Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption
Wa Gao, Tang Li, Haichuan Zhou, Jiu Ju, Heng Yin
Abstract
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 Mt LPMO9L and Mt LPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H 2 O 2 tolerance. Using truncated forms of Mt LPMO9G 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 Mt LPMO9L to create Mt LPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H 2 O 2 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 H 2 O 2 . Importantly, the fusion of CBM resulted in more efficient consumption of H 2 O 2 by LPMO, leading to improved enzymatic activity and reduced autoxidative damage of the copper active center.