Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690

Some aaRSs (aminoacyl-tRNA synthetases) develop editing mechanisms to correct mis-charged tRNA. The CP1 (connective peptide 1) domain of LeuRS (leucyl-tRNA synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690 (5-fluoro-1,3-dihydro-l-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of G/LeuRS (Giardia lamblia LeuRS) has been identified. Similar substitution with the ESI from HsLeuRS (Homo sapiens LeuRS) impeded the leucine activation, aminoacylation and post-transfer editing of the enzyme, but had no effect on the editing specificity toward non-specific amino acids. The(341) in G/LeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNA(Leu) synthesis in vitro and in vivo. The Arg(338) was crucial for tRNA(Leu) charging and the Asp(440) was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg(338) and Asp(440) of G/LeuRS. Interestingly, G/LeuRS was completely resistant to the AN2690, which is an inhibitor of various LeuRSs. The universally conserved aspartate residue in the LeuRS CP1 domains was responsible for the resistance of G/LeuRS and another recently reported AN2690-resistant AaLeuRS (Aquifex aeolicus LeuRS). Our results indicate the functional divergence of some absolutely conserved sites, improve the understanding of the editing function of eukaryotic/archaeal LeuRSs and shed light on the development of a G/LeuRS-specific inhibitor for the treatment of giardiasis.