Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the strategy used for the generation of CYP51(R467K). Site-directed PCR mutagenesis was performed to change the triplet at position 467 from AGA to GGA as described in Materials and Methods. The NsiI-HindIII mutant fragment was ligated into the corresponding region of the wild-type gene in the yeast expression vector YEp51 allowing expression from the GAL10 promoter.
- Fig. 2.
Reduced-carbon monoxide difference spectrum of microsomal cytochrome CYP51(R467K). Shown are the reduced-carbon monoxide difference spectra of microsomal fractions, prepared as described in Materials and Methods, after induction of heterologous expression in yeast transformants containing YEp51 alone (— • —), CYP51 (——), and CYP51 (R467K) (–––).
- Fig. 3.
Comparative analysis of type II binding spectra. The magnitudes of spectra obtained, as described in Materials and Methods, with different concentrations of fluconazole bound to 0.2 nmol of CYP51 (□) or CYP51(R467K) (▵) are shown. Data were reproducible in triplicate experiments.
- Fig. 4.
Inhibition of sterol 14α-demethylase activity. Comparative plot of sterol demethylase activity in the presence of various amounts of fluconazole for CYP51 and CYP51(R467K). P450 was used to examine the inhibitory effects of fluconazole for CYP51 (□) and CYP51(R467K) (○).
- Fig. 5.
Amino acid sequences of P450s in the heme-binding region. Shown are an alignment of CYP51 amino acid sequences from humans, rats, S. cerevisiae (S.C.), Sorghum bicolor (S.B.), Mycobacterium tuberculosis (M.T.), andC. albicans (C.A.) and a comparison to the same region in CYP51(R467K) and various mammalian CYP isoforms involved in foreign compound metabolism. The conserved arginine and lysine residues are in boldface.
