Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

Abstract

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

“…f) 6β-Hydroxylation of testosterone-The assay for testosterone 6β-hydroxylation was performed as described previously [47] (20 μM substrate, 10 nM CYP3A4, 20 nM cytochrome b 5 , 10 min at 28 °C).…”

“…The reference activities that were followed to measure these inhibitory effects were 7-benzyloxyresorufin O-debenzylase [42], paclitaxel 6α-hydroxylase [43], diclofenac 4′-hydroxylase [62] and testosterone 6β-hydroxylase [47] respectively. The substrate concentrations used in these experiments were equal to the Km of the activity followed for each CYP (see Materials and Methods).…”

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

“…The assay for testosterone 6P-hydroxylation (Brian et al, 1990) was carried out exactly as for nifedipine oxidation except that the testosterone concentration was 50 pM and the same HPLC conditions were used for analysis (Renaud et al, 1990). 1-Dehydrotestosterone was used as a standard.…”

“…A fruitful alternative consists of the expression of cloned cDNA, coding for individual forms of P450 enzymes, in an adequate heterologous system. Several human liver P450 from the subfamilies involved in xenobiotic oxidation have already been expressed in different systems, including recombinant simian virus 40 in COS cells and references therein; Romkes et al, 1991;Veronese et al, 1991), recombinant vaccinia viruses in human hepatoma Hep G2 cells (Aoyama et al, 1990a and1990b, and references therein) and recombinant plasmids in the yeast Succharomyces cerevisiae (Yasumori et al, 1989;Brian et al, 1989;Brian et al, 1990;Renaud et al, 1990;Eugster et al, 1990;Ching et al, 1991; Truan, G., Cullin, C., Reisdorf, P., Urban, P. and Pompon, D., unpublished results). P450 NF25 (CYP3A4) is important in pharmacology and toxicology, not only because it is probably the major form of human liver (Guengerich and Turvy, 1991) but also because it is involved in the metabolism of numerous widely used drugs such as nifedipine (Guengerich et al, 1986a), erythromycin and troleandomycin (Renaud et al, 1990 and references therein), quinidine (Guengerich et al, 1986b), cyclosporin A (Kronbach et al, 1988;Aoyama et al, 1989;Combalbert et al, 1989), 1 7a-ethynylestradiol (Guengerich, 1988), midazolam (Kronbach et al, 1989), lidocaine (Bargetzi et al, 1989;Imaoka et al, 1990), and diltiazem (Pichard et al, 1990).…”

“…P450 NF25 (CYP3A4) is important in pharmacology and toxicology, not only because it is probably the major form of human liver (Guengerich and Turvy, 1991) but also because it is involved in the metabolism of numerous widely used drugs such as nifedipine (Guengerich et al, 1986a), erythromycin and troleandomycin (Renaud et al, 1990 and references therein), quinidine (Guengerich et al, 1986b), cyclosporin A (Kronbach et al, 1988;Aoyama et al, 1989;Combalbert et al, 1989), 1 7a-ethynylestradiol (Guengerich, 1988), midazolam (Kronbach et al, 1989), lidocaine (Bargetzi et al, 1989;Imaoka et al, 1990), and diltiazem (Pichard et al, 1990). P450 NF25 was recently functionally expressed in S. cerevisiue (Renaud et al, 1990;Brian et al, 1990). Galactoseinducible expression using GALIO-CYCl, a hybrid promoter composed of the yeast GAL10 gene upstream-activating sequence and the iso-1-cytochrome c gene transcription-initiation sequence (Guarente et al, 1982), allowed relatively good levels of P450 NF25 to be obtained in transformed yeast (approximately 2 -3 nmol/l culture), yeast microsomes to be obtained containing NF25 as the only detectable P450 and in a catalytically active state, and an expression level high enough to allow spectrophotometric binding studies on yeast microsomes (Renaud et al, 1990).…”