Crystal Structures of a Thermophilic Cutinase from Chaetomium thermophilum Reveal Conformational Dynamics of the Catalytic Lid Loop

Microbial cutinases are promising biocatalysts for polymer recycling. Here, we investigated the structural basis of catalytic activation in a thermophilic cutinase from Chaetomium thermophilum (CtCut). Differential scanning calorimetry revealed a three-state thermal unfolding pathway (Tm = 66.4 °C and 69.5 °C), indicating hierarchical stability. To capture distinct conformational states while avoiding affinity-tag artifacts, we employed both tag-free and tagged constructs. We determined apo-structures of wild-type and S136A mutant CtCut at 1.7 Å resolution and a complementary inhibitor complex at 2.65 Å. In the apo-state, a chloride ion coordinated the electrostatically pre-organized active site, while the catalytic H204 adopted a solvent-exposed, inactive loop conformation. In the inhibitor complex, p-nitrophenol displaced the chloride, establishing a characteristic oxyanion hole network. Concomitantly, the “lid” loop transitioned to an open state, with H204 exhibiting pronounced conformational heterogeneity across eight independent molecules. These complementary structures provide structural evidence for conformational dynamics of the catalytic lid loop, consistent with the conformational cycling model previously proposed for a mesophilic homolog.