Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.     

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.     

Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

Cytochrome b5-mediated redox cycling of estrogen

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b5 in redox cycling in order to obtain a full understanding of enzymatic contributions to redox reactions of estrogens. Pure cytochrome P450c and hydrogen peroxide or cumene hydroperoxide oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-quinone (DES Q). This oxidation by H2O2 was doubled by addition of cytochrome b5 to cytochrome P450c (molar ratio of 1:4), but did not proceed with cytochrome b5 alone. The stimulation by cytochrome b5 of the cytochrome P450c-catalyzed oxidation of DES to DES Q occurred via modulation of the Vmax of cytochrome P450c rather than of the Km. DES Q was reduced to DES by purified cytochrome b5 and NADH-dependent cytochrome b5 reductase. Pretreatment of microsomes with an antibody to cytochrome b5 reductase inhibited microsomal NADH-dependent reduction of DES Q to DES by 55%. Cytochrome b5 likely participates in the oxidation of DES to DES Q by interacting with cytochrome P450c and in the reduction of DES Q to DES by interacting with cytochrome b5 reductase. Thus, the study demonstrates that cytochrome b5 plays an active role in biological oxidation and reduction reactions.     

P450 reductase and cytochrome b5 interactions with cytochrome P450: effects on house fly CYP6A1 catalysis

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.     

NADPH- and hydroperoxide-supported 17beta-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.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes

Water-soluble carbodiimide-catalyzed cross-linking of purified cytochrome P-450 LM2, cytochrome b5, and NADPH-cytochrome P-450 reductase was used to identify stable complexes formed between these proteins. High yields of P-450-b5 and P-450 reductase-b5 dimers, and lower yields of P-450 reductase-LM2 dimers were obtained. Substitution of native b5 and P-450 reductase with fully amidinated derivatives showed that LM2 and b5 were cross-linked exclusively through their respective amino and carboxyl groups. However, there appeared to be two complexation sites on the reductase which cross-link to b5 through amino groups and to LM2 through carboxyl groups respectively. A heterotrimer could not be identified following incubation of all three proteins together with EDC.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I(2)) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005 mg/kg Se, and I(2) deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T(4) (TT(4)), total T(3) (TT(3))], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I(2) deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I(2) and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I(2) status.     

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.     

Effects of human cytochrome b5 on CYP3A4 activity and stability in vivo

Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E. coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.     

The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling

Cytochrome P-450cam cationic surface charges at Lys 344, Arg 72, and Lys 392 have been altered by site-directed mutagenesis techniques. The residues at Lys 344 and Arg 72 were previously suggested as salt bridge contacts in the cytochrome b5-cytochrome P-450cam association complex and implicated in the physiological putidaredoxin-cytochrome P-450cam complex [Stayton, P. S., Poulos, T. L., & Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. Mutations to neutralize the basic charge at Arg 72 (R72Q) and to both neutralize and reverse the charge at Lys 344 (K344Q, K344E) resulted in alteration of NADH oxidation rates in the reconstituted physiological electron-transfer system, which is rate limited by putidaredoxin-cytochrome P-450cam electron transfer. The steady-state Vmax values were apparently unperturbed, suggesting that the observed rate differences were largely attributable to Km effects. The Km values observed for the K344Q (24 microM) and K344E (32 microM) mutants are in the direction expected for neutralization and reversal of a salt bridge charge interaction. A control mutation at a basic surface charge located away from the proposed site of interaction, Lys 392 (K392Q), resulted in overall activities quantitated by NADH oxidation rates that are similar to that of wild-type cytochrome P-450cam. Calculation of the cytochrome P-450cam electrostatic field revealed a patch of positive potential at the modeled cytochrome b5 interaction site lying directly above the nearest proximal approach to the buried heme prosthetic group. These results provide experimental and theoretical evidence for the modeled cytochrome P-450cam binding site implicated in both cytochrome b5 and putidaredoxin association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Dilinoleoylphosphatidylcholine decreases ethanol-induced cytochrome P4502E1.

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.