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

## TL;DR

A single enzyme in Toxoplasma gondii can charge two different tRNA types, revealing unique evolutionary adaptations and potential for drug development.

## Contribution

Discovery of a single E-type ProRS in Toxoplasma gondii that can functionally charge both E- and P-type tRNAPro isoacceptors.

## Key findings

- TgProRS efficiently aminoacylates both cytosolic and apicoplast tRNAPro isoacceptors.
- C72/C73 are dispensable for cytosolic tRNAPro charging, but G72/A73 are crucial for apicoplast tRNAPro.
- TgProRS compensates for both cytoplasmic and mitochondrial ProRS activities in yeast.

## 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.

Toxoplasma gondii encodes a single E-type prolyl-tRNA synthetase (TgProRS) but utilizes two distinct tRNAPro isoacceptors: a cytosolic E-type (with C72/C73) and an apicoplast P-type (with G72/A73). TgProRS efficiently charges both isoacceptors and compensates for yeast cytoplasmic and mitochondrial ProRS activities.

Despite being an E-type enzyme, TgProRS efficiently aminoacylates both E- and P-type tRNAsPro.TgProRS selectively charges cytosolic tRNAPro independently of C72/C73, while G72/A73 is crucial for apicoplast tRNAPro.TgProRS’s relaxed specificity and adaptability offer insights into ProRS evolution.

Despite being an E-type enzyme, TgProRS efficiently aminoacylates both E- and P-type tRNAsPro.

TgProRS selectively charges cytosolic tRNAPro independently of C72/C73, while G72/A73 is crucial for apicoplast tRNAPro.

TgProRS’s relaxed specificity and adaptability offer insights into ProRS evolution.

Toxoplasma gondii encodes a single E-type prolyl-tRNA synthetase (TgProRS) but utilizes two distinct tRNAPro isoacceptors: a cytosolic E-type (with C72/C73) and an apicoplast P-type (with G72/A73). TgProRS efficiently charges both isoacceptors and compensates for yeast cytoplasmic and mitochondrial ProRS activities.