Biotechnology is one of the most promising strategies to resolve the global crisis of plastic pollution. A clear understanding of the core enzyme mechanisms in the biotransformation process is critical for rational enzyme engineering and for practical, industrial-scale applications. Herein, we systematically examined and evidenced a largely unexplored piece in the depolymerization mechanism catalyzed by polyethylene terephthalate (PET) hydrolases: their enantioselectivity. We found that all the short-lived tetrahedron intermediates (IM3 and IM8) possess S-type chirality in six representative PET hydrolases. For instance, the binding percentage ratio of pro-S:pro-R is 57:21 in FAST-PETase, while pro-S binding leads to a much lower average energy barrier (5.2 kcal/mol) than pro-R binding (33.1 kcal/mol). Key structural features (e.g. the angle for Ser@H1-His@N1-PET@O2 and distance for His@N1-PET@O2) that significantly modulate the enantioselectivity were identified. The origin of the energy landscape variation between wild-type IsPETase and mutant FAST-PETase was also unveiled via analysis of key features, the distortion/interaction energy, and non-covalent bond interactions. This study supplies the missing piece in the mechanism for depolymerization catalyzed by PET hydrolases, and will aid in the rational design of enzymes for industrial recycling of PET plastic waste.
Keywords: Biological recycling; Enantioselectivity; Enzyme engineering; Hydrolases; Polyethylene terephthalate; Quantum mechanics/molecular mechanics.
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