Enzyme structures
All known PETases (EC number 3.1.1.101) are homologues of cutinases and are therefore annotated as dienelactone hydrolases, lipases, or esterases. In solution, PETases are supposed to be active as monomers30. A total of twelve structures affiliated with different organisms are available in the PDB. For the best characterized examples LCC and Is PETase, multiple entries of variants have been made. All PETases consist of a single domain, the α/β-hydrolase fold, which is formed by a central twisted β-sheet, flanked by two layers of α‑helices 31, and thus belong to a class of small α/β-hydrolases that consist only of the core domain without a mobile lid20. For a few PETase homologues from Bacteroidetes, an additional C-terminal sorting domain for the type IX secretion system has been annotated and was verified in the single structure published (Figure S5, Table 1 ). The type IX secretion system comprises several protein components 32, and the corresponding C-terminal domain was also found in other polymer-active enzymes such as cellulases and endo-1,4-β -glucanases 33–36. PETases share a conserved catalytic triad of serine, histidine, and aspartic acid, and a GX-type oxyanion hole, which stabilizes the reaction intermediate 37. In the PETase homologues, the first oxyanion hole residue X is mostly a conserved aromatic residue such as tyrosine or phenylalanine. The second oxyanion-stabilizing residue is a conserved methionine following the serine of the catalytic triad. For the PETase from I. sakaiensis , several residues were suggested for substrate binding, such as an aromatic clamp formed by the first residue of the oxyanion hole and a second aromatic residue38. In addition to this subsite I, a second subsite II was proposed from the interaction observed in a modelled complex with a PET monomer 39. Variants with increased catalytic activity were designed and tested. The most active enzymes are LCC variants, where the addition of disulfide bonds increased thermostability 40. The two LCC quadruple variants F243[WI]-D238C-S283C-Y127G, which include the additional disulfide bond, are among the most active PETases described to date.