The circular genome of the Plasmodium falciparum 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 xm5s2U, 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 xm5s2U biosynthetic pathway in the apicoplast of P. falciparum. Through comparative genomics, we identified apicoplast-targeted orthologs of key modification enzymes: SufS, MnmA, MnmE, and MnmG. While SufS and MnmA were previously shown to catalyze s2U modifications, we now demonstrate that MnmE and MnmG are essential for apicoplast maintenance. Notably, we found that P. falciparum lacks orthologs of MnmC, MnmL, and MnmM, suggesting that the parasites contain a minimal xm5s2U 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 ΔmnmA parasites. Our data strongly support the presence and essentiality of xm5s2U modifications in apicoplast tRNAs. This study advances our understanding of the minimal requirements for protein translation in the apicoplast organelle.

tRNA modifying enzymes MnmE and MnmG are essential for Plasmodium falciparum apicoplast maintenance