The homochirality of amino acids is vital for the functioning of the translation apparatus. L-Amino acids predominate in proteins and D-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in D-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNAsynthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of D-amino acids recognition. Here, we showed that D-alanine was effectively activated by AlaRS and D-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that D-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes D-aminoacyl-tRNAs, was inactive against D-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.
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