Nitrogenase resurrection and the evolution of a singular enzymatic mechanism
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
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.
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.
Metrics
-
- 3,273
- views
-
- 378
- downloads
-
- 19
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.
-
- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.