Water pollution caused by persistent 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, owing to its strong binding affinity. Although previous experimental studies and state-of-the-art computational techniques have confirmed the successful binding of catechol to the active site of laccase, the detailed dynamics of the interaction, including the conformational changes the enzyme undergoes, rearrangements of active site residues, identification of metastable and binding-competent states, and the associated free energy barriers, remain largely unexplored. In this study, we deciphered the complete binding mechanism of catechol to laccase using a combination of molecular dynamics (MD) simulations, free-energy calculations, Markov state modeling (MSM), and transition path theory (TPT). Our approach identified five distinct macrostates, offering atomic-level insights into the structural and energetic landscape of the laccase-catechol interaction. Critical transition states and intermediates were characterized, emphasizing the role of the active site loop (A161-F162-P163-L164) and a gate mechanism involving neighboring residues. Building on these mechanistic insights, our ongoing work expands the scope to ten aromatic ligands with hydroxyl, amino, and methyl substituents. This broader analysis aims 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 major pathways leading to the active site through sequential and cooperative conformational changes. These findings facilitate the rational engineering of laccase-based biosensors and bioreactors for rapid, sustainable, and efficient wastewater bioremediation.
Anushka Biswas (Sun,) studied this question.