Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

Abstract

The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFecofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active-site sequence features can reliably distinguish extant Mo-nitrogenases from V-and Fe-nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo-nitrogenases. Taxa representing early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo-cofactor in studied organisms, suggesting that early Monitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism-level innovations that characterize the Phanerozoic Eon. K E Y W O R D Sancestral sequence reconstruction, metal cofactor, metalloenzyme, nitrogen fixation, nitrogenase This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Radiation of nitrogen‐metabolizing enzymes across the tree of life tracks environmental transitions in Earth history

“…It is also consistent with phylogenetics work suggesting that molybdenum nitrogenase arose early in the evolution of life on Earth (Raymond et al., 2004). Similarly, phylogenetic reconstructions showing that Mo‐nitrogenases arose before V‐ and Fe‐nitrogenases are consistent with this conclusion (Garcia et al., 2020). It has been argued that nitrogenase was present in LUCA (Weiss et al., 2016), though that has been disputed (Berkemer & McGlynn, 2020; Boyd, et al., 2011; Mus et al., 2019) and our results do not resolve this issue.…”

“…Geochemical approaches, relying on the reconstruction of metabolisms based on the rock record, have suggested that biological nitrogen fixation emerged early (Koehler et al., 2019; Ossa Ossa et al., 2019; Stüeken et al., 2016) and that the nitrogen cycle expanded considerably during the Neoarchean (2.8–2.5 Ga) and Paleoproterozoic (2.5–1.8 Ga) (Garvin et al., 2009a; Godfrey & Falkowski, 2009; Kipp et al., 2018; Koehler et al., 2018; Luo et al., 2018; Zerkle, Poulton, et al., 2017). Phylogenetics studies, relying on sequence data, reconstruct the evolutionary history of genes of interest (e.g., Boyd et al., 2011; Garcia et al., 2020; Jones et al., 2008), and some molecular clock studies have yielded conservative estimates for the approximate timing of an enzyme's origin (Boyd, et al., 2011; Boyd & Peters, 2013; Raymond et al., 2004). While each approach provides valuable insights, they both have weaknesses.…”

Radiation of nitrogen-metabolizing enzymes across the tree of life tracks environmental transitions in Earth history

“…Lastly, though we identified very few iron and vanadium nitrogenases (anf and vnf, respectively) in our datasets, our results tentatively suggest that these nitrogenases radiated across the tree of life later than the iron-molybdenum nitrogenases (nif), which supports conclusions from other phylogenetics studies (Garcia et al, 2020). The early rise of Modependent nitrogenase would have required a source of Mo to act as a cofactor for Mocontaining nitrogenases, implying that nanomolar levels of dissolved Mo (Scott et al, 2008;Reinhard et al, 2013), presumably derived from anoxic weathering or hydrothermal sources of molybdenum to the early ocean, were sufficient for the development of Nif and the proliferation of nitrogen fixers.…”

“…It is also consistent with phylogenetics work suggesting that molybdenum nitrogenase arose early in the evolution of life on Earth (Raymond et al, 2004). Similarly, phylogenetic reconstructions showing that Mo-nitrogenases arose before V-and Fe-nitrogenases are consistent with this conclusion (Garcia et al, 2020). It has been argued that nitrogenase was present in LUCA (Weiss et al, 2016), though that has been disputed (Boyd et al, 2011a;Mus et al, 2019;Berkemer & McGlynn, 2020) and our results do not resolve this issue.…”

“…Phylogenetics studies, relying on sequence data, reconstruct the evolutionary history of genes of interest (e.g. Jones et al, 2008;Boyd et al, 2011b;Garcia et al, 2020), and some molecular clock studies have yielded conservative estimates for the approximate timing of an enzyme's origin (Raymond et al, 2004;Boyd et al, 2011b;Boyd & Peters, 2013). While each approach provides valuable insights, they both have weaknesses.…”