Molecular-Level Mechanistic Insights into PETase-Catalyzed Plastics Hydrolysis from Accurate QM/MM Free Energy Calculations

The enzyme PETase is capable of depolymerizing plastics such as polyethylene terephthalate (PET) at moderate temperatures and demonstrated even higher activity towards polyethylene -2,5- furan dicarboxylate (PEF), opening promising routes for the sustainable upcycling of plastic wastes. To fully exploit the potential of these biocatalytic systems, an atomistically detailed mechanistic understanding of their activity is pivotal. To this end, this study sheds light on two fundamental stages of the catalytic cycle of PET and PEF hydrolysis by PETase—acylation and deacylation—using hybrid QM/MM enhanced sampling molecular dynamics (MD) simulations to capture all relevant dynamic effects. Well-tempered metadynamics simulations at the DFTB3 level are performed along collective variables optimized via linear discriminant analysis, a supervised learning-assisted approach that accounts for the contributions of each potentially relevant degree of freedom. The free energy (FE) profiles indicate that the acylation stage is the rate-limiting step for both PET and PEF degradation, with barriers ≈ 8 kcal/mol and ≈ 4 kcal/mol higher than those obtained for the deacylation step, respectively. Remarkably, substantial mechanistic differences are found. While PET acylation occurs in a concerted manner, with a single energy barrier of ≈ 21 kcal/mol, PEF acylation follows a two-step mechanism where after the first barrier, ≈ 10 kcal/mol high, a metastable intermediate state is formed, which then evolves towards the product once a second barrier of ≈ 2 kcal/mol is overcome. This mechanistic description is consistent with the FE profiles obtained at higher levels of theory (PBE, B3LYP, RI-MP2) via FE perturbation, thus validating the key insights elucidated by metadynamics simulations. Finally, both global and local reactivity descriptors derived from conceptual density functional theory (DFT) suggest that PEF is more electrophilic and susceptible to nucleophilic attack than PET. The results obtained by means of the robust computational protocol adopted here offer for the first time thermodynamic and mechanistic insights into PET and PEF hydrolysis by PETase at the molecular level. This work elucidates the experimentally observed enhanced activity of this enzyme toward PEF, paving the way for further exploration of biocatalytic plastic recycling.