Fig. 6: Ancient divergence between ThrRS-Ed and AlaRS-Ed.

a Maximum likelihood phylogenetic tree of AlaRS cis-Ed, AlaX-L, AlaX-M, AlaX-S and ThrRS-Ed protein sequences from representative organisms belonging to three domain of life reconstructed using IQ-TREE web server (Nguyen et al.⁷⁹). The bootstrap branch support ≥90 are indicated by black circle. b A schematic diagram of AlaRS-Ed showing canonical or the primary (1°) site of Zn²⁺ binding formed by HXXXH/CXGXH motifs and a hypothesised secondary (2°) transient Zn²⁺ binding site, shown as blue shaded area, where the Zn²⁺ migrates to upon substrate binding, depicted by red dashed arrow. The Zn²⁺ ion is hypothesised to migrate between the 1° and 2° sites in cycle, in sync with the catalysis cycle of AlaRS-Ed comprising of substrate binding, substrate hydrolysis and product release. c A schematic model depicting the pre-LUCA evolutionary divergence of AlaRS-Ed and ThrRS-Ed in terms of Zn²⁺ binding strength by virtue of a difference in a residue that is part of hydrophobic shell surrounding the coordination sphere, leading to distinct sensitivity of their activities toward ROS. The distinct sensitivity of AlaRS-Ed and ThrRS-Ed activities toward ROS underscores differential quality control (QC) of aa-tRNA^Ala vs aa-tRNA^Thr that encodes mistranslation landscape of Ala- vs Thr-codons in response to oxidative stress, reinforced by an interplay of potential benefit and penalty associated with mistranslation of respective amino acids.