Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product

Abstract

The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase. The subsite I modifications L93F and Q95Y, derived from LCC, increased the thermal stability, while exchange of H185S, derived from IsPETase, reduced the stability of PHL7. The subsite II residue H130 is suggested to represent an adaptation for high thermal stability, whereas L210 emerged as the main contributor to the observed high PET-hydrolytic activity. Variant L210T showed significantly higher activity, achieving a degradation rate of 20 µm h−1 with amorphous PET films.

On the Binding Mode and Molecular Mechanism of Enzymatic Polyethylene Terephthalate Degradation

“…Taken together, these results suggest that the scPET would be even more closely associated with this catalytic His residue in the further course of the reaction. The binding-site interactions between the PET substrate and residues Y60, M131, and H208 in TfCut2 resembles the situation in PHL7, as seen in its cocrystal structure with TPA …”

“…The bulky sidechains of these two residues form part of a hydrophobic surrounding around the benzene moiety (Figure B), possibly stabilizing the PET substrate in an extended conformation. In mesophilic enzymes such as IsPETase, this conserved Trp residue adopts distinct sidechain conformation, in contrast to those in PHL7 and other thermostable type I polyester hydrolases, in which additional steric hindrance is present by the neighboring His residue (e.g., H185 in PHL7). , …”

“…In mesophilic enzymes such as IsPETase, 63 this conserved Trp residue adopts distinct sidechain conformation, in contrast to those in PHL7 and other thermostable type I polyester hydrolases, in which additional steric hindrance is present by the neighboring His residue (e.g., H185 in PHL7). 11,62 As previously reported, the chain cleavage of PET can be initialized by endo-type random chain scissions, followed by an exo-type depolymerization step. 21,42 Our results confirm close interactions of the oligomeric PET substrate with the subsite I residues of TfCut2 via one repeat unit.…”

“…The binding-site interactions between the PET substrate and residues Y60, M131, and H208 in TfCut2 resembles the situation in PHL7, as seen in its cocrystal structure with TPA. 62 By contrast, the carboxylate carbon of the degradation product DST (δ C = 175.4 ppm) showed no such correlations with H208, but there is evidence that this carbonyl interacts with the indole NH proton (Hε1) of W155 (δ H = 10.8 ppm), indicating that the freed DST is now in close proximity to the subsite I rather than subsite II (Figure 7C). This arrangement is corroborated by its correlations with the OH/CH protons of trehalose molecules (δ H = 4.3 ppm) locking the protein surface.…”

Rate Response of Poly(Ethylene Terephthalate)‐Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase

“…These similarities are also highly evident from a visual comparison of the active site region (Figure S4), as recently reported elsewhere, as well. [54] DuraPETase is by far the enzyme that differs most from the other enzymes (Table S1 and Figure S4), containing 2 areas of insertion (residues 109-111 and 217-219) and a region of deletion (between residues 35 and 36), all of which modify surface loops to some extent. LCC ICCG and LCC are naturally the most similar differing by only four point mutations.…”