Evolutionary remodeling of ubiquinone biosynthesis in Toxoplasma gondii reveals an essential bi-functional...

Apicomplexan parasites like Toxoplasma gondii harbor a divergent mitochondrial proteome, much of which remains uncharacterized despite its essentiality for parasite survival. One such essential pathway is ubiquinone (UQ) biosynthesis. Here, we characterize the UQ synthesis machinery in T. gondii and show that conserved enzymes, TgCoq3 and TgCoq5, are essential for growth and mitochondrial function, and associate in a multi-protein complex. Using proximity labeling and subcellular fractionation, approaches suited to detect low-abundance proteins, we identify TgCoqFMO, a FAD-dependent monooxygenase required for UQ synthesis. Unlike canonical eukaryotic systems that employ multiple monooxygenases to modify specific carbons on the UQ aromatic ring, TgCoqFMO catalyzes two distinct hydroxylation steps, an activity not previously reported in eukaryotes. Molecular docking and chemical screening identified TgCoqFMO inhibitors that impair tachyzoite growth and reduce bradyzoite viability. These findings reveal a divergent UQ biosynthesis pathway with fewer components in apicomplexans and establish TgCoqFMO as a promising antiparasitic target.