Dual-mode recognition of tRNA isoacceptors by Toxoplasma gondii Prolyl-tRNA synthetase.

Prolyl-tRNA synthetases (ProRSs) exhibit diverse domain architectures and motifs, evolving into prokaryotic (P-type) and eukaryotic/archaeal (E-type) variants. Both types exhibit high specificity for the recognition and aminoacylation of their cognate tRNAs. Interestingly, the parasitic eukaryote Toxoplasma gondii encodes a single E-type ProRS (TgProRS) but utilizes two distinct tRNA isoacceptors: a cytosolic E-type (with C72/C73) and an apicoplast P-type (with G72/A73). Our study demonstrates that TgProRS, despite being classified as an E-type enzyme, efficiently charges both tRNA isoacceptors and functionally compensates for yeast cytoplasmic and mitochondrial ProRS activities. Notably, while C72/C73 are dispensable for cytosolic tRNA charging, G72/A73 are crucial for apicoplast tRNA aminoacylation. Furthermore, Mutations in the motif 2 loop selectively affect E- or P-type tRNA recognition. While TgProRS exhibits similar susceptibility to azetidine (a proline mimic) when charging both tRNA types, cytosolic tRNA charging is five times more sensitive to inhibition by halofuginone (a Pro-A76 mimic) compared to apicoplast tRNA charging. These findings underscore TgProRS's dual functionality, showcasing its remarkable evolutionary adaptability and providing valuable insights for developing more selective therapeutic agents.