tRNA modifying enzymes MnmE and MnmG are essential for apicoplast maintenance.

The circular genome of the apicoplast contains a complete minimal set of tRNAs, positioning the apicoplast as an ideal model for studying the fundamental factors required for protein translation. Modifications at tRNA wobble base positions, such as xmsU, are critical for accurate protein translation. These modifications are ubiquitously found in tRNAs decoding two-family box codons ending in A or G in prokaryotes and in eukaryotic organelles. Here, we investigated the xmsU biosynthetic pathway in the apicoplast organelle of . Through comparative genomics, we identified orthologs of enzymes involved in this process: SufS, MnmA, MnmE, and MnmG. While SufS and MnmA were previously shown to catalyze sU modifications, we now show that MnmE and MnmG are apicoplast-localized and contain features required for xmsU biosynthetic activity. Notably, we found that lacks orthologs of MnmC, MnmL, and MnmM, suggesting that the parasites contain a minimal xmsU biosynthetic pathway similar to that found in bacteria with reduced genomes. Deletion of either MnmE or MnmG resulted in apicoplast disruption and parasite death, mimicking the phenotype observed in Δ and Δ parasites. Our data strongly support the presence and essentiality of xmsU modifications in apicoplast tRNAs. This study advances our understanding of the minimal requirements for protein translation in the apicoplast organelle.