More Like this

1. Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins

   https://doi.org/10.1093/gbe/evac031  

   Garcia, Amanda K; Kolaczkowski, Bryan; Kaçar, Betül (March 2022, Genome Biology and Evolution)

   Archibald, John (Ed.)

   Abstract The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over life’s history. Though understanding the early evolution of nitrogen fixation has been a longstanding goal from molecular, biogeochemical, and planetary perspectives, its origins remain enigmatic. In this study, we reconstructed the evolutionary histories of nitrogenases, as well as homologous maturase proteins that participate in the assembly of the nitrogenase active-site cofactor but are not able to fix nitrogen. We combined phylogenetic and ancestral sequence inference with an analysis of predicted functionally divergent sites between nitrogenases and maturases to infer the nitrogen-fixing capabilities of their shared ancestors. Our results provide phylogenetic constraints to the emergence of nitrogen fixation and are consistent with a model wherein nitrogenases emerged from maturase-like predecessors. Though the precise functional role of such a predecessor protein remains speculative, our results highlight evolutionary contingency as a significant factor shaping the evolution of a biogeochemically essential enzyme. 

   more » « less
2. Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

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

   Garcia, Amanda K; Harris, Derek F; Rivier, Alex J; Carruthers, Brooke M; Pinochet-Barros, Azul; Seefeldt, Lance C; Kaçar, Betül (February 2023, eLife)

   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, which is also exhibited by extantA. vinelandiinitrogenase 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. 

   more » « less
3. Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

   https://doi.org/10.1111/1462-2920.14955  

   Luxem, Katja E.; Kraepiel, Anne M. L.; Zhang, Lichun; Waldbauer, Jacob R.; Zhang, Xinning (February 2020, Environmental Microbiology)

   Summary Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)‐only nitrogenase metalloenzymes. Studies with purified enzymes have found that the ‘alternative’ V‐ and Fe‐nitrogenases generally reduce N₂more slowly and produce more byproduct H₂than the Mo‐nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotrophRhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V‐nitrogenase supports growth as fast as the Mo‐nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V‐nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V‐nitrogenase on acetate, the wild‐type strain uses exclusively the Mo‐nitrogenase on both carbon substrates. Notably, the differences in H₂:N₂stoichiometry by alternative nitrogenases (~1.5 for V‐nitrogenase, ~4–7 for Fe‐nitrogenase) and Mo‐nitrogenase (~1) measured here are lower than priorin vitroestimates. These results indicate that the metabolic costs of V‐based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment. 

   more » « less
4. Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase

   https://doi.org/10.1128/aem.01033-23  

   Chanderban, Melissa; Hill, Christopher A.; Dhamad, Ahmed E.; Lessner, Daniel J. (January 2023, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens. 

   more » « less
5. Bioavailability of mineral‐associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

   https://doi.org/10.1111/gbi.12552  

   Srivastava, Shreya; Dong, Hailiang; Baars, Oliver; Sheng, Yizhi (February 2023, Geobiology)

   Abstract Life on Earth depends on N₂‐fixing microbes to make ammonia from atmospheric N₂gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N₂fixation was once believed to have evolved during the Archean‐Proterozoic times using Fe as a cofactor. However, δ¹⁵N values of paleo‐ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo‐oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral‐associated trace metals to a model N₂‐fixing bacteriumAzotobacter vinelandii. N₂fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N₂fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N₂fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral‐associated trace metals are bioavailable as cofactors of nitrogenases to support N₂fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox. 

   more » « less