QM/MM Investigation to Identify the Hallmarks of Superior PET Biodegradation Activity of PETase over Cutinase

Polyethylene terephthalate (PET), the most extensively used plastic, is one of the significant contributors to global plastic pollution. Enzymatic biodegradation of PET using different hydrolases has been previously reported as a promising biodegradation strategy for closed-loop recycling. Among the different hydrolases known to depolymerize PET to its soluble building blocks, the PETase and cutinase family of enzymes have notable PET biodegradation activities. In fact, they exhibit different thermostabilities and efficiencies in hydrolyzing PET polyesters despite sharing high structural similarities. Herein, we employed quantum mechanics/molecular mechanics calculations to identify the key factors necessary for efficient PET hydrolysis. Our results show that in both PETase and cutinase (Tfcut2 as a model system), the PET hydrolysis reaction pathway proceeds through a multi-step process with rate-limiting steps having energy barriers of ∼18.0 and ∼20 kcal/mol for PETase and TfCut2, respectively, which agrees well with the experimental data. A deeper inspection of the structural complexes revealed that the bent conformation adopted by PET and the tighter H-bond interaction between the catalytic triad residues, mediated by the unique disulfide bridge, contribute to the lower barrier (i.e., better catalytic performance) of PETase. The intrinsic molecular features identified in this work will also be useful for rational engineering of more efficient cutinases for PET hydrolysis.