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Cited by 30Synthetic Organic Chemistry MethodsEnzyme Catalysis and ImmobilizationBiochemical and Structural Characterization

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De novo design and evolution of an artificial metathase for cytoplasmic olefin metathesis

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Zou, Zhi, Kalvet, Indrek, Lozhkin, Boris, Morris, Elinor, Zhang, Kailin, Chen, Dongping, Ernst, Marco L., Zhang, Xiang, Baker, David, Ward, Thomas R.·2025·Nature Catalysis
DOI 10.1038/s41929-025-01436-0PMC PMC12638248

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Abstract

Artificial metalloenzymes present a promising avenue for abiotic catalysis within living systems. However, their in vivo application is currently limited by critical challenges, particularly in selecting suitable protein scaffolds capable of binding abiotic cofactors and maintaining catalytic activity in complex media. Here we address these limitations by introducing an artificial metathase—an artificial metalloenzyme designed for ring-closing metathesis—for whole-cell biocatalysis. Our approach integrates a tailored metal cofactor into a hyper-stable, de novo-designed protein. By combining computational design with genetic optimization, a binding affinity ( K D ≤ 0.2 μM) between the protein scaffold and cofactor is achieved through supramolecular anchoring. Directed evolution of the artificial metathase yielded variants exhibiting excellent catalytic performance (turnover number ≥1,000) and biocompatibility. This work represents a pronounced leap in the de novo design and in cellulo engineering of artificial metalloenzymes, paving the way for abiological catalysis in living systems.

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Research works printed but not linked: 67

  1. Hanefeld, U., Hollmann, F. & Paul, C. E. Biocatalysis making waves in organic chemistry. Chem. Soc. Rev. 51, 594–627 (2022). · DOI
  2. Buller, R. et al. From nature to industry: harnessing enzymes for biocatalysis. Science 382, eadh8615 (2023). · DOI
  3. Key, H. M. et al. Beyond iron: iridium-containing P450 enzymes for selective cyclopropanations of structurally diverse alkenes. ACS Cent. Sci. 3, 302–308 (2017). · DOI
  4. Oohora, K., Onoda, A. & Hayashi, T. Hemoproteins reconstituted with artificial metal complexes as biohybrid catalysts. Acc. Chem. Res. 52, 945–954 (2019). · DOI
  5. Mirts, E. N., Bhagi-Damodaran, A. & Lu, Y. Understanding and modulating metalloenzymes with unnatural amino acids, non-native metal ions, and non-native metallocofactors. Acc. Chem. Res. 52, 935–944 (2019). · DOI
  6. Kim, J. K. et al. Elucidating the role of metal ions in carbonic anhydrase catalysis. Nat. Commun. 11, 4557 (2020). · DOI
  7. Shima, S. et al. Reconstitution of [Fe]-hydrogenase using model complexes. Nat. Chem. 7, 995–1002 (2015). · DOI
  8. Sauer, D. F. et al. A highly active biohybrid catalyst for olefin metathesis in water: impact of a hydrophobic cavity in a β-barrel protein. ACS Catal. 5, 7519–7522 (2015). · DOI
  9. Philippart, F. et al. A hybrid ring-opening metathesis polymerization catalyst based on an engineered variant of the β-barrel protein FhuA. Chem. Eur. J. 19, 13865–13871 (2013). · DOI
  10. Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat. Commun. 6, 7789 (2015). · DOI
  11. Yang, H., Srivastava, P., Zhang, C. & Lewis, J. C. A general method for artificial metalloenzyme formation through strain-promoted azide-alkyne cycloaddition. ChemBioChem 15, 223–227 (2014). · DOI
  12. Bos, J., Fusetti, F., Driessen, A. J. M. & Roelfes, G. Enantioselective artificial metalloenzymes by creation of a novel active site at the protein dimer interface. Angew. Chem. Int. Ed. 51, 7472–7475 (2012). · DOI
  13. Podtetenieff, J., Taglieber, A., Bill, E., Reijerse, E. J. & Reetz, M. T. An artificial metalloenzyme: creation of a designed copper binding site in a thermostable protein. Angew. Chem. Int. Ed. 49, 5151–5155 (2010). · DOI
  14. Reig, A. J. et al. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins. Nat. Chem. 4, 900–906 (2012); erratum 4, 1050 (2012). · DOI
  15. Basler, S. et al. Efficient Lewis acid catalysis of an abiological reaction in a de novo protein scaffold. Nat. Chem. 13, 231–235 (2021). · DOI
  16. Dydio, P. et al. An artificial metalloenzyme with the kinetics of native enzymes. Science 354, 102–106 (2016). · DOI
  17. Bos, J., Browne, W. R., Driessen, A. J. M. & Roelfes, G. Supramolecular assembly of artificial metalloenzymes based on the dimeric protein lmrr as promiscuous scaffold. J. Am. Chem. Soc. 137, 9796–9799 (2015). · DOI
  18. Wilson, M. E. & Whitesides, G. M. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(I) moiety. J. Am. Chem. Soc. 100, 306–307 (1978). · DOI
  19. Eda, S. et al. Biocompatibility and therapeutic potential of glycosylated albumin artificial metalloenzymes. Nat. Catal. 2, 780–792 (2019). · DOI
  20. Vornholt, T. et al. Systematic engineering of artificial metalloenzymes for new-to-nature reactions. Sci. Adv. 7, eabe4208 (2021). · DOI
  21. Filice, M. et al. Preparation of an immobilized lipase-palladium artificial metalloenzyme as catalyst in the heck reaction: role of the solid phase. Adv. Synth. Catal. 357, 2687–2696 (2015). · DOI
  22. Lopez, S., Marchi-Delapierre, C., Cavazza, C. & Ménage, S. A selective sulfide oxidation catalyzed by heterogeneous artificial metalloenzymes iron@NikA. Chem. Eur. J. 26, 16633–16638 (2020). · DOI
  23. Martins, F. L., Pordea, A. & Jäger, C. M. Computationally driven design of an artificial metalloenzyme using supramolecular anchoring strategies of iridium complexes to alcohol dehydrogenase. Faraday Discuss. 234, 315–335 (2022). · DOI
  24. Garcia-Sanz, C., de las Rivas, B. & Palomo, J. M. Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity. Nanoscale 16, 6999–7010 (2024). · DOI
  25. Roelfes, G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc. Chem. Res. 52, 545–556 (2019). · DOI
  26. Kato, S., Onoda, A., Schwaneberg, U. & Hayashi, T. Evolutionary engineering of a Cp*Rh(III) complex-linked artificial metalloenzyme with a chimeric beta-barrel protein scaffold. J. Am. Chem. Soc. 145, 8285–8290 (2023). · DOI
  27. Blanco, C. O. & Fogg, D. E. Water-accelerated decomposition of olefin metathesis catalysts. ACS Catal. 13, 1097–1102 (2023). · DOI
  28. Sasmal, P. K., Streu, C. N. & Meggers, E. Metal complex catalysis in living biological systems. Chem. Commun. 49, 1581–1587 (2013). · DOI
  29. Streu, C. & Meggers, E. Ruthenium-induced allylcarbamate cleavage in living cells. Angew. Chem. Int. Ed. 45, 5645–5648 (2006). · DOI
  30. Wittwer, M. et al. Engineering and emerging applications of artificial metalloenzymes with whole cells. Nat. Catal. 4, 814–827 (2021). · DOI
  31. Chordia, S., Narasimhan, S., Paioni, A. L., Baldus, M. & Roelfes, G. In vivo assembly of artificial metalloenzymes and application in whole-cell biocatalysis. Angew. Chem. Int. Ed. 60, 5913–5920 (2021). · DOI
  32. Huang, J. et al. Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme. Nat. Chem. 13, 1186–1191 (2021). · DOI
  33. Gu, Y., Bloomer, B., Liu, Z. N., Clark, D. & Hartwig, J. F. Directed evolution of artificial metalloenzymes in whole cells. Angew. Chem. Int. Ed. 61, e202110519 (2022). · DOI
  34. Liu, Z. N. et al. Assembly and evolution of artificial metalloenzymes within nissle 1917 for enantioselective and site-selective functionalization of C-H and C=C Bonds. J. Am. Chem. Soc. 144, 883–890 (2022). · DOI
  35. Zhao, J. M. et al. Genetic engineering of an artificial metalloenzyme for transfer hydrogenation of a self-immolative substrate in E. coli’s periplasm. J. Am. Chem. Soc. 140, 13171–13175 (2018). · DOI
  36. Stein, A. et al. A dual anchoring strategy for the directed evolution of improved artificial transfer hydrogenases based on carbonic anhydrase. ACS Cent. Sci. 7, 1874–1884 (2021). · DOI
  37. Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016). · DOI
  38. Heinisch, T. et al. surface display of streptavidin for directed evolution of an allylic deallylase. Chem. Sci. 9, 5383–5388 (2018). · DOI
  39. Grimm, A. R. et al. A whole cell display platform for artificial metalloenzymes: poly(phenylacetylene) production with a rhodium-nitrobindin metalloprotein. ACS Catal. 8, 2611–2614 (2018). · DOI
  40. Ogba, O. M., Warner, N. C., O’Leary, D. J. & Grubbs, R. H. Recent advances in ruthenium-based olefin metathesis. Chem. Soc. Rev. 47, 4510–4544 (2018). · DOI
  41. Kim, Y. W., Grossmann, T. N. & Verdine, G. L. Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis. Nat. Protoc. 6, 761–771 (2011). · DOI · Paper page
  42. Blanco, C. O., Cormier, S. K., Koller, A. J., Boros, E. & Fogg, D. E. Olefin metathesis in water: speciation of a leading water-soluble catalyst pinpoints challenges and opportunities for chemical biology. J. Am. Chem. Soc. 147, 9441–9448 (2025). · DOI
  43. Nasibullin, I. et al. Catalytic olefin metathesis in blood. Chem. Sci. 14, 11033–11039 (2023). · DOI
  44. Schunck, N. S. & Mecking, S. In vivo olefin metathesis in microalgae upgrades lipids to building blocks for polymers and chemicals. Angew. Chem. Int. Ed. 61, e202211285 (2022). · DOI
  45. Wu, T. et al. Artificial metalloenzyme assembly in cellular compartments for enhanced catalysis. Nat. Chem. Biol. 21, 779–789 (2025). · DOI
  46. Sabatino, V., Rebelein, J. G. & Ward, T. R. ‘Close-to-Release’: spontaneous bioorthogonal uncaging resulting from ring-closing metathesis. J. Am. Chem. Soc. 141, 17048–17052 (2019). · DOI
  47. Zou, Z. et al. Combining an artificial metathase with a fatty acid decarboxylase in a whole cell for cycloalkene synthesis. Nat. Synth. 3, 1113–1123 (2024). · DOI
  48. An, L. N. et al. Hallucination of closed repeat proteins containing central pockets. Nat. Struct. Mol. Biol. 30, 1755–1760 (2023). · DOI · Paper page
  49. Siegel, J. B. et al. Computational design of an enzyme catalyst for a stereoselective bimolecular diels-alder reaction. Science 329, 309–313 (2010). · DOI
  50. Dou, J. Y. et al. De novo design of a fluorescence-activating β-barrel. Nature 561, 485–491 (2018). · DOI · Paper page
  51. Yeh, A. H. et al. De novo design of luciferases using deep learning. Nature 614, 774–780 (2023). · DOI
  52. Kalvet, I. et al. Design of heme enzymes with a tunable substrate binding pocket adjacent to an open metal coordination site. J. Am. Chem. Soc. 145, 14307–14315 (2023). · DOI
  53. Polizzi, N. F. & DeGrado, W. F. A defined structural unit enables de novo design of small-molecule-binding proteins. Science 369, 1227–1233 (2020). · DOI
  54. Maguire, J. B. et al. Perturbing the energy landscape for improved packing during computational protein design. Proteins 89, 436–449 (2021). · DOI · Paper page
  55. Doyle, L. et al. Rational design of alpha-helical tandem repeat proteins with closed architectures. Nature 528, 585–588 (2015). · DOI
  56. Arnold, F. H. Innovation by evolution: bringing new chemistry to life (Nobel lecture). Angew. Chem. Int. Ed. 58, 14420–14426 (2019). · DOI
  57. Markel, U., Sauer, D. F., Schiffels, J., Okuda, J. & Schwaneberg, U. Towards the evolution of artificial metalloenzymes-a protein engineer’s perspective. Angew. Chem. Int. Ed. 58, 4454–4464 (2019). · DOI
  58. Wang, Y. J. et al. Directed evolution: methodologies and applications. Chem. Rev. 121, 12384–12444 (2021). · DOI
  59. Lynn, D. M., Mohr, B., Grubbs, R. H., Henling, L. M. & Day, M. W. Water-soluble ruthenium alkylidenes: Synthesis, characterization, and application to olefin metathesis in protic solvents. J. Am. Chem. Soc. 122, 6601–6609 (2000). · DOI
  60. Park, H., Zhou, G. F., Baek, M., Baker, D. & DiMaio, F. Force field optimization guided by small molecule crystal lattice data enables consistent sub-angstrom protein–ligand docking. J. Chem. Theory Comput. 17, 2000–2010 (2021). · DOI
  61. Chai Discovery et al. Chai-1: decoding the molecular interactions of life. Preprint at bioRxiv https://doi.org/10.1101/2024.10.10.615955 (2024). · DOI · Paper page
  62. Wohlwend, J. et al. Boltz-1 democratizing biomolecular interaction modeling. Preprint at bioRxiv https://doi.org/10.1101/2024.11.19.624167 (2024). · DOI
  63. Anishchenko, I. et al. Modeling protein–small molecule conformational ensembles with PLACER. Proc. Natl Acad. Sci. 122, e2427161122 (2025). · DOI
  64. Fahey, R. C., Brown, W. C., Adams, W. B. & Worsham, M. B. Occurrence of glutathione in bacteria. J. Bacteriol. 133, 1126–1129 (1978). · DOI
  65. Schafer, F. Q. & Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212 (2001). · DOI · Paper page
  66. Klein, A. S. et al. A de novo metalloenzyme for cerium photoredox catalysis. J. Am. Chem. Soc. 146, 25976–25985 (2024). · DOI
  67. Hou, K. P. et al. De novo design of porphyrin-containing proteins as efficient and stereoselective catalysts. Science 388, 665–670 (2025). · DOI

Metadata

Authors
Zou, Z., Kalvet, I., Lozhkin, B., Morris, E., Zhang, K., Chen, D., Ernst, M. L., Zhang, X., Baker, D., & Ward, T. R.
Affiliations
  • University of Basel (CH)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Baker, David
  • Ward, Thomas R.
ORCID
Venue
Nature Catalysis
Publisher
Springer Science and Business Media LLC
Published
2025-11-03
Volume
8
Issue
11
Pages
1208-1219
ISSN
2520-1158 (ISSN-L)
DOI
10.1038/s41929-025-01436-0
PMC
PMC12638248
OpenAlex
W4415790300
Cited by
30
References
2
Citations by year
2026: 27 · 2025: 3
FWCI
8.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Catalysis (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by)
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (200020_212088)
  • Open Philanthropy Project Improving Protein Design Fund and Human Frontier Science Program Cross-Disciplinary Fellowship
  • Open Philanthropy Project Improving Protein Design Fund
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Synthetic Organic Chemistry Methods
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
artificial metalloenzymes, olefin metathesis, ring-closing metathesis, de novo protein design, protein scaffolds, whole-cell biocatalysis, directed evolution

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