Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins

Abstract

The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over lifes 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. SIGNIFICANCE STATEMENTThe origin of nitrogenase-catalyzed nitrogen fixation was a transformative event in lifes history, garnering long-term study from molecular, biogeochemical, and planetary perspectives. Reconstruction of ancestral nitrogenases suggests that the protein sequence space capable of yielding a nitrogen-fixing enzyme in the past was likely more constrained than previously thought. Specifically, here we show that nitrogenases likely evolved from ancestors that resemble maturases, homologs that today participate in nitrogenase cofactor assembly, contrary to the commonly accepted view that maturases evolved from a nitrogenase ancestor. We further submit that the molecular architecture that may have been required for nitrogenase origins was unlikely to have been shaped by the same environmental drivers often implicated in the evolution of nitrogen fixation. If this decoupling is found to be a recurring pattern in metabolic origins, then the presented results would undercut the common, systems-focused rationale of using ancient environmental conditions to explain the timing of critical and singular biogeochemical innovations in lifes past.