Abstract
D-aminoacyl-tRNA deacylase (DTD), a trans-editing factor found in all bacteria and eukaryotes, removes D-amino acids mischarged on tRNAs. A cross-subunit Gly-cisPro motif forms the mechanistic basis of L-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a “gain of function” through L-chiral selectivity in ATD resulting in the clearing of L-alanine mischarged on tRNAThr(G4•U69) by eukaryotic AlaRS. Animalia genomes enriched in tRNAThr(G4•U69) are in strict association with the presence of ATD, underlining the necessity for a dedicated factor to proofread tRNA misaminoacylation. The study highlights the role of ATD during tRNA gene expansion as a key event associated with the evolution of Animalia.