Expression of human liver cytochrome P 450 IIIA4 in yeast

Cytochrome P ‐450 ( P 450) NF, a member of the P 450 IIIA subfamily, is the major contributor to the oxidation of the calcium‐channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P 450 NF was isolated from a bacteriophage λgt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83 , 8064–8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8–2 [Cullin, C. & Pompon, D. (1988) Gene 65 , 203–217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous – CO spectrum typical of cytochrome P ‐450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P 450‐5 (a form isolated from human liver indistinguishable from P 450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8–2 alone), as revealed by immunoblotting with anti‐ P 450–5 antibodies. On the other hand, antibodies raised in rabbits against human liver P 450 IIC8–10 and rat liver P 450 IA1 and P 450 IIE1 did not recognize yeast‐expressed P 450 NF25. The P 450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast‐expressed P 450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P 450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4‐oxidation, quinidine 3‐hydroxylation and N ‐oxygenation, and N ‐demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH—cytochrome P ‐450 reductase thus couples efficiently with the heterologous P 450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH —cytochrome P ‐450 reductase increased the oxidation rates. Rabbit liver cytochrome b 5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P 450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P ‐450 (lanosterol 14‐demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P ‐450 (30 times less). Thus, yeast microsomes containing P 450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P ‐450.