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.     

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.     

Resveratrol is a selective human cytochrome P450 1A1 inhibitor.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system). Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM. Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD). Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2. The activities of human NADPH-P450 reductase were not significantly changed by resveratrol. In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM. These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans.     

Characterization of rabbit liver P450IIE1 synthesized in transformed yeast cells

cDNA for chimeric protein, P450(3P4), consisting of the amino-terminal 43 residues (the membrane-anchor region) of rabbit P450IIC14 and the remaining 447 residues of rabbit P450IIE1 was constructed, then cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. P450(3P4) thus synthesized in the transformed yeast cells was partially purified, and its spectral and catalytic properties were examined. In the oxidized state P450(3P4) exhibited a high-spin type absorption spectrum even in the absence of a substrate. The reduced CO complex of the P450 showed a Soret absorption maximum at 452 nm. P450(3P4) catalyzed aniline p-hydroxylation, N-nitrosodimethylamine demethylation, benzphetamine N-demethylation, and laurate and caprate (omega-1)-hydroxylation in the reconstituted system containing the P450 and NADPH-P450 reductase. These results indicate that P450(3P4) preparation obtained from the transformed yeast cells has spectral and catalytic characteristics identical with those of P450IIE1 purified from rabbit liver microsomes, confirming the substrate specificity reported of P450IIE1.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5

Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.     

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.     

Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain.

A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.     

Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.     

12(R)-hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium.

Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.     

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

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.     

P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate

In a reconstituted system containing NADPH, dilauroyl-L-3-phosphatidylcholine, and NADPH-cytochrome P-450 reductase purified from rat liver microsomes, cytochrome P-450 (P-450 HFLa) purified from human fetal livers catalyzed the 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate). Addition of cytochrome b5 purified from rat liver microsomes to the reconstituted system resulted in a remarkable increase in the hydroxylase activity. The level of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Antibodies to P-450 HFLa inhibited the 16 alpha-hydroxylation of DHEA-sulfate in a dose-dependent manner. The NH2-terminal amino acid sequence of P-450 HFLa was similar to that of P-450NF (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068). We conclude that P-450 HFLa is a form of cytochrome P-450 involved in the 16 alpha-hydroxylation of DHEA-sulfate.     

Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Summary Photoactivation of cytochrome P450 monooxygenase was studied using a combination of spinach chloroplasts and yeast microsomes containing rat P4501A1/yeast reductase fusion enzyme. Under illumination, in the reaction mixture, NADP was reduced, transferring electrons to the P450/reductase fusion enzyme to convert 7-ethoxycoumarin to 7-hydroxycoumarin.     

Cytochrome P450 IIIA1 (P450p) requires cytochrome b5 and phospholipid with unsaturated fatty acids.

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.