IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v15y2024i1d10.1038_s41467-024-45630-3.html
   My bibliography  Save this article

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
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-024-45630-3
    File Function: Abstract
    Download Restriction: no

    File URL: https://libkey.io/10.1038/s41467-024-45630-3?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    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.
    Full references (including those not matched with items on IDEAS)

    Most related items

    These are the items that most often cite the same works as this one and are cited by the same works as this one.
    1. Linyue Zhang & Edward King & William B. Black & Christian M. Heckmann & Allison Wolder & Youtian Cui & Francis Nicklen & Justin B. Siegel & Ray Luo & Caroline E. Paul & Han Li, 2022. "Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    2. Allwin D. McDonald & Peyton M. Higgins & Andrew R. Buller, 2022. "Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    3. Martin Grønbæk-Thygesen & Vasileios Voutsinos & Kristoffer E. Johansson & Thea K. Schulze & Matteo Cagiada & Line Pedersen & Lene Clausen & Snehal Nariya & Rachel L. Powell & Amelie Stein & Douglas M., 2024. "Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    4. Enrico Orsi & Lennart Schada von Borzyskowski & Stephan Noack & Pablo I. Nikel & Steffen N. Lindner, 2024. "Automated in vivo enzyme engineering accelerates biocatalyst optimization," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    5. Lene Clausen & Vasileios Voutsinos & Matteo Cagiada & Kristoffer E. Johansson & Martin Grønbæk-Thygesen & Snehal Nariya & Rachel L. Powell & Magnus K. N. Have & Vibe H. Oestergaard & Amelie Stein & Do, 2024. "A mutational atlas for Parkin proteostasis," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    6. Dae-yeol Ye & Myung Hyun Noh & Jo Hyun Moon & Alfonsina Milito & Minsun Kim & Jeong Wook Lee & Jae-Seong Yang & Gyoo Yeol Jung, 2022. "Kinetic compartmentalization by unnatural reaction for itaconate production," Nature Communications, Nature, vol. 13(1), pages 1-10, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45630-3. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    If CitEc recognized a bibliographic reference but did not link an item in RePEc to it, you can help with this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.