Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations

Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics/molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases, LCC and PES-H1, along with their highly active variants, LCC^IG and PES-H1^FY. Our findings highlight the impact of semiempirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the “wobbling” tryptophan from T-stacking to parallel π–π interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1^FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes.