Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

J Lipid Res. 2004 Nov;45(11):2000-7. doi: 10.1194/jlr.M400239-JLR200. Epub 2004 Aug 16.


14alpha-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC(50) for fluconazole, suggesting that F145 (conserved only in fungal 14alpha-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antifungal Agents / pharmacology*
  • Binding Sites
  • Catalytic Domain
  • Cytochrome P-450 Enzyme System / chemistry*
  • DNA Primers / chemistry
  • Fluconazole / pharmacology*
  • Fungal Proteins / chemistry*
  • Humans
  • Inhibitory Concentration 50
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Mycobacterium tuberculosis / metabolism
  • Oxidoreductases / chemistry*
  • Protein Binding
  • Protein Conformation
  • Protein Isoforms
  • Sequence Homology, Amino Acid
  • Species Specificity
  • Spectrophotometry
  • Sterol 14-Demethylase
  • Sterols / chemistry*
  • Time Factors


  • Antifungal Agents
  • CYP51A1 protein, human
  • DNA Primers
  • Fungal Proteins
  • Protein Isoforms
  • Sterols
  • Fluconazole
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Sterol 14-Demethylase