Purification and characterization of rat sterol 14-demethylase P450 (CYP51) expressed in Escherichia coli

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosyn thesis 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 cDN A was expressed in Escherichia coli with modification of the N-terminal ami no acid sequence, and the expressed protein (CYP51m) was purified to gel-el ectrophoretic homogenity. The spectrophotometrically determined specific co ntent of CYP51m was 16 nmol/mg protein and the apparent molecular weight wa s estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduc ed CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihy drolanosterol upon reconstitution with NADPH-P450 reductase purified from r at liver microsomes. The apparent K-m and V-max values for lanosterol were 10.5 mu M and 13.9 nmol/min/nmol P450, respectively, and those for 24,25-di hydrolanosterol were 20.0 mu M and 20.0 nmol/min/nmol P450, respectively. T he lanosterol demethylase activity of the reconstituted system of CYP51m wa s inhibited by ketoconazole, itraconazole and fluconazole with apparent IC5 0 values of 0.2, 0.7, and 160 mu M, respectively.