Genomics, Proteomics, Bioinformatics

Structural Evolution Of Nitrogenase Enzymes Over Geologic Time

By Keith Cowing

Status Report

biorxiv.org

April 12, 2025

Filed under amino acid, biorxiv.org, Deep Learning, enzyme, evolution, genomics, Nitrogenase, phylogenetics, protein, protein crystallography

Structural Evolution Of Nitrogenase Enzymes Over Geologic Time — Overview of the methodological pipeline for massive protein structure prediction and experimental crystallographic analysis of extant and ancestral nitrogenase enzymes. A) Nitrogenase phylogeny built from concatenated Nif/Vnf/AnfHDK protein sequences. Clades are labeled according to the nomenclature used by Raymond et al. 2004. (Nif-I: Group I Nif, Nif-II: Group II Nif, Nif-III: Group III Nif). Nif, Vnf, or Anf homologs from select model organisms are labeled with dashed lines (Avin: Azotobacter vinelandii, Cpas: Clostridium pasteurianum, Kpne: Klebsiella pneumoniae, Mace: Methanosarcina acetivorans, Rpal: Rhodopseudomonas palustris). Anc1A/B and Anc2 ancestors targeted for crystallographic analysis are labeled with stars. B) Graphical overview of the pipeline for nitrogenase protein structure prediction and crystallization (see Materials and Methods for further details). Colored rectangles correspond to protein sequences for H, D, and K subunits. For each ancestral node, protein structures were predicted for the most likely ancestral sequence (“ML”) and five alternative sequences (“Alt”) reconstructed based on the site-wise posterior probability distributions in the ancestral sequence. Ancestors Anc1A and Anc2 hybrid enzymes were crystallized containing an ancestral NifD subunit and WT NifH and NifK subunits (WT subunits indicated by lighter color). All predicted structures are publicly available at https://nsdb.bact.wisc.edu. — biorxiv.org

Life on Earth is more than 3.5 billion years old, nearly as old as the age of the planet. Over this vast expanse of time, life and its biomolecules adapted to and triggered profound changes to the Earth s environment.

Certain critical enzymes evolved early in the history of life and have persisted through planetary extremes. While sequence data is widely used to trace evolutionary trajectories, enzyme structure remains an underexplored resource for understanding how proteins evolve over long timescales.

Here, we implement an integrated approach to study nitrogenase, an ancient, globally critical enzyme essential for nitrogen fixation. Despite the ecological diversity of its host microbes, nitrogenase has strict functional limitations, including extreme oxygen sensitivity, energy requirements and substrate availability.

We combined phylogenetics, ancestral sequence reconstruction, protein crystallography and deep-learning based structural prediction, and resurrected three billion years of nitrogenase structural history.

We present the first effort to predict all extant and ancestral structures along the evolutionary tree of an enzyme and present a total of over 5000 structures.

This approach lays the foundation for reconstructing key structural constraints that influence protein evolution and examining ancient enzymes in the context of phylogenetic and environmental change across geological timescales.

Global analyses of nitrogenase DDKK sequence and structural diversity. A) Root Mean Squared Deviation (RMSD) distribution on paired nitrogenase alignments across extant and ML-ancestor pairs. B) Sequence identity and structural similarity (quantified by root mean square deviation (RMSD) of aligned predicted structures) distribution of paired nitrogenase alignments. C) Hierarchical clustering of predicted nitrogenase structures based on structural similarity (RMSD). Each tile in the heatmap corresponds to the RMSD between two nitrogenase structures. — biorxiv.org

Figure 5. Crystal structures of targeted ancestral nitrogenases. A) Nitrogenase protein phylogeny from which nitrogenase ancestral proteins were reconstructed and crystallized for structural characterization. Major clades are labeled following (Garcia et al., 2023). B) Residue-level RMSD between the crystallized ancestral and wild-type (A. vinelandii) NifDK/NifH structures. C) Spatial distribution of ancestral amino acid substitutions relative to WT. Crystallized protein complexes for Anc1A and Anc2 contain ancestral NifD and WT NifH and NifK. Therefore, ancestral substitutions are only in the NifD subunit for these structures. D) Ancestral amino acid substitutions within the NifH-NifDK interface of Anc1B. available under aCC-BY-NC-ND 4.0 International license. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made bioRxiv preprint doi: https://doi.org/10.1101/2024.11.18.623660; this version posted April 10, 2025. The copyright holder for this preprint (which 19 Bound positions of NifH are inferred by alignment with either the nucleotide-free (PDB 2AFH (Tezcan et al., 2005)) or MgATP-bound (PDB 7UT8 (Rutledge et al., 2022)) structures. E-G) Close views of specific, ancestral amino acid substitutions that are inferred to impact NifH418 NifDK interactions. — biorxiv.org

Structural evolution of nitrogenase enzymes over geologic time, biorxiv.org

Astrobiology, evolution, genomics,

Keith Cowing

Explorers Club Fellow, ex-NASA Space Station Payload manager/space biologist, Away Teams, Journalist, Lapsed climber, Synaesthete, Na’Vi-Jedi-Freman-Buddhist-mix, ASL, Devon Island and Everest Base Camp veteran, (he/him) 🖖🏻

Follow on Twitter