NADPH-supported Activity Research Articles

Expression, Purification, and Physical Properties of Recombinant Flavocytochrome Fusion Proteins Containing Rat Cytochrome b5 Linked to NADPH-Cytochrome P450 Reductase by Different Membrane-Binding Segments

Reconstitution of the enzymatic activities using purified microsomal cytochrome P450s (P450) requires the presence of a membrane-binding segment in the mammalian flavoprotein, NADPH--cytochrome P450 reductase (CPR), and the hemeprotein, cytochrome b(5) (b(5)). The mechanism(s) by which the membrane-binding segments of these proteins exert such a critical role in influencing the reconstitution of the NADPH-supported activity of a P450 remains undefined. In the present work we describe the construction, expression, and purification of four different types of recombinant flavocytochromes containing rat b(5) and rat CPR linked by various membrane-binding segments. The physical properties of these artificial fusion proteins have been studied to determine their ability to serve as electron transfer agents. These studies are a prelude to the subsequent study (accompanying paper) evaluating the functional roles of the hydrophobic (membrane-binding) sequences of b(5) and CPR in the reconstitution of P450 activities. The present study shows that the purified recombinant fusion proteins can serve as active electron transport carriers from NADPH to cytochrome c as well as b(5) by intramolecular as well as intermolecular reactions. It is shown here that the electron transport properties of these purified fusion proteins are influenced by high concentrations of KCl, suggesting a role for charged amino acids in protein-protein interactions. The present study illustrates the application of artificial recombinant flavocytochromes as useful proteins for the study of intramolecular electron transport reactions for comparison with intermolecular interactions.

NADPH- and hydroperoxide-supported 17β-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.

Yeast expressed cytochrome P450 2D6 (CYP2D6) exposed on the external face of plasma membrane is functionally competent.

CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma membrane in addition to the regular microsomal location. Present work demonstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner face of plasma membrane. Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH- and cumene hydroperoxide-dependent dextromethorphan demethylation and NADPH-cytochrome c activity confirming previous observations in human hepatocytes. CYP2D6 found on the outside of plasma membrane (by differential immuno-inhibition and acidic shift assays on transformed spheroplasts) was catalytically competent at the cell surface for NADPH-supported activities. Anti-yeast P450-reductase antibodies inhibited neither CYP2D6 nor P450-reductase activities upon incubation with intact spheroplasts. In contrast, both activities were inhibited on isolated plasma membrane fragments. This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase. This finding was confirmed by immunostaining in confocal microscopy. Finally, gene deletion of P450-reductase caused a complete loss of plasma membrane NADPH-supported CYP2D6 activity, which suggests that the reductase participates to some degree in the transmembrane electron transfer chain. This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role.

Mechanistic Studies of 9-Ethynylphenanthrene-Inactivated Cytochrome P450 2B1

The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization-mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995) Arch. Biochem. Biophys. 323, 000-000]. The inactivation by 9EPh resulted in a 90-95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5 or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2 formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2 to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen-iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.

Carbonyl reduction of metyrapone in human liver

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Evidence for two separate β-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other NADPH.

Evidence for two separate β-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other, NADPH.

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes.

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.