Influence of Wobbling\nTryptophan and Mutations on\nPET Degradation Explored by QM/MM Free Energy Calculations

Plastic-degrading enzymes, particularly poly(ethylene\nterephthalate)\n(PET) hydrolases, have garnered significant attention in recent years\nas potential eco-friendly solutions for recycling plastic waste. However,\nunderstanding of their PET-degrading activity and influencing factors\nremains incomplete, impeding the development of uniform approaches\nfor enhancing PET hydrolases for industrial applications. A key aspect\nof PET hydrolase engineering is optimizing the PET-hydrolysis reaction\nby lowering the associated free energy barrier. However, inconsistent\nfindings have complicated these efforts. Therefore, our goal is to\nelucidate various aspects of enzymatic PET degradation by means of\nquantum mechanics/molecular mechanics (QM/MM) reaction simulations\nand analysis, focusing on the initial reaction step, acylation, in\ntwo thermophilic PET hydrolases, LCC and PES-H1, along with their\nhighly active variants, LCC<sup>IG</sup> and PES-H1<sup>FY</sup>.\nOur findings highlight the impact of semiempirical QM methods on proton\ntransfer energies, affecting the distinction between a two-step reaction\ninvolving a metastable tetrahedral intermediate and a one-step reaction.\nMoreover, we uncovered a concerted conformational change involving\nthe orientation of the PET benzene ring, altering its interaction\nwith the side-chain of the “wobbling” tryptophan from\nT-stacking to parallel π–π interactions, a phenomenon\noverlooked in prior research. Our study thus enhances the understanding\nof the acylation mechanism of PET hydrolases, in particular by characterizing\nit for the first time for the promising PES-H1<sup>FY</sup> using\nQM/MM simulations. It also provides insights into selecting a suitable\nQM method and a reaction coordinate, valuable for future studies on\nPET degradation processes.