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

Abstract

Human liver P450 NF25 (CYP3A4) had been previously expressed in Succhuromyces cerevisiue using the inducible GALIO-CYCl promoter and the phosphoglycerate kinase gene terminator [Renaud, J. p., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194,. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72,463 -4721 increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25 -iron-metabolite complexes. %fold, g-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6@-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b, always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25 -iron-metabolite complex. A P450 Fe(I1)-(RNO) complex was obtained upon oxidation of Nhydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase. These results show that specific activities of yeast-expressed P450 NF25 may be artificially low, owing to limiting amounts of the associated microsomal redox proteins and emphasize the importance of controlling the amounts of the different components of the monooxygenase complex in order to optimize these catalytic activities, especially when the expression system is to be used for demonstrating metabolic capacities towards new substrates.

High affinity of ergopeptides for cytochromes P450 3A

“…This activity of P450 NF25 was doubled in the presence of one equivalent of cytochrome b, (data not shown), as previously found for many other activities of this cytochrome (Peyronneau et al, 1992). The two allelic forms of P450 3A4, P450 NF25 and P450 hPCNl, gave similar bromocriptine oxidation activities.…”

“…Expression vector construction, yeast strains, yeast transformation and culture media were previously described (Pompon, 1988;Cullin and Pompon, 1988;Renaud et al, 1990, Peyronneau et al, 1992. Human P450 NF25 cDNA or P450 hPCNl cDNA, which were kindly provided by F. P. Guengerich (Beaune et al, 1986) and F. J.…”

“…adenine (Urban et al, 1990). Yeast microsomes were prepared as previously described (Peyronneau et al, 1992) using glass beads to mechanically disrupt the yeast cell walls. The microsomal pellet was homogenized in 50 mM Tris/HCl pH 7.4 containing 1 mM EDTA and 20% glycerol (by vol.…”

The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

“…An intriguing long standing question has been why is the metabolism of methoxyflurane and a minority of other substrates, such as nifedipine, p-nitroanisole, prostaglandin, lauric acid, n-methylcarbazole, chlorobenzene, p-nitrophenetole, 7-ethoxycoumarin, benzo(a)pyrene, and lidocaine, and testosterone ␤-hydroxylation so stimulated by the presence of cytochrome b 5 while the metabolism of most other substrates, including benzphetamine, is minimally stimulated, not effected, or slightly inhibited in the presence of cytochrome b 5 (Aoyama et al, 1990;Canova-Davis and Waskell, 1984;Hoffman et al, 1989;Okita et al, 1981;Peyronneau et al, 1992;Vatsis et al, 1982).…”

CYTOCHROME P450-Mediated OXIDATION OF GLUCURONIDE DERIVATIVES: EXAMPLE OF ESTRADIOL-17β-GLUCURONIDE OXIDATION TO 2-Hydroxy-Estradiol-17β-Glucuronide BY CYP 2C8

“…Monkey, mouse, and dog male or female human liver microsomes were obtained from XenoTech LLC, Tebu-Bio (Le Perray en Yvelines, France). Yeast-expressed human P450s were prepared as already described and produced by SPI-BIO (Massy, France) (Peyronneau et al, 1992). The mutated CYP 2C8 isoform R241A expressed in yeast was kindly provided by A. Melet (Unité Mixte Recherche 8601, Centre National de la Recherche Scientifique, Paris, France).…”