Purification and Characterization of Rat Sterol 14-Demethylase P450 (CYP51) Expressed in Escherichia coli

Sterol 14-demethylase P460 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, 0., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T, and Yoshida, Y. (1996) J. Biochenu 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 mn, 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 Km and Vmnx values for lanosterol were 10.5 μM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 jjM 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 IC50 values of 0.2, 0.7, and 160 respectively.