Molecular Characterization of Sterol C4-Methyl Oxidase in .

Sterol biosynthesis requires the oxidative removal of two methyl groups from the C-4 position by sterol C-4-demethylase and one methyl group from the C-14 position by sterol C-14-demethylase. In , a CYP5122A1 (Cytochrome P450 family 5122A1) protein was recently identified as the bona fide sterol C-4 methyl oxidase catalyzing the initial steps of C-4-demethylation. Besides CYP5122A1, parasites possess orthologs to ERG25 (ergosterol pathway gene 25), the canonical sterol C-4 methyl oxidase in . To determine the contribution of and in sterol biosynthesis, we assessed the essentiality of these genes in , which causes cutaneous leishmaniasis. Like in , in could only be deleted in the presence of a complementing episome. Even with strong negative selection, chromosomal -null mutants retained the complementing episome in both promastigote and amastigote stages, demonstrating its essentiality. In contrast, the -null mutants were fully viable and replicative in culture and virulent in mice. Deletion and overexpression of ERG25 did not affect the sterol composition, indicating that ERG25 is not required for C-4-demethylation. These findings suggest that CYP5122A1 is the dominant and possibly only sterol C-4 methyl oxidase in , and inhibitors of CYP5122A1 may have strong therapeutic potential against multiple species.