Residue-Position-Dependent Modulation of Urethanase Activity for Polyurethane Depolymerization

Abstract Enzymatic depolymerization of hydrolyzable plastics, such as polyurethane (PU), opens a promising route for the eco-friendly recycling of plastic waste. A comprehensive understanding of the molecular mechanisms governing enzyme-catalyzed PU hydrolysis is crucial for engineering high-performance enzymes. Here, we combined extensive molecular dynamics simulations, hybrid quantum mechanics/molecular mechanics calculations, and experimental mutagenesis to elucidate how residues in distinct spatial regions regulate substrate binding and catalysis in UMG-SP2. Our results reveal that residues in the first shell (e.g., L140) stabilize the substrate in a catalytically productive conformation within the active-site pocket. Three flexible loop regions, H215–L227, L320–D346, and S377–L400, are identified as potential regulators of substrate capture. Furthermore, we quantify the contributions of individual surface-exposed charged residues to the reaction-relevant electric fields in the active site. The reaction mechanism comprises acylation and deacylation stages, with the rate-determining step corresponding to water-assisted nucleophilic attack. Experimental mutagenesis demonstrates that substitutions at residues in different spatial regions selectively modulate substrate binding and catalytic activity, leading to distinct and substrate-dependent effects on low-molecular-weight dicarbamate hydrolysis and PU depolymerization. Collectively, these findings reveal a residue-position-dependent modulation framework for urethanase activity and establish a general mechanism-guided strategy for advancing enzymatic PU depolymerization and recycling.