytic site. Site-directed mutagenesis confirmed that Asp286 and Glu288 within the conserved DxDxE motif are important for enzymatic activity. Combining structural analysis with molecular docking of an SsChiA-chitinopentaose complex model, we propose that SsChiA employs the conserved substrate-assisted retention mechanism characteristic of the GH18 family. Furthermore, on the basis of the asymmetric distribution of aromatic residues in the active center and structural comparisons with known exochitinases, we hypothesize that SsChiA functions as a nonreducing-end exochitinase. This study provides the first mechanistic characterization of the S. scitamineum chitinase SsChiA, establishing a structural framework for the rational design of environmentally friendly fungicides targeting this enzyme.