High-speed atomic force microscopy reveals opposite traffic of processive chitinases impairs α-chitin biodegradation.

The antiparallelly organized α-chitin exhibits greater thermodynamic stability and is more recalcitrant to degradation than its parallel allomorph, β-chitin, thereby impeding the efficient utilization of this natural resource. The processive chitinases usually provide the majority of catalytic potential for chitin biodegradation. Using high-speed atomic force microscopy (HS-AFM), we revealed that the opposite traffic of OfChi-h, the only processive chitinase involved in chitin biodegradation in the insect Ostrinia furnacalis, is a key factor that significantly affects α-chitin degradation. Compared to β-chitin as a substrate, OfChi-h exhibited a much lower catalytic turnover and significantly reduced binding affinity to α-chitin. Our HS-AFM data suggested that, at the single-molecule level, OfChi-h molecules move in opposite directions along α-chitin fibers. Some OfChi-h molecules encounter others in a head-to-head manner, leading to their detachment from the substrate, which in turn impairs catalytic activity and processivity. Our further investigations demonstrated that the insect's strategy to avoid opposite traffic is through a synergistic interaction between OfChi-h and two non-processive endo-acting chitinases. This work reveals the mechanism by which a processive chitinase is impaired by the antiparallel organization of chitin fibers and highlights the synergism between processive and non-processive endo-acting chitinases in the degradation of recalcitrant α-chitin.