An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) 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.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

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.     

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3′-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3′H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide–binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP⁺ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.     

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.     

Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.     

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.     

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.     

Engineering of proteolytically stable NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression in E. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease from Staphylococcus aureus of highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane.     

Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes

Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.     

Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450

A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.     

Functional characterization of medaka CYP3A38 and CYP3A40: kinetics and catalysis by expression in a recombinant baculovirus system

Phylogenic analysis of the teleost genomic lineages has demonstrated the precedent for multiple genome duplications. Among many of the genes duplicated, cytochrome P450 genes have undergone independent diversification, which can be traced to a single ancestral gene. In teleosts, cytochrome P450s, from all major families, have been identified. Among these, the CYP3A family has been cloned in several teleost species and demonstrated to contain multiple paralogs differing in gene expression patterns and tissue distribution. Herein we characterized the catalytic and kinetic activities of two medaka CYP3A paralogs (CYP3A38 and CYP3A40) with benzyloxyresorufin (BFC), a fluorescent 3A-selective substrate, and testosterone, a known metabolic substrate for CYP3A enzymes. Recombinant CYP3A was produced using the baculovirus expression vector system in Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) insect cells and accounted for up to 24% of total cellular protein. Following addition of a heme-albumin conjugate to log phase cells, spectral P450 content reached a maximum of 560 and 2350 pmol/mg microsomal protein for CYP3A38 and CYP3A40, respectively. Incubations containing recombinant CYP3A, human NADPH-cytochrome P-450 oxidoreductase reductase, human cytochrome b5, and a NADPH generation system catalyzed the dealkylation of BFC and hydroxylation of testosterone with a high degree of stereoselectivity. However, efficiencies and specificities were significantly different between the two isoforms. Km and Vmax activities based on BFC-catalysis were 0.116 and 0.363 muM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6beta-, 2beta- and 16beta-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6beta- and 2beta-positions. Putative identification of CYP3A substrate recognition sites (SRS) 1-6 indicates that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 OFRs occur in SRS regions previously known to be associated with steroid hydroxylation. We suggest that differences in kinetics and catalytic activities are a result of amino acid substitutions in SRS regions 1, 3 and 5 within the CYP3A38 and CYP3A40 protein sequence.     

Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 °C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug-drug interaction studies.     

Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.     

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.     

Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli

Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.     

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis: PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES*

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)³ in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.     

Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.