Electrochemistry of cytochromes P450: Analysis of current-voltage characteristics of electrodes with immobilized cytochromes P450 for the screening of substrates and inhibitors

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14α-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 4, pp. 542–549.     

微生物细胞色素P450与胆固醇的生物代谢

细胞色素P450(CYP450)是一类含亚铁血红素的单加氧酶,广泛存在于各类生物体内,参与多种外源物质的代谢和内源物质的转化,如甾类激素、胆汁酸、胆固醇等的代谢。胆固醇是一种环戊烷多氢菲的衍生物,也是人类重要的脂类物质和许多特殊生物活性物质的前体之一,当其过量时会导致高胆固醇血症、动脉粥样硬化、静脉血栓生成等,对机体产生不利的影响。微生物CYP450酶可催化胆固醇的生物代谢,特别是其中的CYP125酶是胆固醇分解代谢起始的关键酶,可用作调节胆固醇代谢的药物靶标。     

Cytochromes P450 and experimental models of drug metabolism

For the development of new drugs, evaluation of drug-drug interactions with already known compounds, as well as for better understanding of metabolism pathways of various toxicants and pollutants, we studied the drug metabolism mediated by cytochromes P450. The experimental approach is based on animal drug-metabolising systems. From the ethical as well as rational reasons, the selection of an appropriate system is crucial. Here, it is necessary to decide on the basis of expected CYP system involved. For CYP1A-mediated pathways, all the commonly used experimental models are appropriate except probably the dog. On the contrary, the dog seems to be suitable for modelling of processes depending on the CYP2D. With CYP2C, which is possibly the most large and complicated subfamily, the systems based on monkey ( Maccacus rhesus ) may be a good representative. The CYP3A seems to be well modelled by pig or minipig CYP3A29. Detailed studies on activities with individual isolated CYP forms are needed to understand in full all aspects of inter-species differences and variations.     

Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the "peroxide shunt" pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites.     

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116–134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.     

Induction of cytochrome(s) P450-dependent drug metabolism in cultured MH1C1 hepatoma cells

A cell line derived from a Morris hepatoma, MH1C1, was examined for its in vitro expression of monooxygenases. These cells were found to contain different forms of cytochrome P450, as shown by the response to inducers, namely phenobarbital (PB), 3-methylcholanthrene (MC) and metyrapone (MP). MH1C1 cell monolayers exposed to PB or MC showed an increase in the concentration of two spectrally distinct forms of cytochrome P450. The PB and MC treatments elicited enzyme activities towards the substrates aminopyrine and benzo(a)pyrene, respectively. The cell treatment with metyrapone led to a simultaneous stimulation of aminopyrine demethylase and benzo(a)pyrene hydroxylase activities, so underlining the peculiar features of this inducer.     

细胞色素P450酶与微生物药物创制北大核心CSCD

细胞色素P450酶广泛存在于动植物和微生物体内,具有底物结构多样性和催化反应类型多样性,在天然产物生物合成中扮演重要作用。P450酶可在温和条件下高选择性地催化结构复杂有机化合物中惰性C-H键的氧化反应,具备化学催化剂难以比拟的优势,因此在微生物制药领域具有广阔的应用空间。本文综述了参与天然产物生物合成的P450酶近年来的研究进展;P450酶的酶工程改造、生物转化实践及其在微生物药物创制方面的应用现状;探讨了P450酶的工业应用瓶颈及其解决途径;并对P450酶未来的应用前景进行了展望。     

Elevated hepatic and depressed renal cytochrome P450 activity in the Tg2576 transgenic mouse model of Alzheimer's disease

Recent studies indicate that the Tg2576 transgenic mouse model of Alzheimer's disease [tg(hAPP)] demonstrates disturbances in plasma glucose and neuroendocrine function reminiscent of Alzheimer's disease (AD). Alterations in any one of these systems can have a profound effect on hepatic cytochrome P450 (CYP) expression. Additionally, the recent discovery that amyloid beta 1-42 can induce the expression of CYP reductase in neuronal cultures further suggests that hepatic CYP-related metabolism may be affected by the expression of mutant human amyloid precursor protein in these tg(hAPP) mice. Therefore, the current study was conducted to investigate the activity and protein content of several CYP isoforms in the livers and kidneys of aged (20-month-old) tg(hAPP) mice. tg(hAPP) mice exhibit significant elevations in hepatic CYP2B, CYP2E1-, CYP3A- and CYP4A-associated activities and CYP4A immunoreactive protein compared with wild-type. In contrast to the liver, a significant depression in renal CYP2E1- and CYP4A-associated activities were demonstrated in tg(hAPP) mice. The presence of the mutant hAPP protein was detected in the brain, kidney and livers of tg(hAPP) mice.     

A Link between cholesterol levels and phenobarbital induction of cytochromes P450

Squalestatin1 (SQ1), a potent inhibitor of squalene synthase produced a dose-dependent induction of cytochromes P450 CYP2H1 and CYP3A37 mRNAs in chicken hepatoma cells. The effect of SQ1 was completely reversed by 25-hydroxycholesterol. Bile acids elicited an induction of CYP3A37 and CYP2H1 mRNA. Bile acids also reduced the phenobarbital induction of CYP2H1 but not of CYP3A37 mRNA. The effects of SQ1 and its reversal by 25-hydroxycholesterol and the effects of bile acids were reproduced in reporter gene assays with a phenobarbital-responsive enhancer unit of CYP2H1. These data suggest that an endogenous molecule related to cholesterol homeostasis regulates induction of drug-inducible CYPs.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Interactions of 8-anilino-1-napthalenesulfonic acid (ANS) and cytochrome P450 2B1: Role of ANS as an effector as well as a reporter group

Interactions of cytochrome P450 2B1 were probed using 8-anilino-1-naphthalenesulfonic acid (ANS), a well known and frequently used reporter group for hydrophobic interactions. Titration of cytochrome P450 2B1 with ANS revealed 6.6 ± 0.2 ANS binding sites per molecule of P450 2B1 with a K_d value of 42 ± 2 M. In our evaluation of the consequences of the binding of ANS to cytochrome P450 2B1, we found that the binding of ANS to P450 2B1 increased benzphetamine demethylation activity by 1.5-fold, indicating a role for ANS as an effector in addition to its role as a reporter group. Kinetic analysis of the effects of ANS on P450 2B1-dependent demethylation activity revealed that ANS increased both the V_max and K_m of the benzphetamine demethylation reaction. ANS stimulation of activity appears not to be due to the replacement or augmentation of the role of lipid since studies of binding and catalytic activities in the presence and absence of added lipid gave the same array of effects. These results demonstrate that ANS can bind to cytochrome P450 2B1 as would be expected of a reporter group probe but show in addition that this probe also acts as an effector molecule stimulating catalytic activity. Thus, results of ANS studies should be viewed with caution since the molecule may play more than one role in its reaction with a protein.Abbreviations ANS 8-anilino-1-naphthalenesulfonic acid - bis-ANS 1,1-bis(4-anilino-5-naphthalenesulfonic acid - DTT dithiothreitol - P450 cytochrome P450     

Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) from Candida bombicola

Candida bombicola is a yeast with at least two appealing features. The species can grow on alkanes when provided as the sole carbon source, and it produces glycolipids, which have several industrial, cosmetic and pharmaceutical applications. Both metabolic processes require in their pathway the activity of cytochrome P450 monooxygenase. This enzyme needs and gets reducing equivalents from NADPH cytochrome P450 reductase (CPR). The CPR gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes an enzyme of 687 amino acids, which shows homology with known CPRs of other species. The functionality of the gene was proven by heterologous expression in Escherichia coli. The recombinant protein exhibited NADPH-dependent cytochrome c reducing activity. Cloning and characterization of this enzyme is an important step in the study of the cytochrome P450 monooxygenase system of Candida bombicola. The GenBank accession number of the sequence described in this article is EF050789.     

Expression of constitutive and inducible cytochrome P450 2E1 in rat brain

Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitrostudies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.     

Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli

Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b₅ (Cyt-b₅) was further coexpressed with CPR, indicating that Cyt-b₅ supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b₅ but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an “alternative” electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

Partial purification of Cytochrome P450 from Helicoverpa armigera (Hübner)

Abstract The cytochrome P450 (Cyt‐P450) proteins from the fat body and midgut of the cotton bollworm, Helicoverpa armigera, were respectively partially purified by a set of purification procedures including differential centrifugation, solubilization of CHAPS, protein precipitation by PEG precipitation and DE‐32 column chromatography. The Cyt‐P450 was detected by methods of CO difference spectrum and SDS‐PAGE. Fraction of detergent solubilized microsomes from the fat body of H. armigera was purified more than 17‐fold. Three protein bands were detected by SDS‐PAGE with molecular masses of 70 600, 63 300 and 571 200Da. It is possible that the proteins with molecular mass of 63 300 and 571 200Da were the isozymes of Cyt‐P450.     

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid–binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the k_cat/K_m values either decreased 5-fold or were equal to the respective free retinoid values. The k_cat/K_m value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in k_cat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.