Human 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen‐dependent diseases (EDDs). Unlike other 17β‐HSD members, 17β‐HSD1 undergoes a significant substrate‐induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17β‐HSD1 in complex with estrone (E1) and cofactor analog NADP⁺, demonstrating critical enzyme‐substrate‐cofactor interactions. These complexes revealed a reversely bound E1 in 17β‐HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His²²¹ is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead‐end complex, which exists widely in NADP(H)‐preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17β‐HSD1 through the formation of a dead‐end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17β‐HSD1 based on the dead‐end inhibition model for efficiently targeting EDDs.Databases: Coordinates and structure factors of 17β‐HSD1‐E1 and 17β‐HSD1‐E1‐NADP⁺ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively.Enzymes: 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) EC 1.1.1.62.

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.