The effect of ionic strength on PETase enzymes: an experimental and computational study

In the last decades, several enzymes capable of hydrolyzing polyethylene terephthalate (PET) under different conditions have been discovered, offering innovative and sustainable solutions for managing plastic waste in the ecosystems. The PETase enzyme from Ideonella Sakaiensis 201-f6 (IsPETase) exerts outstanding catalytic activity at low temperatures, making it the most extensively studied PET-active enzyme of the recent period. However, the discovery of the PETase-like enzyme from the marine-sponge Streptomyces sp. SM14 (PETaseSM14) has introduced a new class of biocatalysts active at high-salt concentrations, whose structural and catalytic properties remain poorly understood. In this investigation, we employ all-atom molecular dynamics (MD) simulations and in-vitro activity assays to highlight the structural and functional characteristics of PETaseSM14 and IsPETase at different NaCl concentrations (150 mM and 900 mM). The results of molecular simulations corroborate the findings from in-vitro activity assays on PET films. They show that, due to loop elongation, IsPETase exhibits a flexible and wide binding site that facilitates substrate accommodation. However, this feature leads to the displacement of catalytic residues and the deactivation of the enzyme, which occurs faster at a high-salt concentration. In contrast, PETaseSM14 exhibits a rigid and smaller binding pocket, which undergoes moderate widening upon salt concentration increasing, thus promoting water and substrate recruitment. Moreover, the assessment of the enzyme adsorption onto a PET slab showed that PETaseSM14, under high-salt conditions, and IsPETase, under low-salt conditions, bind the substrate chains in the same trans:gauche conformational distribution as that found in the amorphous PET sample. Our findings provide novel structural details to guide enzyme engineering tailored to diverse environmental conditions.