Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

  1. Amanda K Garcia
  2. Derek F Harris
  3. Alex J Rivier
  4. Brooke M Carruthers
  5. Azul Pinochet-Barros
  6. Lance Seefeldt
  7. Betül Kaçar  Is a corresponding author
  1. University of Wisconsin-Madison, United States
  2. Utah State University, United States

Abstract

The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, that is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.

Data availability

MATERIALS AVAILABILITYMaterials including bacterial strains and plasmids are available to the scientific community upon request.DATA AND CODE AVAILABILITYPhylogenetic data, including sequence alignments and phylogenetic trees, and the script for ancestral gene codon-optimization are publicly available at https://github.com/kacarlab/garcia_nif2023. All other data are included as source data and supplementary files.

Article and author information

Author details

  1. Amanda K Garcia

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Derek F Harris

    Department of Chemistry and Biochemistry, Utah State University, Logan, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Alex J Rivier

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Brooke M Carruthers

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Azul Pinochet-Barros

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Lance Seefeldt

    Department of Chemistry and Biochemistry, Utah State University, Logan, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6457-9504
  7. Betül Kaçar

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    For correspondence
    bkacar@wisc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0482-2357

Funding

National Aeronautics and Space Administration (19- ICAR19_2-0007)

  • Amanda K Garcia
  • Derek F Harris
  • Alex J Rivier
  • Brooke M Carruthers
  • Azul Pinochet-Barros
  • Lance Seefeldt
  • Betül Kaçar

National Aeronautics and Space Administration (Postdoctoral Fellowship)

  • Amanda K Garcia

University of Wisconsin-Madison

  • Betül Kaçar

Arizona Space Grant Consortium

  • Brooke M Carruthers

National Aeronautics and Space Administration (80NSSC19K1617)

  • Betül Kaçar

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

Reviewing Editor

  1. Christian R Landry, Université Laval, Canada

Version history

  1. Preprint posted: May 19, 2022 (view preprint)
  2. Received: November 18, 2022
  3. Accepted: February 16, 2023
  4. Accepted Manuscript published: February 17, 2023 (version 1)
  5. Version of Record published: March 1, 2023 (version 2)
  6. Version of Record updated: February 6, 2024 (version 3)

Copyright

© 2023, Garcia 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. Amanda K Garcia
  2. Derek F Harris
  3. Alex J Rivier
  4. Brooke M Carruthers
  5. Azul Pinochet-Barros
  6. Lance Seefeldt
  7. Betül Kaçar
(2023)
Nitrogenase resurrection and the evolution of a singular enzymatic mechanism
eLife 12:e85003.
https://doi.org/10.7554/eLife.85003

Share this article

https://doi.org/10.7554/eLife.85003

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