Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption.

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center.