Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues

Abstract

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

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All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Kelsi R Hall

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  2. Katherine J Robins

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5049-4246
  3. Elsie M Williams

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  4. Michelle H Rich

    Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4876-4029
  5. Mark J Calcott

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  6. Janine N Copp

    Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6690-0480
  7. Rory F Little

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  8. Ralf Schwörer

    Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9352-6559
  9. Gary B Evans

    Ferrier Institute, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  10. Wayne M Patrick

    Ferrier Institute, Victoria University of Wellington, Wellington, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  11. David F Ackerley

    School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
    For correspondence
    david.ackerley@vuw.ac.nz
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6188-9902

Funding

Royal Society of New Zealand (15-VUW-037)

  • Wayne M Patrick
  • David F Ackerley

Cancer Society of New Zealand (18.05)

  • Mark J Calcott
  • David F Ackerley

Royal Society of New Zealand (19-VUW-076)

  • Wayne M Patrick
  • David F Ackerley

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Andrei N Lupas, Max Planck Institute for Developmental Biology, Germany

Version history

  1. Received: May 19, 2020
  2. Accepted: November 12, 2020
  3. Accepted Manuscript published: November 13, 2020 (version 1)
  4. Version of Record published: December 15, 2020 (version 2)

Copyright

© 2020, Hall et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Kelsi R Hall
  2. Katherine J Robins
  3. Elsie M Williams
  4. Michelle H Rich
  5. Mark J Calcott
  6. Janine N Copp
  7. Rory F Little
  8. Ralf Schwörer
  9. Gary B Evans
  10. Wayne M Patrick
  11. David F Ackerley
(2020)
Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues
eLife 9:e59081.
https://doi.org/10.7554/eLife.59081

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https://doi.org/10.7554/eLife.59081

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