Fragment Based Active Site Exploration of Urethane Hydrolases Reveals a Diversity of Urethane Binding Modes

<jats:title>Abstract</jats:title> <jats:p>Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme–plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme–plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments.</jats:p> <jats:sec> <jats:title>Synopsis</jats:title> <jats:p>Fragment-based active site exploration of urethanases to elucidate polyurethane binding and cleavage</jats:p> </jats:sec>