Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme.
Cytochrome P-450 (P450) NF, a member of the P450 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 P450 NF was isolated from a bacteriophage lambda gt11 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 P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 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 P450 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 b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 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 P450 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.
Renaud
J P
JP
Centre National de la Recherche Scientifique, Unité de Recherche Associée 400, Paris, France.
Cullin
C
C
Pompon
D
D
Beaune
P
P
Mansuy
D
D
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006801:Humans; D007150:Immunohistochemistry; D018942:Macrolides; D008745:Methylation; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009543:Nifedipine / Q000494:pharmacology; D010084:Oxidation-Reduction; D010957:Plasmids; D011802:Quinidine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013053:Spectrophotometry; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D018942:Macrolides; D003577:Cytochrome P-450 Enzyme System; D009543:Nifedipine; D011802:Quinidine