Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

CAS Key Laboratory of Molecular Recognition and Function Institute of Chemistry Chinese Academy of Sciences Zhongguancun North 1st Street 2 Beijing 100190 China

Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry University of Greifswald Felix Hausdorff Str 4 17487 Greifswald Germany

Department Synthetic and Structural Biochemistry Institute of Biochemistry University of Greifswald Felix Hausdorff Str 4 17487 Greifswald Germany

Helmholtz Zentrum Berlin für Materialien und Energie Hahn Meitner Platz 1 14109 Berlin Germany

Institute of Biological Information Processing Structural Biochemistry Forschungszentrum Jülich Wilhelm Johnen Straße 52428 Jülich Germany

Institute of Theoretical and Computational Chemistry Heinrich Heine University Düsseldorf Universitätsstr 1 40225 Düsseldorf Germany

International Clinical Research Center St Anne's University Hospital Pekarska 53 656 91 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Kamenice 5 C13 625 00 Brno Czech Republic

National Technology Innovation Center of Synthetic Biology 32 West Seventh Avenue Tianjin Airport Economic Area Tianjin 300308 China

State Key Laboratory of Materials Oriented Chemical Engineering College of Biotechnology and Pharmaceutical Engineering Nanjing Tech University Nanjing 211816 China

Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences 32 West Seventh Avenue Tianjin Airport Economic Area Tianjin 300308 China

University of Chinese Academy of Sciences 19A Yuquan Road Beijing 100049 China

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