Human trans-editing enzyme displays tRNA acceptor-stem specificity and relaxed amino acid selectivity.

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNAPro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.