Regular ArticleBaculovirus Expression of Human P450 2E1 and Cytochrome b5: Spectral and Catalytic Properties and Effect of b5 on the Stoichiometry of P450 2E1-Catalyzed Reactions
Abstract
The baculovirus (BV)-insect cell expression system has been successfully used to express high levels of mammalian proteins. Here we report the baculovirus-mediated expression of human P450 2E1 and cytochrome b5 in Sf9 insect cells. The BglI-EcoRI fragment of the human P450 2E1 cDNA (Umeno et al., Biochemistry 27, 1988) was used for the expression of P450 2E1. Human cytochrome b5 cDNA was obtained by the polymerase chain reaction using human liver cDNA as template. Infection of Sf9 insect cells with a recombinant 2E1-BV resulted in the expression of a protein which comigrated with human liver P450 2E1 on SDS gels and cross-reacted with a polyclonal antibody against rat liver P450 2E1. Inclusion of hemin in the cell growth medium greatly enhanced the spectral activity of expressed 2E1. Expression levels of 1.5 nmol/mg cell lysate were obtained after 7 days of infection. However, maximal turnover numbers (min−1) were observed after 48 h of infection. The λmax of the CO:reduced CO difference spectrum of human P450 2E1 was found to be 451.1 nm. In the presence of exogenous hemin, BV-expressed human b5 showed a typical reduced - oxidized difference spectrum with λmax and λmin of 425 and 409 nm, respectively. With saturating levels of purified rat liver NADPH:P450 oxidoreductase and rat liver cytochrome b5 included in the incubations, expressed human P450 2E1 showed high catalytic activity for the metabolism of p-nitrophenol (PNP), ethoxycoumarin, N-nitrosodiethylamine, and N-nitrosodimethylamine (NDMA). The presence of b5; in the incubations increased the activities several-fold. The Km and kcat values for the N-demethylation of NDMA to HCHO in the presence of rat b5 by expressed 2E1 were 36.0 μM and 8.3 min−1, respectively. In the absence of extra added b5, the Km was increased sixfold and the kcat decreased fourfold. In the presence of extra added b5 expressed 2E1 showed a Km for PNP metabolism of 86 μM and a kcat of 7.8 min−1. Simultaneous infection of Sf9 insect cells with both 2E1 and human b5 recombinant BV resulted in a membrane fraction (2E1:b5) containing both proteins at a ratio of b5 to P450 of approximately 1.8:1. The Km and kcat values for NDMA demethylase activity by the 2E1:b5 membrane fraction were similar to those with exogenously added b5. Stoichiometric analysis of the rates of NADPH oxidation and product and H2O2 formation showed that the sum of the HCHO (product) and H2O2 formed could not account for the amount of NADPH utilized which is consistent with a pathway leading to H2O formation. The presence of substrate did not significantly affect the rates of either NADPH oxidation or H2O2 formation. Extra added b5 significantly decreased the rate of NADPH utilization and H2O2 formation by approximately 63 and 86%, respectively, with or without substrate in the incubations. The results show that exogenous b5 decreased H2O2 and H2O formation by an amount which could account for the extra reducing equivalents needed for the increased rate of product formation, suggesting a mechanism for the b5-dependent stimulation.
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Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization
2023, Methods in EnzymologySix cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.
The relationships between cytochromes P450 and H <inf>2</inf> O <inf>2</inf> : Production, reaction, and inhibition
2018, Journal of Inorganic BiochemistryIn this review we address the relationship between cytochromes P450 (P450) and H2O2. This association can affect biology in three distinct ways. First, P450s produce H2O2 as a byproduct either during catalysis or when no substrate is present. This reaction, known as uncoupling, releases reactive oxygen species that may have implications in disease. Second, H2O2 is used as an oxygen-donating co-substrate in peroxygenase and peroxidase reactions catalyzed by P450s. This activity has proven to be important mainly in reactions involving prokaryotic P450s, and investigators have harnessed this reaction with the aim of adaptation for industrial use. Third, H2O2-dependent inhibition of human P450s has been studied in our laboratory, demonstrating heme destruction and also the inactivating oxidation of the heme-thiolate ligand to a sulfenic acid (-SOH). This reversible oxidative modification of P450s may have implications in the prevention of uncoupling and may give new insights into the oxidative regulation of these enzymes. Research has elucidated many of the chemical mechanisms involved in the relationship between P450 and H2O2, but the application to biology is difficult to evaluate. Further studies are needed reveal both the harmful and protective natures of reactive oxygen species in an organismal context.
Structures of human cytochrome P-450 2E1: Insights into the binding of inhibitors and both small molecular weight and fatty acid substrates
2008, Journal of Biological ChemistryCitation Excerpt :Absorbance spectra revealed that the purified CYP2E1 was in a mixed spin state, suggesting that in solution some CYP2E1 protein molecules have water bound to the sixth coordination site of the heme iron, but in other molecules the water is absent, and the heme iron is five-coordinate. Most mammalian P-450s are water-coordinated in the resting state, but this mixed spin state has previously been reported for full-length human CYP2E1, although it has also been isolated as either all high spin or all low spin (37-40). When reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5, the truncated, purified CYP2E1 is catalytically competent in performing two classic CYP2E1 reactions: 2-hydroxylation of p-nitrophenol and 6-hydroxylation of chlorzoxazone (data not shown).
Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates >70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 Å for an indazole complex and 2.6 Å for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed ω-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.
Ethanol oxidation into acetaldehyde by 16 recombinant human cytochrome P450 isoforms: Role of CYP2C isoforms in human liver microsomes
2006, Toxicology LettersThe involvement of cytochromes P450 (CYPs) in the oxidation of ethanol into acetaldehyde was investigated by using 16 recombinant human CYP isoforms. Apparent Km and Vm were determined for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP4A11. All of the tested CYPs, except CYP2A6 and CYP2C18, metabolized ethanol into significant amounts of acetaldehyde and displayed Km values around 10 mM. The significant correlation found between ethanol oxidation and CYP2E1, CYP3A4 and CYP1A2 catalytic activities in a panel of human liver microsomes confirmed the strong implication of these CYPs in ethanol metabolism. The contribution of CYP2C isoforms which are the most abundant in the liver after CYP3A4, was studied using selective inhibitors either with recombinant CYP2C isoforms or in human liver microsomes. Tienilic acid (100 μM) and ticlopidine (20 μM), mechanism-based inhibitors of CYP2C9 and CYP2C19, respectively, decreased ethanol oxidation by 8 ± 1.2% and 7.6 ± 1.6% in human liver microsomal samples while selective inhibitors of CYP2E1 (DEDTC 100 μM), CYP3A4 (TAO 50 μM) and CYP1A2 (furafylline 25 μM) decreased it by 11.9 ± 2.1%, 19.8 ± 1.9% and 16.3 ± 3.9%, respectively. As ethanol can be metabolized by most of CYPs, it helps to explain or predict alcohol–xenobiotics interactions which are of high importance in medical prescription.
Functional characterization of medaka CYP3A38 and CYP3A40: Kinetics and catalysis by expression in a recombinant baculovirus system
2005, Comparative Biochemistry and Physiology - C Toxicology and PharmacologyPhylogenic 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 μM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6β-, 2β- and 16β-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6β- and 2β-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.
RNA expression induced by cisplatin in an organ of Corti-derived immortalized cell line
2004, Hearing ResearchCisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti.
To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase.
As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels.
These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.