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Cited by 4Physical Organic Chemistry and Reaction KineticsEnzyme Catalysis and ImmobilizationEnzyme Production and Characterization

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Insights into the Catalytic Activity of a Metagenome-Derived Urethanase

Open access
Świderek, Katarzyna, Arafet, Kemel, de Sousa Batista, Victor, Grajales-Hernández, Daniel, López-Gallego, Fernando, Moliner, Vicent·2025·Journal of the American Chemical Society
DOI 10.1021/jacs.5c13147PMC PMC12636025

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Abstract

High Resolution Image Download MS PowerPoint Slide The discovery of urethanases shows an opportunity to access the biotechnological recycling of polyurethane-based plastics (PURs), widely used in the manufacture of everyday materials. However, the mechanistic understanding of these enzymes remains under debate. In this work, we report a QM/MM-based mechanistic study of the metagenome-derived urethanase UMG-SP2 catalyzing the degradation of a urethane-like model compound, 4-nitrophenyl benzylcarbamate (pNC). A high-quality structural model generated with AlphaFold2, prior to the availability of the crystal structure, accurately captured the Ser-Ser-Lys catalytic triad characteristic of amidase signature enzymes. Highly accurate constant-pH nonequilibrium molecular dynamics and Monte Carlo (neMD/MC) simulations provided the full titration curve of active site Lys, explaining the need for alkaline media for the enzyme to be active. The generation of the free energy landscape, obtained by means of free energy perturbation methods with the M06-2X DFT functional describing the QM region of the full system, reveals an esterase-like three-step mechanism of UMG-SP2, i.e., acylation, hydrolysis, and decarboxylation, with all steps being kinetically feasible. Our computational results show very good agreement with experimental kinetic data, with a calculated free energy barrier of 21.2 kcal·mol –1 for the rate-determining step compared to 22.9 kcal·mol –1 derived from the experimentally measured turnover frequency (TOF). The present results also open the door for the final decarboxylation occurring in the solution after the release of the product of the hydrolysis step or within the active site. These findings provide an atomistic insight into the urethanase function and establish a robust framework for the future design of biocatalysts targeting polyurethane degradation.

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Crossref · 10.1021/jacs.5c13147

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Figures

1 Overlay of the crystal structure of urethanase UMG-SP2 from an uncultured bacterium with PDB code 8WDW (in gray) and the structure predicted by AlphaFold (in blue). Detail of the active site triad is shown in the center, and acylation step products of the possible amidase (blue arrow) or esterase (red arrow) activity of UMG-SP2 on the 4-nitrophenyl benzylcarbamate (pNC) PUR model compound are schematically shown on the right side.
1 Overlay of the crystal structure of urethanase UMG-SP2 from an uncultured bacterium with PDB code 8WDW (in gray) and the structure predicted by AlphaFold (in blue). Detail of the active site triad is shown in the center, and acylation step products of the possible amidase (blue arrow) or esterase (red arrow) activity of UMG-SP2 on the 4-nitrophenyl benzylcarbamate (pNC) PUR model compound are schematically shown on the right side.CC BY · PubMed Central
2 Titration curves computed for lysine residues Lys75 and Lys188 generated by pH-constant neMD/MC simulations with two different poses of pNC in the active site: Pose 1 (left panel) and Pose 2 (right panel).
2 Titration curves computed for lysine residues Lys75 and Lys188 generated by pH-constant neMD/MC simulations with two different poses of pNC in the active site: Pose 1 (left panel) and Pose 2 (right panel).CC BY · PubMed Central
3 Structural analysis of the initial protein: substrate complexes derived from 1 μs MD simulations. A. Detail of the active site of structures with the two possible poses of the substrate, depending on the relative position between the carbamate group and the catalytic SSK triad (Pose 1 and Pose 2). B. Time evolution of the interatomic distance Oγ(S174)‑C1(Substrate). C. Representative structure of the equilibrated system in Pose 2. Key distances are given in Å. D. Population analysis of interatomic distances defining the key interactions between the substrate and the active site residues.
3 Structural analysis of the initial protein: substrate complexes derived from 1 μs MD simulations. A. Detail of the active site of structures with the two possible poses of the substrate, depending on the relative position between the carbamate group and the catalytic SSK triad (Pose 1 and Pose 2). B. Time evolution of the interatomic distance Oγ(S174)‑C1(Substrate). C. Representative structure of the equilibrated system in Pose 2. Key distances are given in Å. D. Population analysis of interatomic distances defining the key interactions between the substrate and the active site residues.CC BY · PubMed Central
4 Proposed mechanism for the full decomposition of pNC catalyzed by UMG-SP2 as derived from computed M06–2X/MM free energy landscape.
4 Proposed mechanism for the full decomposition of pNC catalyzed by UMG-SP2 as derived from computed M06–2X/MM free energy landscape.CC BY · PubMed Central
5 M06–2X/6–31+G­(d,p)/MM free energy profile of the catalytic mechanism of the substrate 4-nitrophenyl benzylcarbamate hydrolysis catalyzed by UMG-SP2. The orange, green, and blue lines represent the free energy profile for acylation, deacylation, and decarboxylation steps. The gray lines represent the free energy profiles before adding the zero-point vibrational corrections. FES of each step is deposited in the Supporting Information, Figures S19–S21, while energetic values are reported in Tables S8–S10, and estimations of statistical errors in Tables S11–S18.
5 M06–2X/6–31+G­(d,p)/MM free energy profile of the catalytic mechanism of the substrate 4-nitrophenyl benzylcarbamate hydrolysis catalyzed by UMG-SP2. The orange, green, and blue lines represent the free energy profile for acylation, deacylation, and decarboxylation steps. The gray lines represent the free energy profiles before adding the zero-point vibrational corrections. FES of each step is deposited in the Supporting Information, Figures S19–S21, while energetic values are reported in Tables S8–S10, and estimations of statistical errors in Tables S11–S18.CC BY · PubMed Central
6 Kinetic studies in the hydrolysis of pNC driven by the UMG-SP2. A) Reaction scheme of the hydrolysis reaction. (1) 4-nitrophenyl benzylcarbamate, (2) 4-nitrophenol, and (3) Benzylamine. B) UPLC/UV–vis analysis of standards and reaction crudes resulting from the incubation of pNC, (i) Standard of 4-nitrophenol, (ii) standard of benzyl amine, (iii) Initial reaction mixture of hydrolysis (T0). (iv) Reaction at 90 min. (v) Reaction at 120 h. Arrows and numbers in the upper figure indicate the compound detected as referenced in A. C) Reaction course of the hydrolysis of pNC driven by the UMG-SP2. Reaction conditions: 1 mM of 4-nitrophenyl benzylcarbamate, dissolved in 10% of DMSO and 1% of Triton X-100, 30 °C and 1000 rpm at pH 10 in buffer sodium bicarbonate. Activity measurements were performed in triplicate. The gray shadow corresponds to the standard error of each data point.
6 Kinetic studies in the hydrolysis of pNC driven by the UMG-SP2. A) Reaction scheme of the hydrolysis reaction. (1) 4-nitrophenyl benzylcarbamate, (2) 4-nitrophenol, and (3) Benzylamine. B) UPLC/UV–vis analysis of standards and reaction crudes resulting from the incubation of pNC, (i) Standard of 4-nitrophenol, (ii) standard of benzyl amine, (iii) Initial reaction mixture of hydrolysis (T0). (iv) Reaction at 90 min. (v) Reaction at 120 h. Arrows and numbers in the upper figure indicate the compound detected as referenced in A. C) Reaction course of the hydrolysis of pNC driven by the UMG-SP2. Reaction conditions: 1 mM of 4-nitrophenyl benzylcarbamate, dissolved in 10% of DMSO and 1% of Triton X-100, 30 °C and 1000 rpm at pH 10 in buffer sodium bicarbonate. Activity measurements were performed in triplicate. The gray shadow corresponds to the standard error of each data point.CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 5

  1. The New Plastics Economy: Rethinking the future of plastics
  2. 2024, BIOVIA Discovery Studio Visualizer
  3. Case D. A., Amber 2022
  4. 10.1002/1096-987X(200009)21:12<1088::AID-JCC5>3.0.CO;2-8 · DOI
  5. Hehre W. J., 1986, Ab Initio Molecular Orbital Theory

Metadata

Authors
Świderek, K., Arafet, K., de Sousa Batista, V., Grajales-Hernández, D., López-Gallego, F., & Moliner, V.
Affiliations
  • Universitat Jaume I (ES)
  • BioComp Group, Institute of Advanced Materials (INAM)
  • Heterogeneous Biocatalysis Laboratory, Center for Cooperative Research in Biomaterials (CIC biomaGUNE)
Corresponding author
  • Świderek, Katarzyna
  • Moliner, Vicent
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2025-11-05
Volume
147
Issue
46
Pages
42511-42523
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/jacs.5c13147
PMC
PMC12636025
OpenAlex
W4415938173
Cited by
4
References
82
Citations by year
2026: 4
FWCI
1.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Generalitat Valenciana (CIPROM/2021/079, SEJI/2020/007)
  • Ministerio de Ciencia, Innovaci?n y Universidades (CNS2024-154238, EQC2019-006018-P, MDM-2017-0720, PID2019-107098RJ-I00, PID2021-123332OB-C21, RT-134755-T, TED2021-129852B-C21)
  • Ikerbasque, Basque Foundation for Science (019-B1-2023)
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Physical Organic Chemistry and Reaction Kinetics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
Keywords
polyurethane degradation, enzyme mechanism, amidase, acylation, hydrolysis, decarboxylation, active site titration, free energy landscape, rate-determining step, polyurethane recycling, metagenome-derived enzymes
MeSH terms
Amidohydrolases, Catalytic Domain, Metagenome, Biocatalysis, Molecular Dynamics Simulation

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