Mechanistic Insights into Substrate Selectivity and Binding Routes to Laccase for Efficient Wastewater Treatment

Water pollution caused by phenolic contaminants remains a significant challenge to environmental and public health, demanding the need for sustainable solutions. Laccase, a multicopper oxidase from the white-rot fungus (Trametes versicolor), has emerged as a promising green biocatalyst for detecting and degrading catechol, a hazardous phenolic pollutant. Although previous studies have confirmed the successful binding of catechol to the active site of laccase, the detailed dynamics of the interaction, including the conformational changes, identification of metastable states, and free energy barriers, remain unexplored. In this study, we deciphered the complete binding mechanism of catechol to laccase using a combination of Molecular Dynamics simulations, Markov state modeling, and Transition Path Theory (TPT). Our approach identified five macrostates, offering atomic-level insights into the structural and energetic landscape of the laccase-catechol binding. Critical transition states and intermediates were characterized, emphasizing the role of the active site loop (A161-F162-P163-L164). Building on these mechanistic insights, our ongoing work expands the scope to ten ligands with hydroxyl, amino, and methyl substituents, aiming to uncover the molecular basis of substrate selectivity in laccase. Each substrate sampled unbound to bound conformations, with intermediate/metastable states classified into nine macrostates, providing a unified framework reflecting binding preferences to substrate size and functional groups. Furthermore, potential routes identified from TPT analysis were consolidated into four pathways leading to the active site. These findings enable rational design of laccase-based solutions for sustainable wastewater treatment.