Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum

Leishmaniasis is a major health problem that affects populations of similar to 90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14 alpha-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14 alpha-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V-max of similar to 10 and 8min(-1), respectively), it is also found to 14 alpha-demethylate C4-dimethylated lanosterol (V-max = 0.9 min(-1)) and C4-desmethylated 14 alpha-methylzymosterol (V-max = 1.9 min(-1)). Binding parameters with six sterols were tested, with K-d values ranging from 0.25 to 1.4 mu M. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14 alpha-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.