Vidossich_2025_J.Chem.Inf.Model__

Protein engineering of cutinases is a promising strategy for the biocatalytic degradation of non-natural polyesters. We report a mechanistic study addressing the hydrolysis of the aliphatic polyester poly(butylene succinate, or PBS) by the fungal Apergillus oryzae cutinase enzyme. Through atomistic molecular dynamics simulations and advanced alchemical transformations, we reveal how three units of a model PBS substrate fit the active site cleft of the enzyme, interacting with hydrophobic side chains. The substrate ester moiety approaches the Asp-His-Ser catalytic triad, displaying catalytically competent conformations. Acylation and deacylation hydrolytic reactions were modeled according to a canonical esterase mechanism using umbrella sampling simulations at the quantum mechanical/molecular mechanical DFT(B3LYP)/6-31G**/AMBERff level. The free energy profiles of both steps show a high-energy tetrahedral intermediate resulting from the nucleophilic attack on the ester's carboxylic carbon. The free energy barrier of the acylation step is higher (20.2 +/- 0.6 kcal mol(-1)) than that of the deacylation step (13.6 +/- 0.6 kcal mol(-1)). This is likely due to the interaction of the ester's carboxylic oxygen with the oxyanion hole in the reactive conformation of the deacylation step. In contrast, these interactions form as the reaction proceeds during the acylation step. The formation of an additional hydrogen bond interaction with the side chain of Ser48 is crucial to stabilizing the developing charge at the carboxylic oxygen, thus lowering the activation free energy barrier. These mechanistic insights will inform the design of enzyme variants with improved activity for plastic degradation.