Unveiling the enzymatic pathway of UMG-SP2 urethanase: insights into polyurethane degradation at the atomic level

Abstract

The recently discovered metagenomic urethanases UMG-SP1, UMG-SP2, and UMG-SP3 have emerged as promising tools to establish a bio-based recycling approach for polyurethane (PU) waste. These enzymes are capable of hydrolyzing urethane bonds in low molecular weight dicarbamates as well as in thermoplastic PU and the amide bond in polyamide employing a Ser-Ser _cis -Lys triad for catalysis, similar to 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 improving the enzymatic depolymerization of PU plastic materials.

Scheme 1. The chemical structure of the substrate used in this work to mimic a PU segment, di-urethane ethylene 4,4′-methylenedianiline (DUE-MDA).

Fig. 1. The QM/MM model used to study the catalytic mechanism of UMG-SP2. (Left) Cartoon representation of the UMG-SP2:DUE-MDA reactant structure (9929 atoms). The active site residues are represented as grey sticks, whereas the DUE-MDA substrate is shown as orange sticks. Water molecules are represented in red transparent spheres (hydrogen atoms are not shown for clarity purposes). (Right) Close-up of the QM region, composed of 126 atoms, shown in ball-and-stick representation. The PU substrate (DUE-MDA) is colored in orange. The Ser190_nuc-Ser166_cis-Lys91 catalytic triad is labeled in bold.

Fig. 2. Optimized structures of the first catalytic step stationary states of UMG-SP2 (activation of Ser190_nuc). “R”, “TS1”, and “INT1” stand for reactant (A), first transition state (B), and first intermediate (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate (DUE-MDA) is colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Fig. 3. Optimized structures of the second catalytic step stationary states of UMG-SP2 (nucleophilic attack performed by Ser190_nuc). “INT1”, “TS2”, and “INT2-TI” stand for first intermediate (A), second transition state (B), and second intermediate-tetrahedral intermediate (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate (DUE-MDA) is colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Fig. 4. Optimized structures of the third catalytic step stationary states of UMG-SP2 (tetrahedral intermediate breakdown and urethane bond cleavage). “INT2-TI”, “TS3”, and “INT3” stand for second intermediate-tetrahedral intermediate (A), third transition state (B), and third intermediate (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate atoms are colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Fig. 5. Optimized structures of the fourth catalytic step stationary states of UMG-SP2 (proton transfer from Lys91 to Ser166_cis). “INT3”, “TS4”, and “AE” stand for third intermediate (A), fourth transition state (B), and acyl-enzyme (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate atoms are colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Fig. 6. Optimized structures of the fifth catalytic step stationary states of UMG-SP2 (concerted activation of Wat_cat and nucleophilic attack). “AE*”, “TS5”, and “INT5” stand for acyl enzyme/reactant (stage 2, A), fifth transition state (B), and fifth intermediate (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate atoms are colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Fig. 7. Optimized structures of sixth catalytic step stationary states of UMG-SP2 (regeneration of the catalytic triad). “INT5”, “TS6”, and “P” stand for fifth intermediate (A), sixth transition state (B), and product (C), respectively. The most important atoms for this catalytic step are highlighted by a grey shade. The PU substrate atoms are colored in orange. Some QM atoms are depicted as transparent sticks for clarity purposes. Relevant distances are given in Å.

Scheme 2. The complete catalytic mechanism for the cleavage of one urethane bond by UMG-SP2.

Fig. 8. The global Gibbs free energy profile for the cleavage of one urethane bond by UMG-SP2. The presented ΔG values were determined at the B3LYP/6-311+G(2d,2p)-D3(BJ):ff14SB//B3LYP/6-31G(d)-D3(BJ):ff14SB level of theory and are presented in kcal mol⁻¹. The energy profiles of each stage are shown separately. Connecting the Gibbs energy profiles of the two stages requires complex and often inaccurate calculations of the Gibbs energy for the alcohol-leaving group dissociation and active site solvation, that being why we adopted the current representation. Each mechanistic step is indicated in the bottom part of the plot, in dark grey.

Fig. 9. The impact of each surrounding MM residue on the reaction energy barrier as a function of the difference between the distance of the given MM residue to the positive (O_γ(Ser190_nuc)) and the negative sides (O_ester(DUE-MDA)) of the TS3 macrodipole. Positive residues are colored blue, while negative residues are colored red. Polar residues are colored green, and hydrophobic residues are colored white. Only the residues that have a relevant energy contribution (e.g., |1.0| kcal mol⁻¹) are identified. The top half of the graph shows the residues that destabilize TS3, i.e. the most promising targets for mutation.