Rat Cytochrome P450 2B1 Research Articles

Stereoselective Formation of Mono- and Dihydroxylated Polychlorinated Biphenyls by Rat Cytochrome P450 2B1

Changes in atropisomer composition of chiral polychlorinated biphenyls (PCBs) and their mono- and dihydroxylated metabolites (OH- and diOH-PCBs) via rat cytochrome P450 2B1 (CYP2B1) mediated biotransformation were investigated in vitro. Rat CYP2B1 could stereoselectively biotransform chiral PCBs to generate meta-OH-PCBs as the major metabolites after 60 min incubations. Nonracemic enantiomer fractions (EFs: concentration ratios of the (+)-atropisomer or the first-eluting atropisomer over the total concentrations of two atropisomers) of 5-OH-PCBs, were 0.17, 0.20, 0.85, 0.77, and 0.41 for incubations with PCBs 91, 95, 132, 136, and 149, respectively. CYP-mediated stereoselective formation of diOH-PCBs from OH-PCBs was observed for the first time. After 60 min stereoselective biotransformation, the EFs of both 4-OH-PCB 95 and 5-OH-PCB 95 changed from racemic (i.e., 0.50) to 0.62 and 0.46, respectively. These transformations generated statistically nonracemic 4,5-diOH-PCB 95, with EFs of 0.53 and 0.58 for 4-OH-PCB 95 and 5-OH-PCB 95 incubations, respectively. Biotransformation of PCBs 91 and 136 also generated 4,5-diOH-PCB 91 and 4,5-diOH-PCB 136, respectively. These in vitro results were consistent with that observed for stereoselective PCB biotransformation by rat liver microsomes and in vivo. Biotransformation interference between two atropisomers of PCB 136 was investigated for the first time in this study. The biotransformation process of (-)-PCB 136 was significantly disrupted by the presence of (+)-PCB 136 but not the other way around. Thus, stereoselective metabolism of chiral PCBs and OH-PCBs by CYPs is a major mechanism for atropisomer composition change of PCBs and their metabolites in the environment, with the degree of composition change dependent, at least in part, on stereoselective interference of atropisomers with each other at the enzyme level.

Factors Affecting Phase I Stereoselective Biotransformation of Chiral Polychlorinated Biphenyls by Rat Cytochrome P-450 2B1 Isozyme

In vitro incubations of rat cytochrome P-450 (CYP) 2B1 isozyme with three chiral polychlorinated biphenyl (PCB) congeners (PCBs 45, 95, and 132) were performed to investigate factors affecting phase I stereoselective biotransformation. Rat CYP2B1 preferentially biotransformed the second-eluting atropisomers of PCBs 45 and 95 at low substrate concentration ranges (≤15 μM). Biotransformation competition by different congeners was also observed, with increasing competition at higher chlorination. Competition decreased the biotransformation rates of each congener stereoselectively, affecting atropisomeric composition. No atropisomeric enrichment was observed for PCB 132 upon incubation of the racemate. However, under the same conditions, significant differences in biotransformation kinetics were observed in individual atropisomer incubations, indicating that (+)-PCB 132 and (-)-PCB 132 were competitively biotransformed. Homology modeling and docking studies suggested that each atropisomer had different interactions with rat CYP2B1 and could dock with the isozyme at different locations. This is one possible explanation for stereoselective biotransformation and competition of chiral PCBs at the molecular level. Our results suggest that the lack of predictive capability for stereoselectivity of PCBs and other chiral pollutants in biota may be due to competitive and/or inhibitory activities of different substrates, including individual enantiomers of the same compound.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Cytochrome-P450 2B1 gene silencing attenuates puromycin aminonucleoside-induced cytotoxicity in glomerular epithelial cells

Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.

Chiral Polychlorinated Biphenyls Are Biotransformed Enantioselectively by Mammalian Cytochrome P-450 Isozymes to Form Hydroxylated Metabolites

In vitro incubations of purified rat cytochrome P-450 (CYP) 2B1 and human CYP 2B6 were performed to determine if CYP isozymes biotransform polychlorinated biphenyls (PCBs) enantioselectively. Enantioselective metabolism of chiral PCBs 45, 84, 91, 95, 132, and 136 and production of hydroxylated PCB metabolites (OH-PCBs) were observed, while no changes in PCB 183 atropisomer composition were observed for either isozyme. Enantiomer fractions (EFs) of parent PCBs, individually incubated as racemates at 25 ng/mL initial concentration, with rat CYP 2B1 ranged from 0.353 to 0.822. Enantioselectivity was also observed for PCBs 45 (EF = 0.437) and 132 (EF = 0.537) incubated at that concentration with human CYP 2B6. Both atropisomers of chiral PCBs appeared to be biotransformed simultaneously by rat CYP 2B1, except for (+)-PCB 132, but at different rates. Hydroxylated PCBs were identified using gas chromatography-high resolution mass spectrometry for all chiral PCBs enantioselectively transformed by CYPs. These metabolites did not correspond to any commercially available authentic standards, supporting the hypothesis that many unidentified OH-PCBs detected in wildlife may have arisen from in vivo biotransformation of chiral PCBs. A rough estimate suggested that more than half of the total congener metabolized by rat CYP 2B1 was converted to OH-PCBs. Similar concentration decreases were observed for congeners incubated with human CYP 2B6, but less OH-PCBs were formed. Formation of OH-PCBs via an enantioselective OH insertion mechanism was suggested, and may be a source of the unidentified OH-PCBs currently found in the environment.

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer.

Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.

Adenovirus carrying cytochrome P450 4B1 (CYP4B1) under an EGR1 promoter combined with 4-ipomeanol (4-IM) prodrug and radiation

Purpose: To test the in vitro efficacy of the chemotherapeutic alkylating agent prodrug 4-ipomeanol (4-IM) combined with ionizing radiation (IR) in cells transduced with an adenoviral vector carrying a chimeric fusion protein of rabbit cytochrome CYP4B1 and enhanced green fluorescent protein (EGFP) under the control of the radiation-inducible EGR1 promoter. Materials and Methods: Cassette effect was tested by comparing survival fractions determined by low-input MTT assay for HEK293 cells stably transfected with the prodrug-activating fusion protein CYP4B1-EGFP under the control of the EGR1 promoter with that of control HEK293 cells stably transfected with EGFP alone, 8 days after treatment with 4-IM and IR. Radiosensitization by activated 4-IM was tested by comparing survival fractions from clonogenic assays of 9L cells transfected with the CYP4B1-EGFP cassette or EGFP alone, both under a constituitive CMV promoter, after 24 hours of 4-IM exposure. The EGR-4B1-EGFP cassette was inserted into the E1A region of an adenoviral vector, and efficacy compared to adenoviral vector carrying CMV-EGFP (MOI = 100), was tested in U87-MG cells using MTT assay 3 days after treatment with 4-IM and IR. In each case, cells were irradiated at doses of 0, 2, and 4 Gy on day 1, following 4-IM treatment at concentrations of 0, 2.5 and 5.0 μg/ml. Results: MTT assays demonstrated a decrease in the relative survival fractions (survival with 4-IM/survival without 4-IM) of EGR1-CYP4B1-EGFP transfected cells with increasing radiation dosage, but not of the control cells: 88%, 57%, 39% at 0, 2 and 4 Gy for the EGR1-CYP4B1-EGFP cells, and 96%, 87%, 96% at 0, 2 and 4 Gy for the control cells (2.5 μg/ml 4-IM). Clonogenic assays for radiosensitization demonstrated that survival fractions decreased with increasing radiation doses and 4-IM concentrations (for the CYP4B1 bearing cells), but no significant differences in D0 were seen with 4-IM. Of relevance, a similar lack of sensitization was seen in the same model using rat cytochrome P450 2B1 (CYP2B1) to activate cyclophosphamide, but a pronounced effect was seen when this combination was used in a replication competent HSV-1 vector (rRp450). 4-IM (5.0 μg/ml) reduced the relative survival of cells treated with adenovirus carrying EGR-4B1-EGFP to 66 +/− 11% with the addition of 2 Gy IR and to 57 +/− 10% with 4 Gy IR. No effect was seen in cells treated with adenovirus carrying CMV-EGFP. Conclusion: IR potentiates the cytotoxic activity of the EGR1/CYP4B1/4-IM transgene system. Cytochrome P450 activated alkylating agent prodrugs do not appear to themselves sensitize cells to radiation in the models tested, but significant augmentation of IR efficacy can be obtained when these are incorporated into appropriate transgene and vector systems. There may be additional variability between agents and cell lines, as has been reported for temozolomide. We are proceeding to test these gene therapy vectors in murine models.

The Role of Cytochrome 2B1 Substrate Recognition Site Residues 115, 294, 297, 298, and 362 in the Oxidation of Steroids and 7-Alkoxycoumarins

At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16α-hydroxytestosterone:16β-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.

Mechanistic Studies of Cytochrome P450 2B1 Inactivation by Xanthates

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy–ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.

Cis-acting sequences from the rat cytochrome P450 2B1 gene confer pulmonary and phenobarbital-inducible expression in transgenic mice.

Specific cytochrome P450 enzymes show tissue-specific induction, and different regulatory units for expression of these enzymes have been identified. The regulation of the phenobarbital (PB)-inducible P450 genes has been relatively well characterized in terms of PB induction, but less so with regard to tissue-specific expression. CYP2B2 is not expressed in the rat lung, whereas cytochrome P450 2B1 (CYP2B1) is a dominating enzyme in the same tissue. The constitutive expression of CYP2B1 and CYP2B2 in liver is low, but inducible by PB, whereas the pulmonary expression of CYP2B1 is not induced by PB. This indicates utilization of different regulating mechanisms in the two organs. A gene construct consisting of the structural gene for LacZ coupled to a 1.3-kb 5' fragment of the rat CYP2B1 gene was used to generate transgenic mice in order to further elucidate the mechanism behind tissue-specific expression and PB induction of the CYP2B1 gene. Using reverse transcriptase-polymerase chain reaction on total RNA extracted from lung and liver tissue, a lung-specific transcription of the transgene was observed. Transcription of the construct was also observed in livers from PB-treated transgenic animals. By histochemical staining of lung sections with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), we demonstrated expression at the protein level in bronchiolar cells. In conclusion, our results revealed that the region extending to -1. 3 kb in the 5' flanking region of the CYP2B1 gene included sequences that could partly account for the lung-specific transcription of CYP2B1 and the hepatic induction of CYP2B1 transcription by PB.

Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1.

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.

Piperine inhibits aflatoxin B 1-induced cytotoxicity and genotoxicity in V79 Chinese hamster cells genetically engineered to express rat cytochrome P4502B1

We have investigated the potential of piperine for inhibiting the activity of cytochrome P4502B1 and protecting against aflatoxin B 1 (AFB 1) in V79MZr2B1 (r2B1) cells, i.e. V79 Chinese hamster cells engineered for the expression of rat CYP4502B1. The cells were found to contain high activities of 7-methoxycoumarin demethylase (MOCD). Piperine inhibited MOCD in preparations of r2B1 cells with an IC 50 of ≈ 10 μM. The cells in culture dealkylated 7-methoxycoumarin (MOC) to 7-OH-coumarin linearly, at least for 12 h, where piperine produced concentration-dependent inhibition with IC 50 < 30 μM. The time required for maximal inhibition was ≈ 8 h with both 30 and 60 μM concentrations of piperine used. AFB 1 at 0.1–20 μM caused a concentration dependent decrease in the amount of DNA and an increase in the formation of micronuclei (MN). The mycotoxin at 10 μM reduced DNA by ≈ 30% and increased MN appearance by 20-fold against the background level of 7 MN per 500 nuclei. Piperine at 60 μM completely counteracted cytotoxicity and formation of MN by 10 μM AFB 1 and reduced the toxic effects of 20 μM AFB 1 by > 50%. The results suggest that: (i) Piperine is a potent inhibitor of rat CYP4502B1 activity; (ii) AFB 1 is activated by r2B1 cells to cytotoxic and genotoxic metabolites; and (iii) piperine counteracts CYP4502B1 mediated toxicity of AFB 1 in the cells and might, therefore, offer a potent chemopreventive effect against procarcinogens activated by CYP4502B1.

Significance of Glycine 478 in the Metabolism of N-Benzyl-1-aminobenzotriazole to Reactive Intermediates by Cytochrome P450 2B1

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.

Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: relevance to the genotoxicity of kaempferol

Kaempferol is a flavonoid widely distributed in edible plants and has been shown to be genotoxic to V79 cells in the absence of external metabolizing systems. The presence of an external metabolizing system, such as rat liver homogenates (S9 mix), leads to an increase in its genotoxicity, which is attributed to its biotransformation to the more genotoxic flavonoid quercetin, via the cytochrome P450 (CYP) mono-oxygenase system. In the present work we investigated the mechanisms of the genotoxicity of kaempferol further. Special attention has been given to the role of CYP in the genotoxicity of this flavonoid. We studied the induction of mutations in Salmonella typhimurium TA98 in the presence and in the absence of S9 mix and the induction of chromosomal aberrations (CAs) and micronuclei (MN) by kaempferol in V79 cells in the presence and in the absence of S9 mix. To evaluate the role of different CYP in the biotransformation of kaempferol we studied the induction of CAs and MN in V79 cells genetically engineered for the expression of rat CYP 1A1, 1A2 and 2B1. In addition we performed CYP inhibition studies using the above-mentioned indicators and high performance liquid chromatography (HPLC) analysis. The results obtained in this work suggest that rat CYP 1A1 is, among the cytochromes studied, the one that plays the major role in the transformation of kaempferol into quercetin. The relevance of these findings to the human situation is discussed.

Production of a Form-Specific, Inhibitory Antibody Against Rat Cytochrome P450 2B1 Using a Synthetic Peptide Antigen against a Putative Substrate Binding Site

Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1. This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody. The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats. These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes.

Interaction between Cytochrome P450 2B1 and Cytochrome b5: Inhibition by Synthetic Peptides Indicates a Role for P450 Residues Lys-122 and Arg-125

Binding of cytochrome b 5 to rat cytochrome P450 2B1 was inhibited (by 75%) by a synthetic peptide corresponding to P450 residues 116-134. The roles of Lys-122 and Arg-125 were evaluated using peptides in which one or both of these basic residues were replaced with Glu. The Lys-122 substitution nearly abolished while the Arg-125 replacement decreased (by 20%) the inhibitory potential of the peptide. Substitution of both residues resulted in a peptide with no inhibitory activity. These results thus indicate a role for a specific P450 region as well as two basic residues within this region In the cytochrome P450-cytochrome b 5 interaction.

Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites.

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)