Early Nitrogenase Ancestors Encompassed Novel Active Site Diversity

Abstract

Ancestral sequence reconstruction (ASR) infers predicted ancestral states for sites within sequences and can constrain the functions and properties of ancestors of extant protein families. Here, we compare the likely sequences of inferred nitrogenase ancestors to extant nitrogenase sequence diversity. We show that the most-likely combinations of ancestral states for key substrate channel residues are not represented in extant sequence space, and rarely found within a more broadly defined physiochemical space-supporting that the earliest ancestors of extant nitrogenases likely had alternative substrate channel composition. These differences may indicate differing environmental selection pressures acting on nitrogenase substrate specificity in ancient environments. These results highlight ASR's potential as an in silico tool for developing hypotheses about ancestral enzyme functions, as well as improving hypothesis testing through more targeted in vitro and in vivo experiments.

Keywords: Nif; ancestral sequence reconstruction; early life; nitrogenase.

Fig. 1.

Extant nitrogenase sequence diversity. A maximum-likelihood gene tree for the D subunit of extant nitrogenases and outgroup sequences (Nfa, Group IV nitrogenase-like enzymes; Bch, bacteriochlorophyll/protochlorophyllide oxidoreductases), displaying the residue state at each of five variable sites in the Azotobacter vinelandii nifD substrate channel. The last common ancestor evaluated for nitrogenases is marked with a black node; reconstructed site likelihoods for all residues P > 0.10 are shown by residue color in the inset bar chart.

Fig. 2.

Ancestral sequence and state likelihoods. Likelihood ratios, amino acid sequence, and residue physicochemical states are shown for all combinations of the five variable sites (alignment sites 348, 495, 496, 576, and 603) predicted in the nitrogenase substrate channel ASR. “Rare” states occur in no more than two extant sequences.

Fig. 3.

Substitution model effect on nitrogenase ASR. Proportional likelihoods are shown for residue states and physicochemical types at the five variable sites (alignment sites 348, 495, 496, 576, and 603) predicted in the nitrogenase ancestor for three different substitution models: LG + R9; WAG + R10; and BLOSUM62 + R10. Relative amino acid likelihoods for the nitrogenase ancestor were generally conserved across models at sites 495, 496, and 603. At site 576, LG and BLOSUM favor Gly over Ala (88% vs. 12% for LG and 81% vs. 19% for BLOSUM, respectively), whereas WAG favors Ala over Gly (88% vs. 12%, respectively). At site 348, all models recover a plurality of Ala, Glu, Gln, and Lys, with relative probabilities varying between 17.5% and 1.9%. Residue type combinations are robust to model selection across sites.