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

Amanda K Garcia,Bryan Kolaczkowski,Betül Kaçar,Hanon Mcshea

doi:10.1111/gbi.12381

Amanda K Garcia, Bryan Kolaczkowski + Show 2 more

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https://doi.org/10.1111/gbi.12381

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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 FeFe‐cofactor 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 Mo‐nitrogenases 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.

Highlights

All known life requires nitrogen for the synthesis of essential biomolecules, including nucleotides and amino acids
Transcriptional profiling of the three nitrogenase systems in A. vinelandii suggests that FeV- and FeFe-cofactor synthesis relies on several nif genes in addition to vnf and anf genes, respectively
The 177 nitrogenase sequence dataset was finalized to include NifHDK sequences from 256 taxa, AnfHDK 178 sequences from 14 taxa, VnfHDK sequences from 14 taxa, and outgroup light-independent protochlorophyllide oxidoreductase (Bch/ChlLNB) sequences — sharing distant homology with 180 nitrogenases (Boyd, Anbar, et al, 2011; Hu & Ribbe, 2015; Raymond et al, 2004) — from 10 taxa (Appendix S1; additional analyses were performed with an expanded outgroup, Appendix S2). Only one Nif/Anf/VnfHDK sequence set was retained per genus to broaden taxonomic sampling

Summary

Introduction

All known life requires nitrogen for the synthesis of essential biomolecules, including nucleotides and amino acids. Transcriptional profiling of the three nitrogenase systems in A. vinelandii suggests that FeV- and FeFe-cofactor synthesis relies on several nif genes in addition to vnf and anf genes, respectively (Hamilton et al, 2011; Joerger & Bishop, 1988; Kennedy & Dean, 1992) These include nifBEN for most alternative gene clusters, with the exception of certain taxa (including A. vinelandii) that possess vnfEN homologs of nifEN that likely perform a similar biosynthetic function (Boyd, Anbar, et al, 2011; Boyd & Peters, 2013; Hamilton et al, 2011). The 30 active-site residues were identified as those residing within 5 Å of any atom in either the FeMo-cofactor of the A. vinelandii NifD protein (PDB 3U7Q (Spatzal et al, 2011)) or the FeV-cofactor of the A. vinelandii VnfD protein

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Conclusion