Conformational gating of an active-site loop governs phenolic and aromatic amine substrate selectivity in fungal...

Laccases oxidize a broad range of aromatic compounds, yet how conformational dynamics regulate substrate recognition and functional specificity remains incompletely understood. Here, we investigate the molecular determinants of substrate-class selectivity in a fungal laccase by combining long-timescale molecular dynamics simulations, Markov state modeling, transition path analysis, free-energy calculations, and isothermal titration calorimetry. Using representative phenolic and aromatic amine substrates, we show that access to the T1 Cu center is governed by a flexible active-site loop that differentially modulates binding pathways. Phenolic ligands require opening of this loop to achieve sterically accessible binding configurations and proceed through multiple metastable intermediates consistent with a coupled conformational selection-induced-fit mechanism. In contrast, aromatic amines access the active site without loop rearrangement through a pathway dominated by conformational selection. In silico rigidification of the loop selectively disrupts phenolic binding while preserving amine binding, supporting a mechanistic bifurcation encoded by protein dynamics. Protonation states further modulate binding energetics, consistent with isothermal titration calorimetry measurements showing differential affinities among phenolic isomers under acidic conditions. Together, these findings establish conformational gating as a structural mechanism that links protein dynamics to substrate-class-dependent recognition in laccase and provide molecular insight into how flexible active-site elements regulate functional specificity in oxidoreductases.