Conformational Diversity and Allosteric Network Enable Multi-Substrate Recognition in Laccase Enzyme

Laccase is a multicopper oxidase with remarkable potential for environmental remediation due to its ability to degrade a wide range of pollutants. However, the molecular origins of its broad substrate scope have remained elusive. Here, through extensive molecular dynamics (MD) simulations coupled with machine learning (ML) based Markov State Model (MSM), we uncover a previously uncharacterized allosteric pathway that governs laccase's catalytic activity. This pathway functionally couples the dynamics of two key loops at the active site, and its integrity is crucial for enzymatic efficiency. Our analysis of the apo enzyme's conformational free energy landscape reveals that this allostery is part of a cooperative global network, leading to distinct, slowly interconverting conformational states. We demonstrate that different dye substrates bind by selectively recognising these pre-existing states, confirming a conformational selection mechanism. Collectively, these findings provide a dynamic blueprint for laccase's functional versatility and offer a general framework for understanding and engineering other multi-substrate enzymes.