Abstract
Apicomplexan parasites like Toxoplasma gondii harbor a highly divergent mitochondrial proteome, much of which remains uncharacterized despite its essentiality for parasite survival. One such critical 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, forming a multi-protein complex. Using proximity labeling and subcellular fractionation, a strategy suited for detecting proteins of low abundance, we identify TgCoqFAD, a unique FAD-dependent monooxygenase required for UQ synthesis. Unlike canonical eukaryotic systems that employ multiple monooxygenases to modify specific carbons on the UQ aromatic ring, TgCoqFAD catalyzes two distinct hydroxylation steps, an activity not previously reported in eukaryotes. Molecular docking and chemical screening identified TgCoqFAD inhibitors that impair tachyzoite growth and bradyzoite viability. These findings reveal a streamlined and divergent UQ biosynthesis pathway in apicomplexans and establish TgCoqFAD as a promising antiparasitic target.