In this context, the discovery of PET hydrolases (PETases) has emerged as a promising and environmentally friendly approach for plastic biorecycling. These enzymes depolymerize PET into its main constituent monomers, which can then be used to resynthesize virgin PET and thus enable a fully circular plastic recycling process. Most PETases are thermophilic, with optimal activities near the glass transition temperature of PET (~65 ºC) where the polymer chains become more flexible and are prone to enzymatic hydrolysis, due to the inability of PETases to degrade highly crystalline PET. However, a PETase from the mesophilic bacterium Ideonella sakaiensis 201-F6 (IsPETase) was described to degrade amorphous and semi-crystalline PET with high efficiency at 40ºC. While most known PETases have been subjected to protein engineering via rational design or directed evolution, with substantial improvements in thermal stability and depolymerase activity, finding a "one size fits all" solution to improve the catalytic efficiency of any PETase has been elusive. A recent work presented a general mechanism for improving their catalytic activity based on the experimentally observed dihedral torsions of a conformationally dynamic tryptophan side chain in IsPETase, W185 (Figure ), that is highly conserved in PETases in a loop within the active site (hereafter referred to as the W-loop). On a nearby helix there are two residues, S214 and I218 (S/I), which are unique to IsPETase and in all other known PETases correspond to histidine and phenylalanine (H/F), respectively. Crystal structures of IsPETase in the absence of substrates (PDB ID: 5XG0 ) show that the W185 side chain can adopt three conformations, termed A, B, and C as illustrated in Figure , a phenomenon that has been described in the literature as "wobbling". Substrate analogbound enzyme complexes (PDB IDs: 5XH2 13 and 5XH3 ) show that this residue adopts conformer B for substrate binding, where it can form either T-shaped (Figure ), stacked, or parallel displaced interactions with one of the terephthalic rings of the substrate. Comparison of the crystal structure of IsPETase against a thermophilic PETase from a leaf-branch compost metagenome (LCC) suggests that the small size of S214 yields sufficient space for W185 to rotate to accommodate the substrate. Altogether, it is thought that tryptophan motion is relevant for substrate binding and product release. Consistently with this, the S214H mutant of IsPETase shows decreased activity when compared to the wild-type enzyme. We note that there have been multiple elegant computational studies that have helped reveal the mechanisms of PET-degrading enzymes, more global conformational changes involved in catalysis, and contributed to associated engineering effort. However, corresponding studies of the conformational dynamics of this catalytically important tryptophan remain limited. Therefore, to explore the importance of the S/I residues for PETase activity, doubly substituted variants of the strictly conserved H/F residues in several PETases, including the mesophilic BurPL from a Burkholderiales bacterium, and the thermophilic TfCut from Thermobifida fusca and LCC were generated based on the S/I residues unique to IsPETase. All engineered variants have experimentally shown an increase in PET-degrading activity. Conversely, a H/F variant of IsPETase exhibits a decrease in PETase activity. The enhancement in PET hydrolysis caused by the S/I substitution is accompanied by a decrease in the optimal temperature for catalytic activity (Topt), suggesting that the substitutions impact either the local or global stability of these enzymes.