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.