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

Eur. J. Biochem. 207, 109- 116 (1992) 0 FEBS 1992

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5 Marie-Anne PEY RONNEAU ’, Jean-Paul RENAUD I , Gilles TRUAN’, Philippe URBAN ’, Denis POMPON and Daniel MANSUY I Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique,

Universite Paris 5 , France Centre de Genbtique Moltculaire, Centre National de la Recherche Scientifique Associee i I’Universite Paris 6, Gif-sur-Yvette, France

(Received March 11, 1992) - EJB 92 0338

Human liver P450 NF25 (CYP3A4) had been previously expressed in Succhuromyces cerevisiue using the inducible GALIO-CYCl promoter and the phosphoglycerate kinase gene terminator [Re- naud, J. p., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J . Biochem. 194, 889- 8961. 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 N- hydroxyamphetamine, 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.

Correspondence to D. Mansuy, Laboratoire de Chimie et Bio- chimie Pharmacologiques et Toxicologiques, CNRS URA 400,45 rue des Saints-Pkres, F-15270 Paris Cedex 06, France

Fax; 33 1 42 86 04 02. Abbreviations. Cyt. b5, cytochrome b5 ; P450 reductase, NADPH-

P450 reductase; V8, YeDP1/8-2 plasmid; V60, YeDP60 plasmid. Enzymes. NADPH-P450 reductase [EC 1.6.2.41; P450 [EC

1.14.14.1]. Note. The updated recommended nomenclature for P450 species

[Ncbert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., & Waxman, D. J. (1991) DNA 10, 1 - 141 is used throughout the text. The name ‘cytochrome’ has been abandoned according to the Nomenclature Committee of the International Union of Biochem- istry, Nomenclature of electron-transfer proteins, Recommendations 1989 [Eur. J . Biochem. 200, 599-612 (1991)l the appropriate name being ‘haem-thiolate protein’.

P450 form a superfamily of heme-thiolate proteins involved in the primary oxidation of numerous lipophilic com- pounds including endogenous substrates like fatty acids, ster- oids and vitamins, as well as exogenous substrates like drugs, dietary substances and environmental pollutants. The broad substrate specificity is now well understood on the basis of enzyme multiplicity (Gonzalez, 1989). More than 160 cDNA species coding for P450 have been isolated so far and have been classified on the basis of primary amino acid sequence similarities (Nebert et al., 1991). Although the normal fate of xenobiotic oxidation products is excretion, directly or after further conjugation with a polar group, P450 catalysis sometimes yields highly reactive metabolites that can injure cells by altering macromolecular components, leading especially to carcinogenesis (Kadlubar and Hammons, 1987). Other del- eterious effects can arise during the course of xenobiotic oxi-

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dation by human liver P450 due to drug interactions. For example, when two or more drugs are competitively metabolized by the same form of P450 or when a drug specifically induces or inhibits the form responsible for the metabolization of other drugs, this can lead to a reduced or prolonged therapeutic action and potentially toxic side effects. In order to predict hepatic drug metabolism and possible toxic effects and drug interactions, a precise knowledge of the specific activities of individual forms of P450 from human liver toward different classes of substrates is required. For that purpose, numerous groups have studied the properties of human hepatic P450 species using either hepatocytes, liver microsomes or purified enzymes. However, working with human tissues leads to many problems from an ethical and technical point of view, as well as problems of availability, reproducibility and interindividual variability. A fruitful alter- native 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 (Gonzalez, 1989 and references therein; Romkes et al., 1991; Veronese et al., 1991), recombinant vaccinia viruses in human hepatoma Hep G2 cells (Aoyama et al., 1990a and 1990b, 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), mida- zolam (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). Galactose- inducible expression using GALIO-CYCl, a hybrid promoter composed of the yeast GAL10 gene upstream-activating sequence and the iso-1-cytochrome c gene transcription-in- itiation 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). Actually, the main factor limiting the catalytic activity of transformed yeast microsomes was the endogenous NADPH-P450 reductase (P450 reductase) present in yeast but not in significant amounts.

A possible way to obtain more catalytically active yeast strains expressing P450 NF25, could be to coexpress the associated electron-transfer proteins by integration of galactose- inducible expression cassettes into the yeast genome under the control of an efficient and externally controllable promoter. This kind of coexpression in yeast has been recently described and characterized elsewhere (Pompon et al., 1991 ; Truan et al., unpublished results).

Here we report the catalytic activity of P450 NF25 expressed in yeast also overexpressing its own P450 reductase

Table 1. Strains used in this study. (0ver)expression was under the control of the artificial, galactose-inducible GAL10-CYCl promoter.

Strain Strain

type (over) expression phenotype

W(N) Wild(W303) - [URA-, ADE-] W(R) yeast reductase [URA+/-, ADE-] W(R,N) W (R) x W (N) yeast reductase [URA+'-, ADE-] W(B) human cyt. b5 [URA', ADE-] W(B,N) W (B) x W (N) human cyt. b5 [URA', ADE-] W(B,R) W (B) x W (R) yeast reductase

human cyt. b5 [URA', ADE-]

and/or coexpressing human cytochrome bs (cyt. bs) at different levels towards different substrates to show the usefulness of such a coexpression system in determining the effects of changes in the electron-transfer chain within the monooxygenase complex on P450 NF25 activities, and in dramatically increasing the specific activities of this major human hepatic isoform in transformed-yeast microsomes.

MATERIALS AND METHODS

Chemicals and reagents

All molecular biological reagents were of analytical grade. The ingredients for culture media were obtained from Difco (OSI, Paris, France). Nifedipine, nitrendipine, testosterone, 1- dehydrotestosterone and lidocaine were purchased from Sigma (St Louis, MO), and 6P-hydroxytestosterone was obtained from Steraloids (Wilton, NH). NADPH and horse heart cytochrome c were obtained from Boehringer Mann- heim France (Meylan, France). Monoethylglycine xylidide was given by Laboratoires Roger Bellon (Neuilly-sur-Seine, France) and 1 SP-hydroxytestosterone was a gift from Searle (Skokie, IL). N-Hydroxyamphetamine was prepared from 2- nitro-1 -phenylpropene by LiAlH4 reduction (Gilsdorf and Nord, 1952).

Eschevichiu coli strains

supE441, A-) was used for cloning. E. coli strain DH5- 1 (F-, recAl, gyrA96, thi-1, hsdR17,

S. cerevisiue strains (Table 1)

W303-1B (leu2, his3, trpl, ade2-1, ura3, canR, cyr') was constructed by R. Rothstein. This is the 'wild-type' strain W(N). The construction and characterization of the other strains used in this work are reported elsewhere (Pompon et al., 1991; Truan et al., unpublished results):

W(R) (MATE) comes from the stable integration into the genome of W(N) (MATa) of the galactose-inducible GALlO- CYCl promoter at the 5'-end of the yeast P450 reductase gene open reading frame; W(B) (MATa) comes from the stable integration into the genome of W(N) (MAT'), by disruption of the yeast P450 reductase gene, of the human cyt.-b5-coding sequence placed under the control of the GALlO-CYCI promoter and the phosphoglycerate kinase gene terminator; W(R, N) and W(B, N) are diploids [W(R) (MATol)xW(N) (MAT') and W(B) (MATa) x W(N) (MATE) respectively]; W(R, N) contains one copy of the yeast P450 reductase gene

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placed under the control of the GAL10-CYCI promoter and W(B, N) contains one copy of the human cyt.-b5-coding sequence placed under the control of the GALIO-CYCl promoter; W(B, R) is also a diploid [W(B) (MATa)xW(R) (MATa)] which contains one copy of the human cyt.-b,-coding sequence and one copy of the yeast P450 reductase gene, both placed under the control of the GAL10-CYCI promoter.

Expression vectors

The construction of plasmids YeDP1/8 - 2 (V8) (Pompon, 1988; Cullin and Pompon, 1988) and YeDP60 (V60) (Urban et al., 1990) was reported earlier. Plasmid V8 contains a URA3 marker and plasmid V60 carries both the URA3 and ADE2 selection markers. Insertion of NF25 cDNA into V8 to give the expression plasmid NF25-V8 (formerly called pVNF25) w v described in a previous paper (Renaud et al., 1990). Inser- tion of NF25 cDNA into V60 (Truan et al., unpublished results) was achieved by using homologous recombination properties of yeast (Pompon and Nicolas, 1989).

Yeast transformation and culture

NF25-V8 is compatible with W(N), W(R)[URA-1, and W(R, N)[URA-1, while NF25-V60 is compatible with all the strains (Table 1). The transformed yeasts are denoted by the simple juxtaposition of the name of the heterologous P450 cDNA carried on the plasmid and the name of the starting strain. For instance, the strain obtained by transforming W(R) by NF25-V8 is called NF25-W(R). The strain obtained by transforming W(R) by V8 (empty plasmid not containing the foreign cDNA) is called CONTROL-W(R). Transformations were performed according to a modified lithium acetate method (Cullin and Pompon, 1988). Transformed cells were grown at 28 "C in solution A (minimal medium) for V8 (Cullin and Pompon, 1988) or solution B (complete medium) for V60 (Urban et al., 1990). Solution A was 2% (massjvol.) D-galactose, 0.7% (massjvol.) yeast nitrogen base without amino-acids, 0.1 YO (massjvol.) bactocasaminoacids, 0.002% (massivol.) tryptophan and 0.004% (massjvol.) adenine. Solu- tion B contained 2% (massjvol.) D-galactose, 1 YO (massjvol.) yeast extract and 1 YO (massivol.) bactopeptone.

Microsome preparation

After centrifugation, 4 g wet cells were resuspended in 50 ml 50 mM Tris/HCl, pH 7.4, 5 mM EDTA and 100 mM KCl containing 87 pl 2-mercaptoethanol and incubated for 5 min at room temperature. After centrifugation, cells were washed with 50ml of the same buffer, not containing 2- mercaptoethanol, and centrifuged again. The pellet was resuspended in 4 ml cold 50 mM Tris/HCl, pH 7.4, 2 mM EDTA and 1.2 M sorbitol and the volume was adjusted to 25 ml with cold 50 mM Tris/HCl, pH 7.4, 2 mM EDTA and 0.6 M sorbitol buffer. 40 g 0.45 -0.50 mm diameter glass beads (B. Braun, Melsungen, FRG) were added to the suspension and yeast cell walls were disrupted mechanically using a MKS homogenizer (B. Braun, Melsungen, FRG; 4 x 15 s at 4000 rpm) cooled with liquid COz. The beads were removed by glass-filtration and rinsed with 25 ml cold 50 mM Trisj HC1, pH 7.4, 2 mM EDTA and 0.6 M sorbitol. The filtrate was centrifuged at 4°C for 5 min at 1000 g , then for 10 rnin at 14000 g. CaClz was added to the supernatant (15mM final concentration) and the suspension was left on ice for 15 min. Microsomes were spun down by centrifuging at 4°C for

15 min at 14000 g , resuspended in a minimum volume (approximately 2 - 3 ml) cold 50 mM TrisjHCI, pH 7.4, 1 mM EDTA and 20% glycerol and kept at - 80 "C for months.

Quantitation of the different enzymes in microsomal fractions

The total microsomal protein concentration was determined according to the method of Lowry et al. (1951). Total P450 was measured according to Omura and Sat0 (1964). Cyt. b5 was quantified from the reduced versus oxidized difference spectrum of microsomes, using a differential absorption coefficient 48424-409 of 185mM-' . cm-' (Omura and Sato, 1964). NADPH-P450 reductase activity was expressed as the rate of cytochrome c reduction under slightly modified conditions from a previously described protocol (Urban et al., 1990). A solution containing 0.1 mg cytochrome c (approximately 8 nmol) in 50 mM Tris/HCl, pH 7.4, and 1 mM EDTA (final volume 940 p1) was divided equally between both cuvettes of a Kontron 820 spectrophotometer. 20 p1 fresh NADPH (12 mM in distilled water) was added to the sample cuvette, while the same volume of distilled water was added to the reference cuvette. Reduction was initiated by the addition to both cuvettes of 10 pl of a suspension containing 10 pg microsomal protein (only 1 pg for microsomes from P450-reductase-overexpressing yeasts). The absorbance change at 550nm was monitored at 20°C and the rate of cytochrome c reduction was calculated using an absorption coefficient of 21 mM-' . cm-'.

Catalytic activity studies

Rabbit liver cyt. b5 was purified according to Strittmatter et al. (1978).

Nifedipine oxidation was performed as previously described (Renaud et al., 1990).

The assay for lidocaine oxidation essentially followed the procedure of Oda et al. (1989). Typical incubations included lidocaine (0.5 pmol, added from a fresh 0.1 M stock solution in water/acetonitrile (99.5 : OS), yeast microsomes containing P450 NF25 (lOOpmol), NADPH (0.25 pmol) and 50mM TrisjHCl, pH 7.4 and 1 mM EDTA in a final volume of 0.5 ml. When rabbit liver cyt. b5 (1 mol/mol P450 from a 20 pM stock solution) was included, microsomes were incubated with cyt. b5 for 20 rnin on ice, then with the substrate for 3 min at 37°C before addition of NADPH. The reaction proceeded for 15 rnin at 37°C and was then stopped by the addition of 50 pl 1 M NaOH. The reaction mixture was extracted twice with CHzClz (800 pl and 500 p1 portions). The combined organic phases were evaporated to dryness under a N2 stream at 30°C. The residue was dissolved in 600 p1 of the mobile phase of HPLC and 100 pl were injected onto a Nucleosil CI8 reverse- phase HPLC column (5 p particle size, 4.6 mmx25 cm, Socikte Franqaise de Chromatographie sur Colonne, Neuilly- Plaisance, France) placed in line following a 1-cm-long octyldecylsilyl guard column, using 20 mM potassium phos- phate, pH 3.0 acetonitrile (9: 1) as the eluent (flow rate 1 ml/ min). The metabolites were monitored at 219 nm.

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.

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Formation of a P450 - NF25-Fe(lI) - nitrosoalkane complex followed by difference visible spectroscopy

Yeast microsomes were suspended in 100 mM Tris/HCl, pH 7.4 and 1 mM EDTA (P450 NF25 final concentration 200 nM), rabbit cyt. b, from a 20 pM stock solution was added (final concentration 200 nM) and the suspension (1 ml) was equally divided between both cuvettes of a Kontron 820 spectrophotometer. After recording the baseline, 1 p1 fresh N-hydroxyamphetamine (0.01 M in dimethylsulfoxide) was added to the sample cuvette, the same volume of solvent being added to the reference cuvette. 10 pl fresh NADPH (12 mM in water) was added to both cuvettes and difference spectra were recorded over 380 - 500 nm at different times. Measure- ments were carried out at 20°C.

RESULTS

Expression of P450 NF25 in yeast

The yeast strains used in this study have been engineered by genomic modifications of the W303 - 1B strain, considered here the 'wild-type' strain and denoted W(N). They contain a modified yeast P450 reductase gene and/or the human liver cyt. b5 coding sequence, stably integrated into the yeast genome under the control of the galactose-inducible GALIO- CYCI promoter, and are called W(R), W(B), W(R, N), W(B, N) and W(B, R). The construction of these strains and their transformation with some human and mouse P450-expressing plasmids have been described elsewhere (Pompon et al., 1991 ; Truan et al., unpublished results). NF25 cDNA was inserted in the expression vectors V8 (Pompon, 1988; Cullin and Pom- pon, 1988) and V60 (Urban et al., 1990) as described in Ma- terials and Methods and the resulting expression plasmids NF25-V8 and NF25-V60 were used to transform the various yeast strains. The compatibility between expression vectors and strains depends on whether the genetic markers present on the plasmid complement the yeast gene deficiencies or not (Table 1).

Transformants were selected and grown in galactose-containing medium. Plasmid V8 contains a URA3 marker that maintains a selective pressure for the plasmid in ura3-deficient strains grown in uracile-free minimal medium (solution A). When transformed with V8-type plasmids, yeast cells could be grown in solution A up to absorbancies of 2 - 3, and P450- NF25 specific contents of approximately 50 - 100 pmol/mg microsomal protein were obtained. When the cultures reached higher densities, the P450 specific content decreased very rap- idly, probably because most cells were no longer viable (D. Pompon, unpublished observations). Maximum yields of 1 - 2 nmol P450/1 culture could be achieved in this way. Plasmid V60, which carries both the URA3 and ADE2 selection markers, allowed transformed yeast cells to grow either in solution A without adenine or in solution B, since this complete medium contains limited amounts of adenine, as previously explained (Urban et al., 1990). When transformed with V60-type plasmids, yeast cells could be grown in solution B, with absorbancies up to 20-30 without a decrease in P450- NF25 specific content in microsomes (approximately 50 - 150 pmol/mg microsomal protein) as was demonstrated for mouse P450 1Al (Urban et al., 1990). This specific content was in the same order of magnitude as with V8, but the amounts of P450 NF25 produced/l culture were increased by a factor of five (approximately 5 - 10 nmol/l culture). The other advantage is the lower cost of complete culture medium.

Table 2. NADPH-P450 reductase content in microsomes from transformed yeast cells. P450 NF25 content was always in the range 50- 150 pmol/mg microsomal protein. Cyt. b5 content was in the range 100- 300 pmol/mg microsomal protein but it was not possible to distinguish yeast cyt. b5 and human cyt. b5 in microsomes from NF25-W(B, N), NF25-W(B, R), and NF25-W(B) with the available techniques (see Materials and Methods). The reductase activity shows the mean values of a t least two independent duplicate determinations. The standard deviation was approximately 10%.

Microsomes Reductase activity

nmol cytochrome c reduced . (mg protein)-' . min-'

60 1000 2000

25 690

< O . l

All transformed yeast cells were found to be viable on plates for months and no genetic instability was detected with the diploid cells since similar P450 levels and monooxygenase activities were found after restreaking several times. It should be emphasized that the expression cassettes in W(R) and W(B) were inserted at the yeast reductase gene locus with opposite orientations so as to limit the probability of recombination events within the diploid cells W(B, R) (Truan et al., unpublished results).

Study of the coexpression of P450 NF25, NADPH-P450 reductase and cytochrome b5 in yeast

The total P450 content, estimated by the Fe(I1)-CO versus Fe(I1) difference spectrum of microsomes according to the method of Omura and Sat0 (1964), was, in all NF25-transformed strains, in the range 50 - 150 pmol/mg microsomal protein. As described previously (Cullin and Pompon, 1988; Pompon, 1988; Urban et al., 1990; Renaud et al., 1990), the level of endogenous P450 was always found to be negligible and the level of expressed P450 NF25 was directly measured as the total P450 in microsomes. In fact, only in CONTROL- W(B) microsomes could significant amounts of endogenous yeast P450 be detected. As the human cyt.-b5 coding sequence has precisely been integrated into the yeast genome at the P450 reductase locus in W(B), an assumption was made that the deletion of the yeast P450 reductase gene was responsible for the induction of some endogenous P450 (Pompon, unpublished observations). This could explain the unusually high P450 content in NF25-W(B) microsomes (approximately 300 pmol/mg protein). Actually, in that case, P450 NF25 rep- resented only a small proportion of the total P450, which was estimated to be approximately 20 - 30% from the maximum amplitude of the difference spectrum obtained upon addition of dihydroergotamine, a specific substrate showing a high affinity for P450 NF25 (Renaud et al., 1990 and references therein), to NF25-W(B) microsomes (not shown). This had no consequences on the following comparison of the catalytic activities in microsomes from the various strains, since NF25- W(B) microsomes showed no catalytic activity.

In contrast, the yeast P450 reductase activity in microsomes from the different strains (assayed as the NADPH- cytochrome c reductase activity), showed considerable vari- ations (Table 2). In NF25-W(N) microsomes, the basic re-

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ductase activity level was 60 nmol cytochrome c reduced . min-' . (mgprotein)-'. In NF25-W(R) microsomes, because of the endogenous P450 reductase overexpression, the reductase activity reached 2000 nmol cytochrome c reduced . min-' . (mg protein)-', 33 times as much as in NF25-W(N) microsomes. As expected, no reductase activity was found in NF25-W(B) microsomes due to the deletion of the P450 reductase gene. The P450 reductase levels found in NF25- W(R, N) and NF25-W(B, R) microsomes were about half of that found in NF25-W(R) microsomes. This is in good agreement with the fact the two former strains are diploids containing a single copy of the yeast P450 reductase gene placed under the control of the GALIO-CYC1 promoter and should thus show intermediate levels of P450 reductase overexpression. In NF25-W(B, N) microsomes, the P450 reductase level was about half of that found in NF25-W(N) microsomes. Consistently, NF25-W(B, N) is a diploid which contains only one copy of the wild-type P450 reductase gene.

The cyt. b5 content of the various yeast strains was determined from the reduced versus oxidized difference spectrum of microsomes (Omura and Sato,1964). Only the total (yeast and human) amount could be measured this way. The total cyt. b5 content in microsomes was in the range 100 - 300 pmol/ mg protein, and there was no marked increase with the strains coexpressing human cyt. b5. In fact, the respective contri- butions of human and yeast cyt. b5 cannot be distinguished with the available techniques and the possible effects of yeast P450 reductase overexpression and of human cyt. b5 coexpression on the levels of yeast cyt. b5 expression are not known. Human cyt. b5 expression could only be deduced from the observed catalytic activities of NF25-W(B, N) and NF25- W(B, R) microsomes.

Influence of NADPH-P450 reductase and cytochrome b5 coexpression on P450 NF25 catalytic activities

Nifedipine, lidocaine and testosterone have been reported to be metabolized mainly by P450 NF25 in human liver. This isoform catalyzes the 1 ,Coxidation of the dihydropydirine ring of nifedipine to yield the corresponding pyridine (Guengerich et al., 1986a), the mono-N-deethylation of lidocaine to give monoethylglycine xylidide (Bargetzi et al., 1989; Imaoka et al., 1990), and the 6P-hydroxylation of testosterone (Kremers et al., 1981; Waxman et al., 1988). NF25- containing yeast microsomes have also been shown to catalyze nifedipine 1,4-oxidation (Brian et al., 1990; Renaud et al., 1990) and testosterone 61-hydroxylation (Brian et al., 1990). In the present work, the catalytic activities of P450 NF25- containing yeast microsomes from the engineered strains overexpressing yeast P450 reductase and/or expressing human cyt. b5 have been determined for nifedipine, testosterone, and lidocaine (Table 3).

With microsomes from the transformed 'wild-type' strain, NF25-W(N), relatively low-turnover numbers (0.04 - 0.2 min- ') were found for all tested substrates without addition of purified rabbit liver cyt. b5. Catalytic activities one order of magnitude higher were found with NF25-W(R) microsomes, thus clearly showing the beneficial effect of yeast P450 reductase overexpression. This demonstrates the effective coup- ling between the heterologous P450 NF25 and the endogenous yeast P450 reductase that was already assumed from the small, but significant, activities found in NF25-W(N). With CON- TROL-W(R), where plasmid V8 without any insert was used to transform W(R), negligible activities were found except in the case of lidocaine for which the background activity

reached the level found with NF25-W(N) microsomes. How- ever, this blank activity was still 10-times lower than that found with NF25-W(R) microsomes, in which the P450 reductase level is comparable to that found in CONTROL- W(R) microsomes. This might be due to a small endogenous N-dealkylation activity that could be stimulated by the high levels of P450 reductase in W(R). With NF25-W(R, N) microsomes, intermediate activities between those found with NF25-W(N) and NF25-W(R) microsomes were observed, in good agreement with the intermediate level of reductase activity (Table 2). As expected, NF25-W(B) microsomes, which lack any NADPH-dependent P450 reductase activity, com- pletely failed to oxidize the three substrates. This emphasizes the absolute requirement of a NADPH-P450 reductase able to transfer electrons from NADPH to a given P450 for monooxygenase activity to occur when O2 is the source of oxygen atoms. NF25-W(B, N) microsomes contain a low level of yeast P450 reductase, actually about half the level found in NF25- W(N) microsomes (Table 2). Nevertheless, the observed turnovers were higher than with the latter microsomes, indicating a stimulating effect of the presence of human cyt. b5 on all the tested activities. This also proved the coexpression of human cyt. b5 in NF25-W(B, N) to be in sufficiently high amounts to observe an efficient activating effect. This stimulating effect of human cyt. b5 was also demonstrated in the comparison between the activities measured with NF25-W(B, R) and NF25-W(R, N) microsomes; although the former contained a slightly lower level of P450 reductase (Table 2), they showed 2-4-fold higher activities than the latter. NF25-W(B, R) microsomes also showed 5 - 7-fold higher activities than NF25-W(B, N) microsomes, which is consistent with the overexpression of yeast P450 reductase.

Table 3 also shows the effects of the addition of exogenous, purified rabbit liver cyt. b5 on the activities of the different microsomes. The resulting enhancement seems to be stronger when the level of P450 reductase is lower. With NF25-W(B) microsomes, there was still no detectable activity since no P450 reductase was present. From NF25-W(N) microsomes to NF25-W(R, N) microsomes and to NF25-W(R) microsomes, the enhancement factor decreased from seven to three and to two in the case of nifedipine 1,Coxidation. For testosterone, addition of rabbit cyt. b5 also showed a greater effect when yeast P450 reductase was not overexpressed, i.e. with NF25-W(N) microsomes. In the case of lidocaine, a similar tendency was observed except that for NF25-W(N) microsomes, rabbit cyt. b5 increased the turnover number by a factor of two only, but this was due to the fact that the blank level was not negligible with this substrate and thus masked the real contribution of the P450-dependent oxidation. Lastly, for the three tested activities, addition of rabbit cyt. b5 had no effect with either NF25-W(B, N) or NF25-W(B, R) microsomes, probably because the coexpressed human cyt. b5 has already reached a saturating level.

Formation of a P450 Fe(I1)-nitrosoalkane complex

P450-NF25-containing yeast microsomes were previously used to study the binding of various substrates to this isoform by differential spectroscopy and to measure the corresponding dissociation constants (Renaud et al., 1990). Such yeast microsomes should also be used to study the formation of P450- iron-metabolite complexes. In that case, the substrate must first be oxidized to a metabolite exhibiting a high affinity for P450, generally in its ferrous state. Therefore, in order to study the formation of such complexes, yeast microsomes

114

Table 3. Nifedipine 1,4-oxidation, lidocaine Ndeethylation and testosterone 6P-hydroxylation catalyzed by yeast-expressed NF25. Experimental conditions are described in the Materials and Methods. Microsomes were prepared from yeast cells grown in solution B except for NF25- W(N) and NF25-W(R) (grown in solution A). 1 molar equivalents of purified rabbit liver cyt. b5 were added relative to P450 NF25. All values are from at least two duplicate determinations with standard deviations always 5 5-10%.

Microsomes Rabbit Nifedipine cyt. b5 1,4-oxidation

Lidocaine Testosterone N-deethylation 6P-hydroxylation

~- ~ ~~

nmol . (nmol P450)-' . min-'

Control-W(R) -

NF25-W(N) -

NF24-W(R,N) -

NF25-W(R) -

NF25-W(B) -

NF25-W(B,N) -

NF25-W(B,R) -

+ + f

+ + + +

CO.01 < 0.01

0.2 1.4 0.5 1.5 1.6 3.1

co.01 < 0.01

1.8 1.6

0.1 0.1 0.1 0.2 0.3 1 .I 0.9 2.0

< 0.01 <O.OI

0.2 0.2 0.8 0.8

< 0.01 <0.01

0.04 0.1 0.5 0.8 1 .o 1.9

< 0.01 < 0.01

0.2 0.2 1.2 1.1

400 450 500 Wavelength (nm)

Fig. 1. Formation of P450-NF25 - Fe(I1) - nitrosoalkane complex upon oxidation of N-hydroxyamphetamine. (-) in the presence of NF25- W(R) microsomes and externally added rabbit liver cyt. b 5 ; (---) in the presence of NF25-W(N) microsomes and externally added rabbit liver cyt. b5 (see Materials and Methods for experimental details).

showing the highest oxidizing activities appear to be the best tool. The formation of inhibitory P450-Fe(II)-nitrosoalkane complexes has been found to occur during the in vivo and in uitro metabolism of certain drugs containing an amine func- tion like macrolide antibiotics (Mansuy, 1987). Some of these complexes are easily formed in vitro with rat liver microsomes by in-situ oxidation of the corresponding N-hydroxylamines by P450 in the presence of NADPH and O2 (Franklin, 1974) and are characterized by a Soret peak around 456 nm (Mansuy et al., 1976; Mansuy et al., 1977). Reaction of NF25-W(N) microsomes with N-hydroxyamphetamine in the presence of NADPH and of externally added rabbit liver cyt. b5 led to the progressive appearance of a peak at 456 nm (Fig. 1). Complex

formation was maximum after 20 min and its amount was estimated to be only approximately 10% of the starting P450 NF25, assuming a molar absorption coefficient of 65 mM-l . cm-' for this complex (Franklin, 1974). When the same experiment was performed with NF25-W(R) microsomes, more than 80% of P450 NF25 was transformed into a P450- Fe(I1)-nitrosoalkane complex within 20 min (Fig. 1). Omis- sion of rabbit cyt. b5 from the incubation mixture led to an approximately 40% transformation of starting P450 NF25 after 20 min and no further complex formation occured upon subsequent addition of rabbit cyt. b5 (not shown).

DISCUSSION

Several groups are now involved in the study of heterologously expressed human P450 species and discrepanc- ies between the results from different laboratories sometimes occur. This may be explained mainly due to the use of different expression systems, with many varying parameters including the nature of the host cell and of the expression vector, stable versus transient expression, etc. All of these parameters may affect the levels of the electron-transfer proteins associated to P450 within the monooxygenase complex, namely P450 reductase and cyt. b5. As P450 catalytic activities are critically dependent on the presence of these proteins, insufficient levels of these redox components could be a severe limiting factor on P450 activities. Comparing specific activities of individual forms of P450 thus requires at least the precise knowledge of the P450 reductaselcyt. b5 status in the P450 preparation in each case in order to draw valid conclusions. To avoid this difficulty, heterologously expressed P450 can also be purified to be used in reconstituted systems (Brian et al., 1990), with the advantage of the presence of only one isoform. However, the reorganization of the monooxygenase complex within added lipids is not always correctly achieved and many authors have reported problems in the particular case of P450 species from the 3A subfamily (Waxman et al., 1985; Kawano et al., 1987; Imaoka et al., 1988; Gemzik et al., 1990; Halvorson et al., 1990; Brian et al., 1990). This has been

115

explained by progressive inactivation of P450 3A forms during the purification protocol.

In this work, we have studied the influence of yeast P450 reductase overexpression and human cyt. bs coexpression on some catalytic activities of yeast-expressed human liver P450 3A4, the monooxygenase complex being directly reconstituted in yeast microsomes. The data reported in Table 3 first confirm that yeast P450 reductase is able to couple efficiently with human liver P450 NF25 in yeast microsomes, as previously suggested by the existence of catalytic activities in yeast only expressing P450 NF25 (Brian et al., 1990; Renaud et al., 1990). Overexpression of P450 reductase in a transformed yeast strain expressing P450 NF25, NF25-W(R), led to a spectacu- lar 33-fold increase of the NADPH-dependent reductase activity of yeast microsomes towards cytochrome c (Table 2) as well as 8-fold, 9-fold and 25-fold increases in the monooxygenase activities of these microsomes towards nifedipine, lidocaine and testosterone, respectively (Table 3). Intermedi- ate NADPH-dependent reductase activity and rate increases were observed with NF25-W(R, N) microsomes, in good agreement with the intermediate level of P450 reductase overexpression. Lastly, the results in Table 3 show that yeast P450 reductase is absolutely required for P450 NF25 catalytic activities in yeast microsomes, as its complete absence in NF25-W(B) led to the lack of any NADPH-supported monooxygenase activity.

The effect of addition of purified rabbit liver cyt. bs to the different microsomes on the catalytic activities was also examined. For the three substrates, addition of rabbit cyt. b5 in the incubation mixture always had a stimulating effect on the catalytic rates with either NF25-W(N), NF25-W(R, N) or NF25-W(R) microsomes. Furthermore, there was a tendency for higher rate increases with lower P450 reductase levels, as was found for nifedipine 1,4-0xidation and testosterone 6fl- hydroxylation catalyzed by NF25-W(N) microsomes. Human cyt. b5 coexpression in NF25-W(B, N) and NF25-W(B, R) could be ascertained from the comparison of the catalytic activities. First, NF25-W(B, N) microsomes, which contain a lower level of P450 reductase, showed 2 - 5-fold higher activities than NF25-W(N) microsomes. Similarly, NF25-W(B, R) microsomes showed about 2.5-fold higher activities than NF25-W(R, N) microsomes and about the same activities as NF25-W(R) microsomes. Finally, the addition of rabbit cyt. b5 to NF25-W(B, N) or NF25-W(B, R) microsomes showed no further stimulating effect on the three tested activities, suggesting that human cyt. b5 had already reached a saturating level. The enhancement of nifedipine oxidase activity by human cyt. h5 (Guengerich et al., 1986a) and of lidocaine N- deethylation by rat cyt. bs (Imaoka et al., 1990) had already been reported for purified P450 3A4 in reconstituted systems. The results presented here show that both human and rabbit cyt. b5 enhance P450 NF25-catalyzed nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6b-hydroxylation.

Finally, the system that afforded the highest catalytic turnovers in all cases was the association of NF25-W(R) microsomes with externally added purified rabbit liver cyt. bs. The activities thus obtained were about two-time as high as those found with NF25-W(B, R) microsomes in the presence of rabbit cyt. bs. This suggests that the P450 reductase level is the most critical factor for optimizing P450 NF25 catalytic activities. In particular, when one wants to study the formation of P450-metabolite complexes, which requires the monooxy- genation of the substrate, NF25-W(R) microsomes and rabbit

efficient in the formation of the inhibitory P450 NF25-Fe(II)- nitrosoalkane complex upon the oxidation of N-hydroxyamphetamine, as it led to an almost complete transformation of P450 NF25 into this complex within 20 min (Fig. 1). The presence of rabbit cyt. b5 was also beneficial in this case. This is the first report of the formation of such an inhibitory P450 - iron-metabolite complex from an in situ oxidation of a N- hydroxylamine by a given human liver P450. Yeast strains expressing individual forms of human P450 should be very useful for predicting inhibitory complex formation after metabolization of a given drug and its possible consequences for drug interactions. Such drug interactions involving macrolide antibiotics and P450 3A4 have been widely docu- mented (Ludden, 1985).

In conclusion, the advantages of the coexpression in yeast of human P450 NF25 and associated electron-transfer proteins are numerous.

a) Microsomes from such engineered strains directly contain the different components of the monooxygenase complex within the same endoplasmic reticulum membrane. For the study of human P450 3A4 specifically, it is particularly important to have in hand such a ‘self-reconstituted’ system.

b) Yeast-expressed P450-NF25 catalytic activities are greatly enhanced through overexpression of yeast endogenous P450 reductase and can be further increased by coexpression of human cyt. b5 or by the addition of purified rabbit cyt. b5 to the transformed yeast microsomes.

c) This yeast expression system can be used for spectro- scopic studies not only for determining the binding affinities of substrates to individual forms of human liver P450 but also for predicting the formation of inhibitory P450 -iron- metabolite complexes.

This work was supported in part by a grant No. 910309 from the Institut National de la Santk et de la Recherche MPdicale. We thank Dr Veronique Lauriault for carefully reading the manuscript.

REFERENCES Aoyama, T., Yamano, S., Waxman, D. J., Lapenson, D. P., Meyer,

U. A,, Fischer, V., Tyndale, R., Inaba, T., Kalow, W., Gelboin, H. V. &Gonzalez, F. J. (1989)J. Biol. Chem. 264,10388-10395.

Aoyama, T., Nagata, K., Yamazoe, Y., Kato, R., Matsunaga, E., Gelboin, H. V. & Gonzalez, F. J . (1990a) Endocrinology 126,

Aoyama, T., Yamano, S., Guzelian, P. S., Gelboin, H. V. & Gonzalez, F. J. (1990b) Proc. Natl. Acad. Sci. U S A 87,4790-4793.

Bargetzi, M. J., Aoyama, T., Gonzalez, F. J. & Meyer, U. A. (1989) Clin. Pharmacol. Ther.46, 521 - 527.

Brian, W. R., Srivastava, P. K., Umbenhauer, D. R., Lloyd, R. S. & Guengerich, F. P. (1989) Biochemistry 28, 4993 -4999.

Brian, W. R., Sari, M. A,, Iwasaki, M., Shimada, T., Kaminsky, L. S.& Guengerich, F. P. (1990) Biochemistry 29, 11 280 - 11 292.

Ching, M. S., Lennard, M. S., Tucker, G. T., Woods, H. F., Kelly, D. E.& Kelly, S. L. (1991) Biochem. Pharmacol. 42, 753-758.

Combalbert, J., Fabre, I., Fabre, G., Dalet, I., Derancourt, J., Cano, J. P. & Maurel, P. (1989) Drug Metab. Dispos. 17, 197-207.

Cullin, C. & Pompon, D. (1988) Gene (Amst.) 65,203-217. Eugster, H. P., Sengstag, C., Meyer, U. A,, Hinnen, A. & Wurgler, F.

Franklin, M. R. (1974) Mol. Pharrnacol. 10,975-985. Gemzik, B., Halvorson, M. R. & Parkinson, A. (1990) J. Steroid

Gilsdorf, R. T. & Nord, F. F. (1952) J. Am. Chem. SOC. 74, 1837-

Gonzalez, F. J. (1989) Pharmacol. Rev. 40,243-288. Guarente, L., Yocum, R. R. & Gifford, P. (1982) Proc. Natl. Acad.

3101 -3106.

E. (1990) Biochem. Biophys. Res. Comm. 172,737-744.

Biochem. 35,429-440.

1843.

cyt. h5 appears to be the’best system.’It was found to be very Sci. USA 79, 7410-7414.

116

Guengerich, F. P. (1988) Mol. Pharmacol. 33, 500-508. Guengerich, F. P. & Turvy, C. G. (1991) J . Pharmacol. Exp. Ther.

Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff, T. & Waxman, D. J. (1986a) J. Biol. Chem. 261, 5051 -5060.

Guengerich, F. P., Muller-Enoch, D. & Blair, I. A. (1986b) Mol. Pharmacol. 30,287 - 295.

Halvorson, M., Greenway, D., Eberhart, D., Fitzgerald, K. & Parkinson, A. (1990) Arch. Biochem. Biophys. 277, 166- 180.

Imaoka, S., Terano, Y. & Funae, Y. (1988) J. Biochem. (Tokyo) 104,

Imaoka, S., Enomoto, K., Oda, Y., Asada, A,, Fujimori, M., Shimada, T., Fujita, S., Guengerich, F. P. & Funae, Y. (1990) J . Pharmacol. Exp. Ther. 255, 1385-1391.

Kadlubar, F. F. & Hammons, G. J. (1987) in Mammalian cytochromes P450 (F. P. Guengerich, ed.), vol. 2, pp. 81 - 130, CRC Press, Boca Raton, FL.

Kawano, S., Kamataki, T., Yasumori, T., Yamazoe, Y. & Kato, R. (1987) J . Biochem. (Tokyo) 102, 493-501.

Kremers, P., Beaune, P., Cresteil, T., De Graeve, J., Columelli, S., Leroux, J. P. & Gielen, J. E. (1981) Eur. J . Biochem. 118, 599- 606.

Kronbach, T.. Fischer, V. & Meyer, U. A. (1988) Clin. Pharmacol. Ther. 43,630-635.

Kronbach, T., Mathys, D., Umeno, M., Gonzalez, F. J. & Meyer, U. A. (1989) Mol. Pharmacol. 36, 89 -96.

Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275.

Ludden, T. M. (1985) Clin. Pharmacokinet. 10,63-79. Mansuy, D. (1987) Pharmacol. & Ther. 33,41-45. Mansuy, D., Beaune, P., Chottard, J. C., Bartoli, J. F. & Gans, P.

(1976) Biochem. Pharmacol. 25, 609-612.

256,1189-1194.

481 -487.

Mansuy, D., Gans, P., Chottard, J. C. & Bartoli, J. F. (1977) Eur. J . Biochem. 76,607 - 61 5.

Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R. & Waxman, D. J. (1991) DNA 10,l-14.

Oda, Y., Imaoka, S., Nakahira, Y., Asada, A,, Fujimori, M., Fujita, S. & Funae, Y. (1989) Biochem. Pharmacol. 38,4439-4444.

Omura, T. & Sato, R. (1964) J. Biol. Chem. 239,2370-2378. Pichard, L., Gillet, G., Fabre, I., Dalet-Beluche, I., Bonfils, C.,

Thenot, J. P. & Maurel, P. (1990) Drug Metab. Dispos. 18, 71 1 - 719.

Pompon, D. (1988) Eur. J. Biochem. 177,285-293. Pompon, D. & Nicolas, A. (1989) Gene (Amst.) 83, 15-24. Pompon, D., Urban, P., Cullin, C., Truan, G. & Slonimski, P. (1991)

Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. & Mansuy, D.

Romkes, M., Faletto, M. B., Blaisdell, J. A., Raucy, J. L. & Goldstein,

Strittmatter, P., Fleming, P., Connors, M. & Corcoran, D. (1978)

Urban, P., Cullin, C. & Pompon, D. (1990) Biochimie 72,463-472. Veronese, M. E., Mackenzie, P. I., Doecke, C. J., McManus, M. E.,

Miners, J. 0. & Birkett, D. J. (1991) Biochem. Biophys. Res. Comm.

Waxman, D. J., Dannan, G. A. & Guengerich, F. P. (1985) Biochemis-

Waxman, D. J., Attisano, C., Guengerich, F. P. & Lapenson, D. P.

Yasumori, T., Murayama, N., Yamazoe, Y., Abe, A,, Nogi, Y.,

Pending French Patent No. 91 08 884.

(1990) Eur. J. Biochem. 194, 889-896.

J. A. (1991) Biochemistry 30, 3247- 3255.

Methods Enzymol. 52,97 - 101.

175, 1112-1118.

try 24,4409-4417.

(1988) Arch. Biochem. Biophys. 263, 424-436.

Fukasawa, T. & Kato, R. (1989) Mol. Pharmacol. 35,443-449.

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