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-1-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of GlLeuRS (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. Thr341 in GlLeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was crucial for tRNALeu charging and the Asp440 was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg338 and Asp440 of GlLeuRS. Interestingly, GlLeuRS 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 GlLeuRS 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 GlLeuRS-specific inhibitor for the treatment of giardiasis.
Zhou
Xiao-Long
XL
State Key Laboratory of Molecular Biology and Graduate School of the Chinese Academy of Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, People's Republic of China.
Tan
Min
M
Wang
Meng
M
Chen
Xin
X
Wang
En-Duo
ED
D001896:Boron Compounds / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005872:Giardia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D046249:Transfer RNA Aminoacylation
D001896:Boron Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D007935:Leucine-tRNA Ligase; C512998:tavaborole