Dual-mode recognition of tRNA^Pro 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^Pro 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^Pro isoacceptors and functionally compensates for yeast cytoplasmic and mitochondrial ProRS activities. Notably, while C72/C73 are dispensable for cytosolic tRNA^Pro charging, G72/A73 are crucial for apicoplast tRNA^Pro aminoacylation. Furthermore, Mutations in the motif 2 loop selectively affect E- or P-type tRNA^Pro recognition. While TgProRS exhibits similar susceptibility to azetidine (a proline mimic) when charging both tRNA^Pro types, cytosolic tRNA^Pro charging is five times more sensitive to inhibition by halofuginone (a Pro-A76 mimic) compared to apicoplast tRNA^Pro charging. These findings underscore TgProRS's dual functionality, showcasing its remarkable evolutionary adaptability and providing valuable insights for developing more selective therapeutic agents.