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 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.
Nitahara
Y
Y
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo, 192-8577, Japan.
Aoyama
Y
Y
Horiuchi
T
T
Noshiro
M
M
Yoshida
Y
Y
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase
D000935:Antifungal Agents; C548418:CYP51A1 protein, human; C548417:Cyp51 protein, rat; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase