Prochiral Selectivity in Enzymatic Polyethylene Terephthalate Depolymerization Revealed by Computational Modeling

Enzyme catalysis has shown its great power in dealing with global poly(ethylene terephthalate) (PET) waste. However, it is still challenging to design a super enzyme that can treat the sheer volume of worldwide PET waste. Without a complete understanding of the catalytic mechanism, it will be difficult to reach this important goal. Here, we systematically study the PET depolymerization mechanism catalyzed by structurally different hydrolases. The role of fleeting chiral intermediates was proved to be crucial. We observed different prochiral selectivities among these PET hydrolases. While most hydrolases favor Si-face binding, a few hydrolases (e.g., Humicola insolens cutinase) mainly adapt Re-face binding. Interestingly, we found that Si-face binding leads to higher activity than Re-face binding in all of the studied hydrolases. This Si-face selectivity originates from the difficulty of proton transfer from catalytic histidine residue to the substrate and the less stability of the oxyanion hole. Since the Si-face binding ratio ranges from 0 to 95%, we infer that all these hydrolases are not perfectly evolved to degrade PET. Our in silico results highlight that enlarging binding site residues (e.g., Leu66 and Asn69) will enhance enzymatic depolymerization, which was further confirmed by our in vitro experiments where both Leu66Phe and Asn69Phe show significantly increased PET hydrolysis activity. Hopefully, this work will aid the future rational design of super enzymes to fight PET pollution.