The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.