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

^*Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146; and ^‡Departments of Molecular Biology and Biochemistry, Physiology, and Biophysics, and the Program in Macromolecular Structure, University of California, Irvine, CA 92617-3900

Edited by Johann Deisenhofer, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 17, 2001 (received for review November 26, 2000)

Abstract

Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. 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-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a 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 P450BM3. Although a channel analogous to that in P450BM3 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/product-out openings in CYP51. Mapping mutations identified in Candida 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 for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Footnotes

↵ † To whom reprint requests should be addressed. E-mail: podustlm{at}ctrvax.vanderbilt.edu.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 1E9X and 1EA1).
Abbreviations:

4-PI,

4-phenylimidazole;

FLU,

fluconazole