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Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase

Nature Catalysis (2024)Cite this article

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A preprint version of the article is available at bioRxiv.

Abstract

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a β-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme’s conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

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Fig. 1: Positive epistasis drives the evolution of OXA-48.
Fig. 2: Kinetic changes drive the evolution of OXA-48.
Fig. 3: Evolution of a catalytically superior ensemble.

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Data availability

Cryogenic crystal structures are deposited at the Protein Data Bank under the PDB IDs 8PEA (F72L), 8PEB (Q5) and 8PEC (Q5-CAZ). Jupyter notebooks and input files required to replicate the MD simulations and analyses of the OXA-48 variants, as well as trajectories and MD snapshots, are available on the University of Bristol Research Data Storage Facility (RDSF) at https://doi.org/10.5523/bris.phtj9yrbdkrq2t6n53k84evkg. The repository furthermore contains PDB structures of all ensemble refinements presented in this work. All other data are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

MD simulations were conducted using the computational facilities of the Advanced Computing Research Centre, University of Bristol. C.F. thanks the PhD schools NFIF, IBA and Biocat for their funding. H.A.B. thanks the SNSF for funding (P5R5PB_210999, PZ00P3_208691 and P400PB_194329). M.W.v.d.K. thanks BBSRC for funding (BB/M026280/1). N.T. thanks the Canadian Institute of Health Research (CIHR) for the project grant (AWD-019305). H.-K.S.L. thanks the Centre for New Antibacterial Strategies for the project grant. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation programme (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M. A.J.M. and H.A.B. also thank BBSRC (grant no. BB/R016445/1) and EPSRC (EP/M013219/1 and EP/M022609/1) for funding. This work was carried out using the computational facilities of the Advanced Computing Research Centre, University of Bristol (http://www.bris.ac.uk/acrc/).

Author information

Author notes

  1. These authors contributed equally: Christopher Fröhlich, H. Adrian Bunzel.

Authors and Affiliations

  1. Department of Pharmacy, UiT The Arctic University of Norway, Tromsø, Norway

    Christopher Fröhlich & Pål J. Johnsen

  2. Department of Biosystem Science and Engineering, ETH Zurich, Basel, Switzerland

    H. Adrian Bunzel

  3. Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, UK

    H. Adrian Bunzel, Adrian J. Mulholland & Marc W. van der Kamp

  4. School of Biochemistry, University of Bristol, Bristol, UK

    H. Adrian Bunzel & Marc W. van der Kamp

  5. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Karol Buda & Nobuhiko Tokuriki

  6. Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway

    Hanna-Kirsti S. Leiros

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Contributions

C.F., H.A.B. and N.T. conceived the study. C.F. performed directed evolution, selection and cloning and assayed dose–response curves. C.F. and K.B. expressed and purified enzymes. C.F. determined thermostabilities. K.B., C.F. and H.A.B. assayed enzyme kinetics. K.B. and N.T. performed the statistical analysis. H.A.B. performed, and H.A.B., A.J.M. and M.W.v.d.K. analysed, the MD simulations. C.F. crystallized proteins, and C.F. and H.-K.S.L. solved structures and refined structures. C.F., H.A.B., P.J.J. and N.T. wrote the paper with input from all co-authors.

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Correspondence to Christopher Fröhlich or Nobuhiko Tokuriki.

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Nature Catalysis thanks Canan Atilgan, Christos Karamitros, Alejandro J. Vila and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Fröhlich, C., Bunzel, H.A., Buda, K. et al. Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase. Nat Catal (2024). https://doi.org/10.1038/s41929-024-01117-4

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