Prochiral Selectivity in Enzymatic Polyethylene TerephthalateDepolymerization Revealed by Computational Modeling

Enzyme catalysis has shown its great power indealing with global poly(ethylene terephthalate)(PET)wasteHowever, it is still challenging to design a super enzyme that cantreat the sheer volume ofworldwide PET waste.Without acomplete understanding ofthe catalytic mechanism, it will bedifcult to reach this important goal. Here, we systematically studythe PET depolymerization mechanism catalyzed by structuralhdiferent hydrolases. The role of feeting chiral intermediates wasproved to be crucial, We observed different prochiral selectivitiesamong these PET hydrolases. While most hydrolases favor Si-facebinding, a few hydrolases(e.g,, Humicola insolens cutinase) mainlyadapt Re-face binding, Interestingly, we found that Siface bindingleads to higher activity than Re-face binding in all of the studied hydrolases. This Si-face selectivity originates from the dificulty ofproton transfer from catalytic histidine residue to the substrate and the less stability of the oxyanion hole, Since the Si-face bindingratio ranges from 0 to 9$%, we infer that all these hydrolases are not perfectly evolved to degrade PET. Our in silico results highlightthat enlarging binding site residues (e.g, Leu66 and Asn69) will enhance enzymatic depolymerization, which was further confirmedby our in vitro experiments where both Leu66Phe and Asn69phe show sienifcantly increased PE'T hvdrolvsis activity, Hopefully.this work will aid the future rational design of super enzymes to fght PET pollution.