The Catalytic Mechanism of the Plastic-Degrading Enzyme Urethanase UMG-SP2

The recently discovered metagenomic urethanases UMG-SP1, UMG-SP2, and UMG-SP3 have emerged as promising tools to improve existing chemical processes for polyurethane (PU) waste recycling. These enzymes are capable of breaking down urethane bonds in low molecular weight dicarbamates using the Ser-Sercis-Lys triad for catalysis, similar to other members of the amidase signature protein superfamily. Understanding the catalytic mechanism of these urethanases is crucial for enhancing their enzymatic activity and improving PU bio-recycling processes. In this study, we employed hybrid quantum mechanics/molecular mechanics methods to delve into the catalytic machinery of the UMG-SP2 urethanase in breaking down a model PU substrate. Our results indicate that the reaction proceeds in two stages: STAGE 1 acylation, in which the enzyme becomes covalently bound to the PU substrate, releasing an alcohol-leaving group; STAGE 2 - deacylation, in which a catalytic water hydrolyzes the enzyme:ligand covalent adduct, releasing the product in the form of a highly unstable carbamic acid, expected to rapidly decompose into an amine and carbon dioxide. We found that STAGE 1 comprises the rate-limiting step of the overall reaction, consisting of the cleavage of the substrate’s urethane bond by its ester moiety and the release of the alcohol-leaving group (overall Gibbs activation energy of 20.8 kcal·mol-1). Lastly, we identified point mutations that are expected to enhance the enzyme's turnover for the hydrolysis of urethane bonds by stabilizing the macrodipole of the rate-limiting transition state. These findings expand our current knowledge of urethanases and homolog enzymes from the amidase signature superfamily, paving the way for future research on the enzymatic depolymerization of PU plastic materials.