Abstract
Polyurethane (PUR), the fifth most prevalent synthetic polymer, substantially contributes to the global plastic waste problem. Biotechnology-based recycling methods have recently emerged as innovative solutions to plastic waste disposal and sparked interest among scientific communities and industrial stakeholders in discovering and designing highly active plastic-degrading enzymes. Here, the ligand-free crystal structure of UMG-SP2, a metagenome-derived urethanase with depolymerization activities, at 2.59 Å resolution, as well as its (co-)structures bound to a suicide hydrolase inhibitor and a short-chain carbamate substrate at 2.16 and 2.40 Å resolutions, respectively, is reported. Structural analysis and molecular dynamics simulations reveal that the flexible loop L3 consisting of residues 219-226 is crucial for regulating the hydrolytic activity of UMG-SP2. The semi-rational redesign of UMG-SP2 reveals superior variants, A141G and Q399A, exhibiting over 30.7- and 7.4-fold increased activities on polyester-PUR and a methylene diamine derivative of PUR, respectively, compared to the wild-type enzyme. These findings advance the understanding of the structure-function relationship of PUR-hydrolyzing enzymes, which hold great promise for developing effective industrial PUR recycling processes and mitigating the environmental footprint of plastic waste.