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