Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade

Abstract

Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6,779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade Ø. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.

Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

“…28 We previously applied evo-velocity analysis on the full RNR sequences to confirm our interpretation of the RNR phylogeny. 3 Consistent with SSN analysis, class II and III cone sequences (Figure 2c, red and yellow) cluster together and form a hub in the evo-velocity embedded sequence space, with class I cone sequences extending from the central hub (Figure 2c, blue). When colored by "pseudotime" (a proxy measure for phylogenetic depth) (Figure 2d), evo-velocity predicts the hub region to be the most ancestral, supporting our hypothesis that ATPcones in extant species have a single origin that resembles the class II and III cones.…”

“…Recently, we reported the first large‐scale phylogenetic inference of the RNR family by using the conserved α/β barrel core to align the highly diverse sequences (Figure 1 ). 3 Our inference, consisting of 6,779 α sequences, showed the divergence of the three biochemical classes and additionally identified a small, phylogenetically distinct “class Ø” clade, consisting of the most minimal RNRs known to‐date (Figure S1 ). In the work presented here, we perform detailed analyses on insertions and extensions around the core barrel to further understand the mechanisms by which the RNRs may have diversified.…”

“…From a biochemical perspective, they are best classified by the type of radical cofactor used to generate the catalytic thiyl radical (classes I, II, III). In our phylogenetic work, 3 all 20 inferences (10 replicates each for the sequence evolution models LG + R10 and WAG + R10) reconstructed these major biochemical classes as monophyletic clades (Figure 1b ). However, biochemical subclasses do not necessarily correspond to phylogenetic clades.…”

“…Loops 1–3 (dark green, red, blue) are involved in allosteric regulation, and the gray secondary structure elements are involved in substrate binding (starred). (b) Previously published unrooted phylogeny of 6,779 RNR α sequences 3 . Host organisms of structurally characterized RNRs are shown in circles.…”

“…(b) Previously published unrooted phylogeny of 6,779 RNR α sequences. 3 Host organisms of structurally characterized RNRs are shown in circles. In clockwise order, class I: Fj, Flavobacterium johnsoniae; Au, Actinobacillus ureae; Ec, Escherichia coli ; Ng, Neisseria gonorrhea; Bs, Bacillus subtilis; St: Salmonella typhimurium; Aa, Aquifex aeolicus; Pa, Pseudomonas aeruginosa ; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens ; class II: Ll, Lactobacillus leichmannii; Tm, Thermotoga maritima; Rs, Rhodobacter sphaeroides ; and class III: T4 , Bacteriophage T4; Tm, Thermotoga maritima .…”