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Unlocking Lewis acid catalysis in non-haem enzymes for an abiotic ene reaction

Nature Catalysis (2025)Cite this article

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Abstract

Lewis acid catalysis is a powerful tool in organic synthesis. However, biocatalytic Lewis acid catalysis has been limited in its reaction scope and diversity, constraining its synthetic utility. In this study, we expand the scope of biocatalytic Lewis acid catalysis by integrating abiotic ene reactions into metalloenzymatic catalysis. We found that substituting the iron centre with copper enabled SadA, a non-haem iron hydroxylase from Burkholderia ambifaria, to catalyse abiotic Conia-ene reactions. A high-throughput screening platform based on fluorogenic click chemistry was developed to optimize this abiotic transformation. Using this platform, directed evolution was used to generate variants that produced a range of Conia-ene cyclization products with stereogenic quaternary carbon centres, achieving up to 99% yield, 250 total turnovers and 99% enantiomeric excess. Mechanistic studies suggested that the reaction proceeded through a dual activation mechanism, where the Cu(II) centre activated both the ketoester and alkyne moieties.

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Fig. 1: Conceptualization of abiological metalloenzymatic Conia-ene reactions.
Fig. 2: Discovery and optimization of metalloenzymatic Conia-ene reaction.
Fig. 3: Substrate scope of enzymatic Conia-ene reaction catalysed by SadA-ene.
Fig. 4: Mechanistic studies.

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Data availability

Data supporting the findings of this study are available in the paper and the Supplementary Information. All biological materials, such as plasmids generated in this study, are available from the corresponding authors upon request.

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Acknowledgements

We thank P. Mortimer and the Johns Hopkins University (JHU) Mass Spectrometry Facility for analytical support. Financial support was provided by the JHU and the David and Lucile Packard Foundation (to X. Huang). S.L.J.M. is grateful to the NSF (grant no. CHE 2106417) for support. This work was also supported by the National Natural Science Foundation of China (grant nos. 22371256, 21978272) (to Y.Y.) and the Fundamental Research Funds for the Provincial Universities of Zhejiang (grant no. RF-C2022006) (to Y.Y.). X.H. thanks National Key R&D Program of China (grant no. 2022YFA1504301), the National Natural Science Foundation of China (grant nos. 22122109 and 22271253), Zhejiang Provincial Natural Science Foundation of China under grant no. LDQ23B020002 and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (grant no. SN-ZJU-SIAS-006) for support. This work was also supported by the Generalitat de Catalunya AGAUR grant no. 2021SGR00623 project (to M.G.-B.) and the Spanish MICINN (Ministerio de Ciencia e Innovación) grant nos. PID2019-111300GA-I00, PID2022-141676NB-I00 and TED2021-130173B-C42 projects, and grant no. RYC 2020-028628-I (to M.G.-B.).

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Author notes

  1. These authors contributed equally: Xinpeng Mu, Xinyuan Ji, Xiahe Chen.

Authors and Affiliations

  1. Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Xinpeng Mu, Xinyuan Ji, Jinyan Rui, Lucie T. M. Goldberg & Xiongyi Huang

  2. College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China

    Xiahe Chen & Yunfang Yang

  3. Center of Chemistry for Frontier Technologies, Department of Chemistry, State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou, China

    Hongli Wu & Xin Hong

  4. Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA

    Madison M. Worth, Abigail D. Reitz & Sarah L. J. Michel

  5. Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, Campus Montilivi, Girona, Spain

    Marc Garcia-Borràs

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  1. Xinpeng Mu
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Contributions

X. Huang conceived the project. X. Huang and X.M. designed the experiments. X.M. performed initial screening. X.M. and X.J. performed directed evolution experiments and results analysis. X.M., X.J., L.T.M.G. and J.R. performed substrate scope study. X.J., L.T.M.G., M.M.W. and A.D.R. performed ICP-MS analysis under the guidance of S.L.J.M. Y.Y. directed the computational studies. X. Hong, X.C. and H.W. performed DFT calculations. X.C. performed molecular dynamics simulation studies under the guidance of Y.Y. Results analyses of all computational data were performed by M.G.-B., Y.Y., X. Hong, X.C. and H.W., with input from X. Huang. X. Huang wrote the manuscript with input from all other authors. Y.Y. and X. Hong wrote the computational section of the manuscript and the Supplementary Information.

Corresponding authors

Correspondence to Yunfang Yang or Xiongyi Huang.

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Competing interests

A provisional patent application has been under preparation through Johns Hopkins University based on the results presented herein with X. Huang, J.R., X.J. and X.M. as inventors (JHU reference no. C18543). The other authors declare no competing interests.

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Nature Catalysis thanks Bernhard Hauer, Ru Jiang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Methods, Tables 1–14, Figs. 1–14, Notes 1–14 and References.

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Supplementary Data 1

Coordinates from molecular dynamics simulations.

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Mu, X., Ji, X., Chen, X. et al. Unlocking Lewis acid catalysis in non-haem enzymes for an abiotic ene reaction. Nat Catal (2025). https://doi.org/10.1038/s41929-025-01350-5

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