Mechanism of Polyester Hydrolysis by Marine Bacterium PE-H Enzyme: an Atomistic and Thermodynamic Characterization

Polyethylene terephthalate (PET) is a widely used plastic due to its durability and adaptability; however, its resistance to natural degradation has led to severe accumulation in the environment. Recently, a PET-degrading marine bacterium, Pseudomonas aestusnigri, was identified and proposed for possible use in sustainable plastic recycling, particularly the PE-H enzyme, which hydrolyses PET with MHET as the main hydrolysis product. In this work, we investigate the reaction mechanism of PE-H through umbrella sampling hybrid quantum mechanics/molecular mechanics molecular dynamics simulations at the PBE/AMBER level. Our results show a two-stage reaction pathway: acylation and deacylation, both of which proceed stepwise via tetrahedral intermediate formation. We identified deacylation as the rate-limiting step with a free energy barrier of approximately 10.6 kcal·mol^-1, which is relatively lower than the barrier of other PET hydrolyses. Our analysis suggests that structural features promoting oxyanion hole formation or enhancing substrate accommodation contribute to lower free energy barrier and promote catalysis. We highlight the role of the S171, H249 and D217 triad responsible for the catalysis of proton transfer and nucleophilic attack reactions, and of F98 and M172 responsible for the formation of the oxyanion hole contributing to the stabilization of tetrahedral intermediates formed along the path. These findings provide mechanistic insights into PE-H catalysis and suggest structural factors that could be extended to other enzymes, providing a basis for future studies to understand enzymatic plastic degradation.