Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

摘要

Cytochrome P45O 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, ob- tusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydro- lanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-A crystal structures re- ported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tubercufosis (MTCYP51) are the first struc- tures of an authentic P45O drug target. MTCYP51 exhibits the P45O fold with the exception of two striking differencesa bent I helix and an open conformation of BC loop-that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P45OBM3. Al- though a channel analogous to that in P45OBM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/prod- uct-out openings in CYP51. Mapping mutatians identified in Can- dida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis far rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.