The accuracy of translation is crucial for ensuring cellular homeostasis and maintaining cellular integrity. Noteworthy, the homochirality of synthesized proteins is determined by their composition: L-amino acids and achiral glycine. In contrast, D-amino acids are the components of bacterial cell walls and regulatory molecules in the nervous and humoral system in eukaryotes, etc., and their other physiological roles in biological systems have been recently evaluated. Therefore, ensuring of the correct selection of an appropriate amino acid enantiomer is critical for protein biosynthesis. However, it is worth noting that the role of cis-editing factors (editing domains of aminoacyl-tRNA synthetases) in establishing of amino acid stereoselectivity during translation remains poorly investigated. Therefore, there is a necessity to study the stereospecificity of ARSases among different structural classes to elucidate the mechanisms of L-aminoacyl-tRNA selection. Hitherto, the potential of ARSases to edit D-aminoacyl-tRNA by their editing domains was unexplored. In addition, the mechanism of D-aminoacyl-tRNAs hydrolysis by D-aminoacyl-tRNA-deacylase (DTD) has not been extensively verified by experimental studies. Therefore, in our work we aimed to study the role of two ARSases, representatives of classes I (TyrRS) and II (AlaRS), in the maintenance of stereospecificity in translation apparatus, and to elucidate the mechanism of hydrolysis of tRNAs, activated with D-amino acids, by DTD. Taking into consideration the uniqueness of TyrRS in the recognition of Tyr enantiomers and the ability to attach L-Tyr to both the 2′- and 3′-hydroxyl groups of the terminal (A76) adenosine of tRNATyr, the selection of this synthetase is logical. The attachment sites of D-Tyr have not been clarified yet. In turn, AlaRS is a promising representative of class II of ARSases; it may mistakenly activate achiral Gly and Ser and it also contains an editing domain for correction of these errors. However, whether AlaRS could also activate the D-Ala and D-serine and edit noncognate D-aminoacylated substrates remained unknown. Analysis of the kinetic parameters (kcat and Km) of activation of homologous and non-homologous amino acids in the ATP-PPi exchange reaction revealed that Thermus thermophilus TyrRS did not show any discrimination between D- and L-Tyr (discrimination factor was only 1:24). The absence of significant distinction of substrates was also observed at the stage of aminoacyl-tRNATyr formation (1:19). Regarding AlaRS, it was shown for the first time that it may activate D-Ala and D-Ser with discrimination levels of 1:467 and 1:180645, as well as to attach them to tRNAAla. The activation levels of Gly (1:193) and L-Ser (1:237) for AlaRS are consistent with those obtained for the mutant form of E. coli AlaRS (C666A), editing-deficient, and reach the ratio 1:207 and 1:107, respectively. Activation of D-Ala and D-Ser was also checked on E. coli AlaRS (C666A). These data overlap with the data on T. thermophilus AlaRS. Therefore, we found that D-amino acids have different levels of affinity and activation in class I and II ARSases. Using modified at 2′- and 3′-positions of terminal adenosine of ribose 2′- and 3′-dA76 tRNATyr and analysis of kinetic studies we elucidated the primary attachment site of D-Tyr to tRNATyr and determined the role of last one during the hydrolysis of D-Tyr-tRNATyr. We found that the 2′-OH group of tRNATyr plays a dual role: on one hand, it is a D-Tyr attachment site by TyrRS and on the other, it is necessary for hydrolysis of D-aminoacyl-tRNA by DTD. Analysis of T. thermophilus DTD activity in deacylation reactions of A76/2′d/3′d-tRNATyr substrates confirmed the importance of both hydroxyl groups of tRNATyr for hydrolysis of D-Tyr-tRNATyr. The data of computer simulations of D-Tyr-tRNATyr in complex with DTD and proposed quantum-chemical model of hydrolysis of these complexes are in full agreement with experimental studies. We found that the key factor for hydrolysis of D-aminoacyl-tRNA by DTD is the presence of the 2′-OH group of the A76 residue of D-Tyr-tRNATyr and two water molecules. We also showed the importance of the basic carbonyl groups of amino acids Gly137-Pro138 and Ala127-His128 of the enzyme. In biochemical testing with [32P]-labelled Gly/L-Ala/D-Ala-tRNAAla we also found that AlaRS was able to perform effective hydrolysis of D-Ala-tRNAAla unlike DTD, which did not exhibit any hydrolytic ability. In contrast, Gly/L-Ala-tRNAAla was hydrolyzed by both synthetase and deacylase. Thus, we established the role of the AlaRS editing domain in providing amino acid stereospecific selection. In general, it was shown that class II ARSases can also activate D-amino acids and edit the errors of enantioselective selection. The mechanism of hydrolysis of D-aminoacyl-tRNA by DTD was identified and the role of tRNA in this catalysis was demonstrated.
