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Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing

Author

Listed:
  • Rosario Vanella

    (University of Basel
    ETH Zurich)

  • Christoph Küng

    (University of Basel
    ETH Zurich)

  • Alexandre A. Schoepfer

    (University of Basel
    École Polytechnique Fédérale de Lausanne (EPFL)
    École Polytechnique Fédérale de Lausanne (EPFL))

  • Vanni Doffini

    (University of Basel
    ETH Zurich)

  • Jin Ren

    (University of Basel
    ETH Zurich)

  • Michael A. Nash

    (University of Basel
    ETH Zurich
    Molecular Systems Engineering
    Swiss Nanoscience Institute)

Abstract

Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We use enzyme proximity sequencing to analyze how 6399 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase from Rhodotorula gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. Enzyme proximity sequencing can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.

Suggested Citation

  • Rosario Vanella & Christoph Küng & Alexandre A. Schoepfer & Vanni Doffini & Jin Ren & Michael A. Nash, 2024. "Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45630-3
    DOI: 10.1038/s41467-024-45630-3
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    References listed on IDEAS

    as
    1. Emily E. Wrenbeck & Laura R. Azouz & Timothy A. Whitehead, 2017. "Single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded," Nature Communications, Nature, vol. 8(1), pages 1-10, August.
    2. Matteo Cagiada & Sandro Bottaro & Søren Lindemose & Signe M. Schenstrøm & Amelie Stein & Rasmus Hartmann-Petersen & Kresten Lindorff-Larsen, 2023. "Discovering functionally important sites in proteins," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    3. Gordon Rix & Ella J. Watkins-Dulaney & Patrick J. Almhjell & Christina E. Boville & Frances H. Arnold & Chang C. Liu, 2020. "Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
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