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Sample records for cytochrome p450 monooxygenases

  1. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  2. Effects of bromocriptine on hepatic cytochrome P-450 monooxygenase system.

    PubMed

    Moochhala, S M; Lee, E J; Hu, G T; Koh, O S; Becket, G

    1989-02-01

    We have evaluated the in vitro effects of bromocriptine (Br), on the hepatic cytochrome P-450 monooxygenase system of rats pretreated with saline phenobarbitone (PB) and beta-naphthoflavone (BNF). Br inhibited ethoxyresorufin O-dealkylase (EROD) activity in liver microsomes of rats pretreated with saline and PB but not in BNF pretreated animals. Maximum inhibition of EROD activity by Br in the microsomes of saline and PB pretreated rats were 50%-60% of the control. In contrast, a dual effect was observed on aminopyrine N-demethylase activity (APD) by Br in microsomes of saline, PB and BNF pretreated rats. At a low concentration (25 microM), Br inhibited the activity of APD to a similar extent in all pretreatment groups; however, with higher concentrations of Br (50 microM to 300 microM), enhancement of APD activity was observed. Br (300 microM) increased the APD activity to 2-3 times the control level in microsomes of rats pretreated with saline, PB or BNF. Spectral studies revealed a Type II binding of Br to cytochrome P-450 from microsomes of saline and PB pretreated rats. A reverse type I binding was observed for BNF induced microsomes. In addition, Br also enhanced NADPH cytochrome c (P-450) reductase activity to a similar extent in all pretreatment groups. These results suggest that the inhibition of EROD activity may be due to direct binding by Br to certain isozymes of cytochrome P-450 and that the enhancing effect of Br on APD activity may be in part due to the activation of the NADPH cytochrome c reductase component of the cytochrome P-450 monooxygenase system. PMID:2499727

  3. Terpene hydroxylation with microbial cytochrome P450 monooxygenases.

    PubMed

    Janocha, Simon; Schmitz, Daniela; Bernhardt, Rita

    2015-01-01

    Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes. PMID:25682070

  4. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  5. Regulation of cytochrome P-450 monooxygenases in the mouse

    SciTech Connect

    Kelley, M.F.

    1986-01-01

    Recently, the compound 1,4-bis(2-(3,4-dichloropyridyloxy)) benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16..cap alpha..-carbonitrile (PCN) and (4) the binding of (/sup 3/H) TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2..cap alpha..-, 6..beta..- and 15..beta..- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of (/sup 3/H) TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of (/sup 3/H) TCPOBOP to cytosolic marcomolecular elements.

  6. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s.

    PubMed

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E; Nelson, David R; Tuszynski, Jack A; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria -42; fungi -19; plant -28; animal -22; plant and animal -1 and common P450 family -1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study's results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  7. Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are a group of versatile redox proteins that mediate the biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds which act as plant defense agents. To determine if cytochrome P450 monooxygenases are involved in defense response to...

  8. Selective Usage of Transcription Initiation and Polyadenylation Sites in Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  9. Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.

    PubMed

    Ichinose, Hirofumi; Michizoe, Junji; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

    2004-06-22

    Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media. PMID:15986701

  10. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    PubMed Central

    2010-01-01

    Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is

  11. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

    PubMed

    Brummund, Jan; Müller, Monika; Schmitges, Thomas; Kaluzna, Iwona; Mink, Daniel; Hilterhaus, Lutz; Liese, Andreas

    2016-09-10

    Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450. PMID:27396939

  12. Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase.

    PubMed

    Jennewein, S; Rithner, C D; Williams, R M; Croteau, R B

    2001-11-20

    A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species. PMID:11707604

  13. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  14. Induction of cytochrome P450 1A1 and monooxygenase activity in Tilapia by sediment extract

    SciTech Connect

    Ueng, Y.F.; Ueng, T.H.; Liu, T.Y.

    1995-01-01

    Cytochrome P450 (P450)-dependent monooxygenases of fishes are inducible by a variety of environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Induction of fish monoxygenases may serve as a biological monitor for PAH- and PCB-types of environmental chemicals. Many studies have demonstrated environmental induction of fish monooxygenases using various experimental approaches. However, relatively few studies have been conducted using fish treated with contaminated river sediment extracts. Damsui River is the largest river in the north of Taiwan. The lower section of the river in the Taipei Metropolitan area is heavily polluted by industrial and municipal wastes. Tilapia (Oreochromis mossambicus) is one of the few species of fish that occur in the polluted river. Previous field studies showed that the levels of P450 1A1, benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in tilapia collected at Fu-Ho Bridge, a polluted section of Damsui River, were higher than respective levels in fish collected from an unpolluted section. These results suggested that tilapia caught at the polluted site were exposed to substances similar in action to PAHs and PCBs, because these chemical pollutants are potent inducers of P450 1A1. PAHs and PCBs are persistent compounds that can accumulate in sediment. Tilapia are occasionally associated with the bottom and could ingest chemically contaminated sediment. In the present study, we determined the induction properties of monooxygenases using tilapia treated with extract of sediment collected from a polluted section of Damsui River. The present study demonstrates that Damsui River sediment extract has the ability to induce hepatic P450 1A1 and dependent monooxygenase activities in tilapia. 17 refs., 2 figs., 2 tabs.

  15. Genomewide annotation and comparative genomics of cytochrome P450 monooxygenases (P450s) in the polypore species Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora.

    PubMed

    Syed, Khajamohiddin; Nelson, David R; Riley, Robert; Yadav, Jagjit S

    2013-01-01

    Genomewide annotation of cytochrome P450 monooxygenases (P450s) in three white-rot species of the fungal order Polyporales, namely Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora, revealed a large contingent of P450 genes (P450ome) in their genomes. A total of 199 P450 genes in B. adusta and 209 P450 genes each in Ganoderma sp. and P. brevispora were identified. These P450omes were classified into families and subfamilies as follows: B. adusta (39 families, 86 subfamilies), Ganoderma sp. (41 families, 105 subfamilies) and P. brevispora (42 families, 111 subfamilies). Of note, the B. adusta genome lacked the CYP505 family (P450foxy), a group of P450-CPR fusion proteins. The three polypore species revealed differential enrichment of individual P450 families in their genomes. The largest CYP families in the three genomes were CYP5144 (67 P450s), CYP5359 (46 P450s) and CYP5344 (43 P450s) in B. adusta, Ganoderma sp. and P. brevispora, respectively. Our analyses showed that tandem gene duplications led to expansions in certain P450 families. An estimated 33% (72 P450s), 28% (55 P450s) and 23% (49 P450s) of P450ome genes were duplicated in P. brevispora, B. adusta and Ganoderma sp., respectively. Family-wise comparative analysis revealed that 22 CYP families are common across the three Polypore species. Comparative P450ome analysis with Ganoderma lucidum revealed the presence of 143 orthologs and 56 paralogs in Ganoderma sp. Multiple P450s were found near the characteristic biosynthetic genes for secondary metabolites, namely polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene cyclase and terpene synthase in the three genomes, suggesting a likely role of these P450s in secondary metabolism in these Polyporales. Overall, the three species had a richer P450 diversity both in terms of the P450 genes and P450 subfamilies as compared to the model white-rot and brown-rot polypore species Phanerochaete chrysosporium and Postia placenta. PMID

  16. Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus.

    PubMed

    Chan, Hiang Hao; Wajidi, Mustafa Fadzil Farid; Zairi, Jaal

    2014-01-01

    Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450. PMID:25399430

  17. Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics.

    PubMed

    David, J P; Boyer, S; Mesneau, A; Ball, A; Ranson, H; Dauphin-Villemant, C

    2006-05-01

    The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed. PMID:16651188

  18. Cytochrome P-450-dependent monooxygenases in olfactory epithelium of dogs: possible role in tumorigenicity

    SciTech Connect

    Dahl, A.R.; Hadley, W.M.; Hahn, F.F.; Benson, J.M.; McClellan, R.O.

    1982-04-02

    The respiratory tract epithelium of dogs, from the nose to the lungs, was examined for cytochrome P-450 and associated biotransformation activities. In the ethmoturbinates, where olfactory epithelium is located, the amount of cytochrome P-450 was comparable to that in the liver, when measured on the basis of activity per milligram of microsomal protein. The rest of the nasal region also contained large quantities of cytochrome P-450. The presence of these enzymes in the nose may be important in chemical-induced tumorigenesis. The nasal carcinogen hexamethylphosphoramide was shown to be metabolized by nasal microsomal enzymes to another nasal carcinogen, formaldehyde.

  19. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    PubMed

    Qhanya, Lehlohonolo Benedict; Matowane, Godfrey; Chen, Wanping; Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  20. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

    PubMed Central

    Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  1. Systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi.

    PubMed

    Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

    2014-01-01

    Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin's theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our

  2. (+)-Abscisic Acid 8′-Hydroxylase Is a Cytochrome P450 Monooxygenase1

    PubMed Central

    Krochko, Joan E.; Abrams, Garth D.; Loewen, Mary K.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Abscisic acid (ABA) 8′-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8′-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8′-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10−6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8′-hydroxylase is a cytochrome P450 monooxygenase. PMID:9808729

  3. Identification of Cytochrome P450 monooxygenases in Diaphornia citri, an economically important psyllid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty P450’s in the CYP monooxygenases were identified in the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The psyllid is responsible for the transmission of Candidatus Liberibacter asiaticus, the causative agent of huanglongbing ( HLB), also known as citrus greening dise...

  4. Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

    PubMed

    Hrycay, Eugene G; Bandiera, Stelvio M

    2015-01-01

    This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex. PMID:26002730

  5. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    PubMed

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  6. Fungal Cytochrome P450 Monooxygenases: Their Distribution, Structure, Functions, Family Expansion, and Evolutionary Origin

    PubMed Central

    Chen, Wanping; Lee, Mi-Kyung; Jefcoate, Colin; Kim, Sun-Chang; Chen, Fusheng; Yu, Jae-Hyuk

    2014-01-01

    Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi. PMID:24966179

  7. Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium.

    PubMed

    Hu, Jie; Okawa, Hiroto; Yamamoto, Kentaro; Oyama, Kazuhiko; Mitomi, Masaaki; Anzai, Hiroyuki

    2011-03-01

    Pyripyropenes are potent inhibitors of acyl-CoA:cholesterol acyltransferase, which were initially discovered to be produced by Aspergillus fumigatus. Recently, Penicillium coprobium PF1169 has also found to produce pyripyropene A (PyA), which exhibits insecticidal properties. Pyripyropenes are natural hybrid products of both terpenoid and polyketide origin. In our research, based on data generated using the Genome Sequencer FLX for P. coprobium PF1169, we predicted the biosynthetic gene cluster of PyA by blast analysis comparing with polyketide synthase and prenyltransferase of other species. By screening the genomic fosmid library, nine open reading frames (ppb1 to ppb9) related to the biosynthesis of PyA were deduced. Among them, two cytochrome P450 monooxygenase genes (ppb3 and ppb4) were separately introduced into the model fungus A. oryzae. Bioconversion of certain predicted intermediates in the transformants has elucidated the manner of hydroxylation in the biosynthetic pathway by the expressed products of these two genes (P450-1 and P450-2). That is, P450-1 exhibits monooxygenase activity and plays the hydroxylation role at C-11 of pyripyropene E. While P450-2 plays an active role in the hydroxylation of C-7 and C-13 of pyripyropene O. PMID:21224862

  8. Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

    PubMed

    Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2015-11-01

    The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. PMID:26234643

  9. Cytochromes p450.

    PubMed

    Werck-Reichhart, Danièle; Bak, Søren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  10. Cytochromes P450

    PubMed Central

    Werck-Reichhart, Danièle; Bak, Søren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  11. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    SciTech Connect

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  12. CYP709B3, a cytochrome P450 monooxygenase gene involved in salt tolerance in Arabidopsis thaliana

    PubMed Central

    2013-01-01

    Background Within the Arabidopsis genome, there are 272 cytochrome P450 monooxygenase (P450) genes. However, the biological functions of the majority of these P450s remain unknown. The CYP709B family of P450s includes three gene members, CYP709B1, CYP709B2 and CYP709B3, which have high amino acid sequence similarity and lack reports elucidating biological functions. Results We identified T-DNA insertion-based null mutants of the CYP709B subfamily of genes. No obvious morphological phenotypes were exhibited under normal growth conditions. When the responses to ABA and salt stress were studied in these mutants, only the cyp709b3 mutant showed sensitivity to ABA and salt during germination. Under moderate salt treatment (150 mM NaCl), cyp709b3 showed a higher percentage of damaged seedlings, indicating a lower tolerance to salt stress. CYP709B3 was highly expressed in all analyzed tissues and especially high in seedlings and leaves. In contrast, CYP709B1 and CYP709B2 were highly expressed in siliques, but were at very low levels in other tissues. Under salt stress condition, CYP709B3 gene expression was induced after 24 hr and remained at high expression level. Expression of the wild type CYP709B3 gene in the cyp709b3 mutant fully complemented the salt intolerant phenotype. Furthermore, metabolite profiling analysis revealed some differences between wild type and cyp709b3 mutant plants, supporting the salt intolerance phenotype of the cyp709b3 mutant. Conclusions These results suggest that CYP709B3 plays a role in ABA and salt stress response and provides evidence to support the functions of cytochrome P450 enzymes in plant stress response. PMID:24164720

  13. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  14. Cytochromes p450.

    PubMed

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  15. Cytochrome P450 database.

    PubMed

    Lisitsa, A V; Gusev, S A; Karuzina, I I; Archakov, A I; Koymans, L

    2001-01-01

    This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope. PMID:11769119

  16. Identification of universal selectivity-determining positions in cytochrome P450 monooxygenases by systematic sequence-based literature mining.

    PubMed

    Gricman, Łukasz; Vogel, Constantin; Pleiss, Jürgen

    2015-09-01

    Cytochrome P450 monooxygenases (CYPs) are a large, highly diverse protein family with a common fold. The sequences, structures, and functions of CYPs have been extensively studied resulting in more than 53,000 scientific articles. A sequence-based literature mining algorithm was designed to systematically analyze this wealth of information on SNPs, designed mutations, structural interactions, or functional roles of individual residues. Structurally corresponding positions in different CYPs were compared and universal selectivity-determining positions were identified. Based on the Cytochrome P450 Engineering Database (www.CYPED.BioCatNet.de) and a standard numbering scheme for all CYPs, 4000 residues in 168 CYPs mentioned in 2400 articles could be assigned to 440 structurally corresponding standard positions of the CYP fold, covering 96% of all standard positions. Seventeen individual standard positions were mentioned in the context of more than 32 different CYPs. The majority of these most frequently mentioned positions are located on the six substrate recognition sites and are involved in control of selectivity, such as the well-studied position 87 in CYP102A1 (P450(BM-3)) which was mentioned in the articles on 63 different CYPs. The recurrent citation of the 17 frequently mentioned positions for different CYPs suggests their universal functional relevance. PMID:26033392

  17. Cytochromes P450: Roles in Diseases*

    PubMed Central

    Pikuleva, Irina A.; Waterman, Michael R.

    2013-01-01

    The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s. PMID:23632021

  18. Role of Cytochrome P450 Monooxygenase in Carcinogen and Chemotherapeutic Drug Metabolism.

    PubMed

    Wahlang, B; Falkner, K Cameron; Cave, Matt C; Prough, Russell A

    2015-01-01

    The purpose of this chapter is to provide insight into which human cytochromes P450 (CYPs) may be involved in metabolism of chemical carcinogens and anticancer drugs. A historical overview of this field and the development of literature using relevant animal models and expressed human CYPs have provided information about which specific CYPs may be involved in carcinogen metabolism. Definition of the biochemical properties of CYP activity came from several groups who studied the reaction stoichiometry of butter yellow and benzo[α]pyrene, including their role in induction of these enzyme systems. This chapter will list as much as is known about the human CYPs involved in carcinogen and anticancer drug metabolism, as well as summarize studies with rodent CYPs. A review of three major classes of anticancer drugs and their metabolism in humans is covered for cyclophosphamide, procarbazine, and anthracycline antibiotics, cancer chemotherapeutic compounds extensively metabolized by CYPs. The emerging information about human CYP gene polymorphisms as well as other enzymes involved in foreign compound metabolism provides considerable information about how these genetic variants affect carcinogen and anticancer drug metabolism. With information available from individual's genomic sequences, consideration of populations who may be at risk due to environmental exposure to carcinogens or how to optimize their cancer therapy regimens to enhance efficacy of the anticancer drugs appears to be an important field of study to benefit individuals in the future. PMID:26233902

  19. Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization.

    PubMed

    Griffiths, Scott; Mesarich, Carl H; Saccomanno, Benedetta; Vaisberg, Abraham; De Wit, Pierre J G M; Cox, Russell; Collemare, Jérôme

    2016-06-21

    Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families. PMID:27274078

  20. Alterations of cytochrome P450-dependent monooxygenase activities in Eriocheir japonicus in response to water pollution.

    PubMed Central

    Ishizuka, M; Hoshi, H; Minamoto, N; Masuda, M; Kazusaka, A; Fujita, S

    1996-01-01

    Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the field studies, crabs collected from the river with the highest concentration of PAHs exhibited the highest levels of CYP, the highest activities of benzo[a]pyrene 3-hydroxylase, imipramine 2-hydroxylase, bunitrolol 4-hydroxylase, ethoxycoumarin O-deethylase, and the ability to metabolically activate benzo[a]pyrene, but erythromycin N-demethylase activity was not induced. The correlation between PAH levels and drug-metabolizing enzyme activities in female crabs were not as marked as in male crabs. The levels and activities of CYP did not appear to reflect the concentrations of organochlorines and polychlorinated biphenyl congeners (PCBs) studied in the fat of crab hepatopancreas. Images Figure 1. Figure 2. A Figure 2. B Figure 2. C Figure 3. Figure 4. A Figure 4. B Figure 5. A Figure 5. B Figure 5. C Figure 5. D Figure 5. E Figure 5. F Figure 6. PMID:8841764

  1. Acute and subacute effects of miconazole nitrate on hepatic styrene oxide hydrolase and cytochrome P-450-dependent monooxygenase activities in male and female AKR/J mice.

    PubMed

    James, M O

    1988-08-01

    The imidazole-containing anti-fungal drug, miconazole nitrate, was shown to enhance hepatic microsomal styrene oxide hydrolase and inhibit several cytochrome P-450-dependent monooxygenase activities in the AKR/J mouse. Miconazole was a more potent inhibitor of cytochrome P-450-dependent monooxygenase activities in microsomes from male than female mice, and inhibitory potency also varied with substrate. When administered in vivo miconazole nitrate stimulated epoxide hydrolase activity, but had a substrate-dependent biphasic effect on cytochrome P-450-dependent monooxygenase activities. Monooxygenase activities with benzo[a]pyrene and benzphetamine were inhibited to varying degrees in liver homogenate and hepatic microsomes from mice sacrificed 45 min after miconazole administration. After repeated administration of miconazole, liver weight, microsomal protein yield and cytochrome P-450 were increased, as were specific monooxygenase activities with ethoxycoumarin and ethoxyresorufin, but benzphetamine N-demethylase activity was decreased. These results suggested that a metabolite of miconazole was responsible for the inhibition of benzphetamine N-demethylase. It was of special interest that ethoxyresorufin O-deethylase activity was induced in the AKR/J mouse by miconazole, since the AKR/J mouse is not responsive to induction by aromatic hydrocarbons. PMID:3394155

  2. Regiochemical variations in reactions of methylcubane with tert-butoxyl radical, cytochrome P-450 enzymes, and a methane monooxygenase system

    SciTech Connect

    Choi, S.Y.; Hollenberg, P.F.; Newcomb, M.; Putt, D.A.; Eaton, P.E.; Upadhyaya, S.P.; Xiong, Y.; Liu, K.E.; Lippard, S.J.

    1996-07-17

    Reactions of methylcubane (1) with the tert-butoxyl radical (t-BuO{sup .}), with cytochrome P-450 enzymes, and with a methane monooxygenase (MMO) system have been studied. 2-Methylcubanecarboxylic acid (9b) is a new compound prepared from cubanecarboxylic acid. The key synthetic reactions were (1) metalation and subsequent iodination of the 2-position of (diisopropylcarbamoyl)cubane to effect the initial functionalization, (2) lithium-for-iodine exchange and methylation followed by reduction to give 2-methyl-l-[(diisopropylamino)methyl]-cubane, and (3) dimethyldioxirane oxidation of this amine to give 9b. Reaction of 1 with t-BuO{sup .} in the presence of 2,2,5,5-tetramethylisoindole-N-oxyl radical (TMIO{sup .}) at 40-55{degree}C gave mainly cube-substituted products in confirmation of the report that hydrogen atom abstraction by the electrophilic alkoxyl radical at low temperature occurs at the cubyl C-H positions. In a competition experiment at 42{degree}C, methylcubane was at least 3.5 times more reactive toward t-BuO{sup .} than cyclohexane, indicating that the cubyl positions in 1 are >= 40 times more reactive than the methyl positions in 1 (per hydrogen) toward the alkoxyl radical. Oxidation of 1 by enzymes gave alcohol products that were converted to their acetate derivatives for identification and quantitation. Microsomal cytochrome P-450 enzymes from rat and the rat purified P-450 isozyme CYP2B1 hydroxylated 1 at all positions, whereas the reconstituted MMO system from Methylococcus capsulatus (Bath) hydroxylated l only at the methyl position. 78 refs., 1 tab.

  3. Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

    SciTech Connect

    Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

    1985-02-01

    The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H/sub 2/O/sub 2/ such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.

  4. Evaluating cytochrome P450 in birds by monooxygenases and immunohistochemistry: possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2000-01-01

    Six month old Lesser Scaup and nestling Tree Swallows were injected intraperitoneally with beta-naphthoflavone (BNF) or vehicle. Nestling Tree Swallows were also collected from five sites with differing levels of contaminants. Liver samples were taken and stored at -80C until microsome preparation and monooxygenase (MO) assay. Skin and heart samples were placed in buffered formalin until immunohistochemical (IMHC) analysis for cytochrome P4501A (CYP1A). Scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 20- to 65-fold increases in four MOs. Responses of two of the four MOs were as high at 20 mg/kg as at 100mg/kg. There was no IMHC response in the vehicle-injected ducks, while in skin the IMHC response was the same for both dose levels of BNF and in heart there was response in two of four samples at 20 mg/kg and in all five samples at 100mg/kg. Tree Swallows injected with BNF at 100, but not at 20 mg/kg showed significant increases (ca.5-fold) in two MO activities. There was no IMHC response in control swallows. In skin and heart there were IMHC responses in one of five swallows at 20 mg/kg and four of five swallows at 100mg/kg. There was poor correlation between individual skin IMHC responses and MO activities and PCB concentrations in 47 field-collected Tree Swallow samples, but 14 of the 16 skin samples with positive IMHC responses were from the location with the highest MO activities and PCB concentrations. Although present data do not allow construction of significant dose response curves, the responses in skin make it well worth continuing study on this potential nonlethal technique for biomonitoring contaminant exposure of birds.

  5. A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

    PubMed

    Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

    2015-09-01

    Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. PMID:25968601

  6. A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus.

    PubMed Central

    St-Pierre, B.; De Luca, V.

    1995-01-01

    Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation. These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light. The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway. However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C. roseus cell-suspension cultures. These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms. PMID:12228585

  7. Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases.

    PubMed

    Kolrep, Franziska; Hessel, Stefanie; These, Anja; Ehlers, Anke; Rein, Kathleen; Lampen, Alfonso

    2016-08-01

    The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded. PMID:26374342

  8. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    PubMed Central

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  9. Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance

    PubMed Central

    Liu, Simu; Bartnikas, Lisa M.; Volko, Sigrid M.; Ausubel, Frederick M.; Tang, Dingzhong

    2016-01-01

    Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew. PMID:26973671

  10. Cytochrome P-450 monooxygenase systems in aquatic species: Carcinogen metabolism and biomarkers for carcinogen and pollutant exposure

    SciTech Connect

    Stegeman, J.J. ); Lech, J.J. )

    1991-01-01

    High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters. The potential for using P-450 induction to detect direct-acting carcinogens and tumor promoters that are noninducers is limited, although such compounds can be expected to co-occur with pollutants that are inducers.

  11. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene.

    PubMed

    Cankar, Katarina; van Houwelingen, Adèle; Bosch, Dirk; Sonke, Theo; Bouwmeester, Harro; Beekwilder, Jules

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene. PMID:21115006

  12. Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

    PubMed Central

    Jawallapersand, Poojah; Mashele, Samson Sitheni; Kovačič, Lidija; Stojan, Jure; Komel, Radovan; Pakala, Suresh Babu; Kraševec, Nada; Syed, Khajamohiddin

    2014-01-01

    Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative

  13. Identification of cytochrome P450 monooxygenase genes and their expression profiles in cyhalothrin-treated Colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Shi, Xiao-Qin; Kong, Ye; Zhou, Li-Tao; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2013-11-01

    Based on a Leptinotarsa decemlineata transcriptome dataset and the GenBank sequences, a total of 74 cytochrome P450 monooxygenase genes (Cyps) were identified. These genes fell into CYP2 clan, mitochondrial clan, CYP3 clan and CYP4 clan, and were classified into 19 families and 35 subfamilies according to standard nomenclature. Two new families were discovered in CYP4 clan, and were named CYP412 and CYP413 respectively. Four new families that were recently discovered in Tribolium castaneum, including mitochondrial family CYP353, CYP3 clan families CYP345 and CYP347, and CYP4 clan family CYP350, were also found in L. decemlineata. The phylogenetic trees of CYPs from L. decemlineata and other representative insect species were constructed, and these trees provided evolutionary insight for the genetic distance. Our results facilitate further researches to understand the functions and evolution of L. decemlineata Cyp genes. In order to find cyhalothrin-inducible Cyp genes, the expression levels of Cyps belonging to CYP12, CYP6, CYP9 and CYP4 families were determined by quantitative reverse transcriptase-PCR in cyhalothrin-treated and control fourth-instar larvae. Nine Cyp genes, i.e., Cyp12H2, Cyp6BH2, Cyp6BJ1, Cyp6BQ17, Cyp6EG1, Cyp6EH1, Cyp6EJ1 Cyp4BN13v1 and Cyp4BN15, were highly expressed in cyhalothrin-treated larvae. These CYPs are the candidates that are involved in cyhalothrin detoxification. PMID:24267698

  14. Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis

    SciTech Connect

    Tokai, Takeshi; Koshino, Hiroyuki; Takahashi-Ando, Naoko; Sato, Masayuki; Fujimura, Makoto; Kimura, Makoto . E-mail: mkimura@riken.jp

    2007-02-09

    Fusarium Tri4 encodes a cytochrome P450 monooxygenase (CYP) for hydroxylation at C-2 of First committed intermediate trichodiene (TDN) in the biosynthesis of trichothecenes. To examine whether this CYP further participates in subsequent oxygenation steps leading to isotrichotriol (4), we engineered Saccharomyces cerevisiae for de novo production of the early intermediates by introducing cDNAs of Fusarium graminearum Tri5 (FgTri5 encoding TDN synthase) and Tri4 (FgTri4). From a culture of the engineered yeast grown on induction medium (final pH 2.7), we identified two intermediates, 2{alpha}-hydroxytrichodiene (1) and 12,13-epoxy-9,10-trichoene-2{alpha}-ol (2), and a small amount of non-Fusarium trichothecene 12,13-epoxytrichothec-9-ene (EPT). Other intermediates isotrichodiol (3) and 4 were identified in the transgenic yeasts grown on phosphate-buffered induction medium (final pH 5.5-6.0). When Trichothecium roseum Tri4 (TrTri4) was used in place of FgTri4, 4 was not detected in the culture. The three intermediates, 1, 2, and 3, were converted to 4,15-diacetylnivalenol (4,15-diANIV) when fed to a toxin-deficient mutant of F. graminearum with the FgTri4 {sup +} genetic background (viz., by introducing a FgTri5 {sup -} mutation), but were not metabolized by an FgTri4 {sup -} mutant. These results provide unambiguous evidence that FgTri4 encodes a multifunctional CYP for epoxidation at C-12,13, hydroxylation at C-11, and hydroxylation at C-3 in addition to hydroxylation at C-2.

  15. Cooperative properties of cytochromes P450

    PubMed Central

    Denisov, Ilia G.; Frank, Daniel J.; Sligar, Stephen G.

    2009-01-01

    Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes. PMID:19555717

  16. Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel

    PubMed Central

    Tan, Cheau Yuaan; Hirakawa, Hidehiko; Nagamune, Teruyuki

    2015-01-01

    Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking “proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins” (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD+. The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions. PMID:25733255

  17. Metabolism of fluperlapine by cytochrome P450-dependent and flavin-dependent monooxygenases in continuous cultures of rat and human cells.

    PubMed

    Fischer, V; Wiebel, F J

    1990-04-15

    The metabolism of fluperlapine, a neuroleptic dibenzazepine derivative with a N-methyl-piperazinyl substituent, was investigated in continuous cultures of rat and human cells which express various cytochrome P450-dependent monooxygenase activities. The differentiated rat hepatoma cells H4IIEC3/G- and their variants 2sFou and FGC-5 metabolized fluperlapine predominantly by N-oxygenation and only to a minor degree by N-demethylation or glucuronidation of primary phenolic products. Total fluperlapine metabolism in dedifferentiated rat hepatoma cells H5 and partially differentiated human hepatoma cells HepG2 was much smaller than in the differentiated rat hepatoma lines. This was primarily attributable to their low capacity for N-oxygenation. Human lung adenocarcinoma lines NCI-H322 and NCI-H358 formed only trace amounts of fluperlapine N-oxide. Pretreatment of 2sFou cells with benz(a)anthracene, phenobarbital or dexamethasone markedly increased the formation of N-demethylated and glucuronidated products but did not affect the rate of N-oxide formation. Guanethidine and cysteamine, inhibitors of flavin-dependent monooxygenase activity, reduced fluperlapine N-oxidation more strongly than aldrin epoxidation, a marker for cytochrome P450 activity. In contrast, n-octylamine inhibited aldrin epoxidation but was without effect on fluperlapine N-oxygenation. The results suggest that certain cells in continuous culture are capable of expressing flavin-dependent monooxygenase(s) in addition to cytochrome P450-containing monooxygenases. Such cells may offer useful systems for studying the oxidative metabolism of a broad spectrum of xenobiotics and analysing the importance of the two oxygenation reactions for the biological effects of their substrates. PMID:2322314

  18. Structural Diversity of Eukaryotic Membrane Cytochrome P450s*

    PubMed Central

    Johnson, Eric F.; Stout, C. David

    2013-01-01

    X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance. PMID:23632020

  19. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  20. Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide by the Cytochrome P450 Monooxygenases: Physiological and Pharmacological Implications

    PubMed Central

    Walker, Vyvyca J.; Hollenberg, Paul F.

    2010-01-01

    Arachidonoyl ethanolamide (anandamide) is an endogenous amide of arachidonic acid and an important signaling mediator of the endocannabinoid system. Given its numerous roles in maintaining normal physiological function and modulating pathophysiological responses throughout the body, the endocannabinoid system is an important pharmacological target amenable to manipulation directly by cannabinoid receptor ligands or indirectly by drugs that alter endocannabinoid synthesis and inactivation. The latter approach has the possible advantage of more selectivity, thus there is the potential for fewer untoward effects like those that are traditionally associated with cannabinoid receptor ligands. In that regard, inhibitors of the principal inactivating enzyme for anandamide, fatty acid amide hydrolase (FAAH), are currently in development for the treatment of pain and inflammation. However, several pathways involved in anandamide synthesis, metabolism, and inactivation all need to be taken into account when evaluating the effects of FAAH inhibitors and similar agents in preclinical models and assessing their clinical potential. Anandamide undergoes oxidation by several human cytochrome P450 (P450) enzymes, including CYP3A4, CYP4F2, CYP4X1, and the highly polymorphic CYP2D6, forming numerous structurally diverse lipids, which are likely to have important physiological roles, as evidenced by the demonstration that a P450-derived epoxide of anandamide is a potent agonist for the cannabinoid receptor 2. The focus of this review is to emphasize the need for a better understanding of the P450-mediated pathways of the metabolism of anandamide, because these are likely to be important in mediating endocannabinoid signaling as well as the pharmacological responses to endocannabinoid-targeting drugs. PMID:20133390

  1. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  2. P450monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium

    PubMed Central

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-01-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system (LDS), mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosporium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed. PMID:22624627

  3. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

    PubMed Central

    Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

    2005-01-01

    4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

  4. Mannitol Stress Directs Flavonoid Metabolism toward Synthesis of Flavones via Differential Regulation of Two Cytochrome P450 Monooxygenases in Coleus forskohlii.

    PubMed

    Awasthi, Praveen; Gupta, Ajai Prakash; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

    2016-01-01

    Cytochrome P450 monooxygenases (CYP450s) are known to play important roles in biosynthesis of all secondary metabolites, including flavonoids. Despite this, few CYP450s have been functionally characterized in model plants and roles of fewer CYP450s are known in non-model, medicinal, and aromatic plants. Our study in Coleus forskohlii indicates that flavone synthase (CYP93B) and flavonoid 3' monooxygenase (CYP706C) are key enzymes positioned at a metabolic junction, to execute the biosynthesis of different sub-classes of flavonoids (flavones, flavonol, anthocynanin, isoflavones etc.) from a common precursor. Such branch points are favored targets for artificially modulating the metabolic flux toward specific metabolites, through genetic manipulation or use of elicitors that differentially impact the expression of branch point genes. Genkwanin, the only flavone reported from C. forskohlii, is known to possess anti-inflammatory activity. It is biosynthesized from the general flavonoid precursor: naringenin. Two differentially expressed cytochrome P450 genes (CfCYP93B, CfCYP706C), exhibiting maximum expression in leaf tissues, were isolated from C. forskohlii. Mannitol treatment resulted in increased expression of CfCYP93B and decrease in expression of CfCYP706C. Metabolite quantification data showed that genkwanin content increased and anthocyanin levels decreased in response to mannitol treatment. Alignment, phylogenetic analysis, modeling, and molecular docking analysis of protein sequences suggested that CfCYP93B may be involved in conversion of naringenin to flavones (possibly genkwanin via apigenin), while CfCYP706C may act on common precursors of flavonoid metabolism and channel the substrate toward production of flavonols or anthocynanins. Decrease in expression of CfCYP706C and increase in accumulation of genkwanin suggested that mannitol treatment may possibly lead to accumulation of genkwanin via suppression of a competitive branch of flavonoids in C

  5. Mannitol Stress Directs Flavonoid Metabolism toward Synthesis of Flavones via Differential Regulation of Two Cytochrome P450 Monooxygenases in Coleus forskohlii

    PubMed Central

    Awasthi, Praveen; Gupta, Ajai Prakash; Bedi, Yashbir S.; Vishwakarma, Ram A.; Gandhi, Sumit G.

    2016-01-01

    Cytochrome P450 monooxygenases (CYP450s) are known to play important roles in biosynthesis of all secondary metabolites, including flavonoids. Despite this, few CYP450s have been functionally characterized in model plants and roles of fewer CYP450s are known in non-model, medicinal, and aromatic plants. Our study in Coleus forskohlii indicates that flavone synthase (CYP93B) and flavonoid 3′ monooxygenase (CYP706C) are key enzymes positioned at a metabolic junction, to execute the biosynthesis of different sub-classes of flavonoids (flavones, flavonol, anthocynanin, isoflavones etc.) from a common precursor. Such branch points are favored targets for artificially modulating the metabolic flux toward specific metabolites, through genetic manipulation or use of elicitors that differentially impact the expression of branch point genes. Genkwanin, the only flavone reported from C. forskohlii, is known to possess anti-inflammatory activity. It is biosynthesized from the general flavonoid precursor: naringenin. Two differentially expressed cytochrome P450 genes (CfCYP93B, CfCYP706C), exhibiting maximum expression in leaf tissues, were isolated from C. forskohlii. Mannitol treatment resulted in increased expression of CfCYP93B and decrease in expression of CfCYP706C. Metabolite quantification data showed that genkwanin content increased and anthocyanin levels decreased in response to mannitol treatment. Alignment, phylogenetic analysis, modeling, and molecular docking analysis of protein sequences suggested that CfCYP93B may be involved in conversion of naringenin to flavones (possibly genkwanin via apigenin), while CfCYP706C may act on common precursors of flavonoid metabolism and channel the substrate toward production of flavonols or anthocynanins. Decrease in expression of CfCYP706C and increase in accumulation of genkwanin suggested that mannitol treatment may possibly lead to accumulation of genkwanin via suppression of a competitive branch of flavonoids in C

  6. avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus.

    PubMed Central

    Yu, J; Chang, P K; Cary, J W; Bhatnagar, D; Cleveland, T E

    1997-01-01

    Recent studies have shown that at least 17 genes involved in the aflatoxin biosynthetic pathway are clustered within a 75-kb DNA fragment in the genome of Aspergillus parasiticus. Several additional transcripts have also been mapped to this gene cluster. A gene, avnA (previously named ord-1), corresponding to one of the two transcripts identified earlier between the ver-1 and omtA genes on the gene cluster was sequenced. The nucleotide sequence of the avnA gene contains a coding region for a protein of 495 amino acids with a calculated molecular mass of 56.3 kDa. The gene consists of three exons and two introns. Disruption of the avnA gene in the wild-type aflatoxigenic A. parasiticus strain (SU1-N3) resulted in a nonaflatoxigenic mutant which accumulated a bright yellow pigment. Thin-layer chromatographic studies with six different solvent systems showed that the migration patterns of the accumulated metabolite were identical to those of averantin, a known aflatoxin precursor. Precursor feeding studies with this mutant showed that norsolorinic acid and averantin were not converted to aflatoxin whereas 5'-hydroxyaverantin, averufanin, averufin, versicolorin A. sterigmatocystin, and O-methylsterigmatocystin were converted to aflatoxins. Southern blot analysis of the wild-type strain and avnA-disrupted mutant strain indicated that the avnA gene was disrupted in the mutant strain. A search of the GenBank database for similarity indicated that the avnA gene encodes a cytochrome P-450-type monooxygenase, and it has been assigned to a new P-450 gene family named CYP60A1. We have therefore concluded that the avnA gene encodes a fungal cytochrome P-450-type enzyme which is involved in the conversion of averantin to averufin in the aflatoxin biosynthetic pathway in A. parasiticus. PMID:9097431

  7. Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

    PubMed

    Gambaro, Sabrina E; Robert, Maria C; Tiribelli, Claudio; Gazzin, Silvia

    2016-02-01

    In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage. PMID:25370011

  8. An assessment of cadmium toxicity on cytochrome P-450 and flavin monooxygenase-mediated metabolic pathways of dimethylaniline in male rabbits

    SciTech Connect

    Anjum, F.; Raman, A.; Shakoori, A.R.; Gorrod, J.W. )

    1992-07-01

    Cadmium is an environmental pollutant and its effect on the in vitro metabolism of N,N-dimethylaniline (DMA) using male rabbits was investigated. Activities of cytochrome P-450 and FMO-dependent monooxygenases were studied using hepatic microsomes. Following CdCl2 (i.p.) administration (6 mg/kg/day for 6 days), both DMA-N-oxidation and DMA-N-demethylation decreased by 86%. The effects of CdCl2 on the phenobarbitone (PB)-induced form of P-450 were also studied. Intraperitoneal pretreatment of rabbits with PB (5 mg/kg/day for 5 days) increased N-demethylation by 82%, while N-oxidation decreased by 49%. Both reactions decreased significantly on additional treatment with CdCl2. Promethazine (5 mg/kg/day for 5 days) did not produce any change in the activities of either enzyme. The enzymes remained unaffected by CdCl2 treatment in promethazine-pretreated animals thus confirming its role as a hepatoprotective agent.

  9. Polymorphism of human cytochrome P-450.

    PubMed

    Guengerich, F P; Umbenhauer, D R; Churchill, P F; Beaune, P H; Böcker, R; Knodell, R G; Martin, M V; Lloyd, R S

    1987-03-01

    The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3577206

  10. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

  11. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  12. Transcriptional Regulation of Grape Cytochrome P450 Gene Expression in Response to Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  13. [Cytochrome P450 enzymes and microbial drug development - A review].

    PubMed

    Li, Zhong; Zhang, Wei; Li, Shengying

    2016-03-01

    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes. PMID:27382792

  14. Cytochrome P450: taming a wild type enzyme

    PubMed Central

    Jung, Sang Taek; Lauchli, Ryan; Arnold, Frances H

    2011-01-01

    Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable'--they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability. PMID:21411308

  15. Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms.

    PubMed

    Karande, Rohan; Debor, Linde; Salamanca, Diego; Bogdahn, Fabian; Engesser, Karl-Heinrich; Buehler, Katja; Schmid, Andreas

    2016-01-01

    The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g L tube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile, and have low solubility in water. PMID:26153144

  16. Formaldehyde production promoted by rat nasal cytochrome P-450-dependent monooxygenases with nasal decongestants, essences, solvents, air pollutants, nicotine, and cocaine as substrates

    SciTech Connect

    Dahl, A.R.; Hadley, W.M.

    1983-02-01

    To identify compounds which might be metabolized to formaldehyde in the nasal cavity, 32 potential substrates for cytochrome P-450-dependent monooxygenases were screened with rat nasal and, for comparison, liver microsomes. Tested substrates included 6 nasal decongestants, cocaine, nicotine, 9 essences, 3 potential air pollutants, and 12 solvents. Each test substrate, with the possible exception of the air pollutants, contained one or more N-methyl, O-methyl, or S-methyl groups. Eighteen of the tested materials were metabolized to produce formaldehyde by nasal microsomes. Five substrates, namely, the solvents HMPA and dimethylaniline, cocaine, and the essences dimethyl anthranilate and p-methoxyacetophenone, were metabolized to produce formaldehyde at rates exceeding 1000 pmol/mg microsomal protein/min by nasal microsomes. Eight substrates, including four nasal decongestants, nicotine, and an extract of diesel exhaust particles, were metabolized to produce formaldehyde at rates of 200 to 1000 pmol/mg microsomal protein/min. Five other substrates were metabolized to formaldehyde at detectable rates. The results indicate that a variety of materials which often come in contact with the nasal mucosa can be metabolized to formaldehyde by nasal enzymes. The released formaldehyde may influence the irritancy of inhaled compounds and has been suggested to play a role in the tumorigenicity of some compounds.

  17. Induction of cytochrome P450-associated monooxygenases in northern leopard frogs, Rana pipiens, by 3,3',4,4',5-pentachlorobiphenyl

    USGS Publications Warehouse

    Huang, Y.-W.; Melancon, M.J.; Jung, R.E.; Karasov, W.H.

    1998-01-01

    Northern leopard frogs (Rana pipiens) were injected intraperitoneally either with a solution of polychlorinated biphenyl (PCB) 126 in corn oil at a concentration of 0.2, 0.7, 2.3 and 7.8 mg/kg body weight or with corn oil alone. Appropriate assay conditions with hepatic microsomes were determined for four cytochrome P450-associated monooxygenases: ethoxyresorufin-O-dealkylase (EROD), methoxy-ROD (MROD), benzyloxy-ROD (BROD) and pentoxy-ROD (PROD). One week after PCB administration, the specific activities of EROD, MROD, BROD and PROD were not elevated at doses ? 0.7 mg/kg (p > 0.05), but were significantly increased at doses ? 2.3 mg/kg compared to the control groups (p < 0.05). The increased activity of these four enzymes ranged from 3to 6.4fold relative to control levels. The increased activities were maintained for at least four weeks. Due to a lack of induction at low doses of PCB 126, which were still relatively high compared to currentlyknown environmental concentrations, we suspect that EROD, MROD, BROD, and PROD activities are not sensitive biomarkers for coplanar PCB exposure in leopard frogs.

  18. Induction of cytochrome P450-associated monooxygenases in northern leopard frogs, Rana pipiens, by 3,3{prime},4,4{prime},5-pentachlorobiphenyl

    SciTech Connect

    Huang, Y.; Jung, R.E.; Karasov, W.H.; Melancon, M.J.

    1998-08-01

    In the past decade, biochemical and physiological characteristics such as hepatic detoxifying system. DNA adducts, thyroid malfunction, and acetylcholinesterase inhibition have been used extensively as biomarkers for contaminant exposure. Northern leopard frogs (Rana pipiens) were injected intraperitoneally either with a solution of polychlorinated biphenyl (PCB) 126 m corn oil at a concentration of 0.2, 0.7, 2.3, or 7.8 mg/kg body weight or with corn oil alone. Appropriate assay conditions with hepatic microsomes were determined for four cytochrome P450-associated monooxygenases: ethoxyresorufin-O-dealkylase (EROD), methoxy-ROD (MROD), benzyloxy-ROD (BROD), and pentoxy-ROD (PROD). One week after PCB administration, the specific activities of EROD, MROD, BROD, and PROD were not elevated at doses {le}0.7 mg/kg (p > 0.05) but were significantly increased at doses {ge}2.3 mg/kg compared to the control groups (p < 0.05). The increased activities of these four enzymes were 3 to 6.4 times those in the control groups. The increased activities were maintained for at least 4 weeks. Because of a lack of induction at low doses of PCB 126, which were still relatively high compared to currently known environmental concentration, the authors suspect that EROD, MROD, BROD, and PROD activities are not sensitive biomarkers for coplanar PCB exposure in leopard frogs.

  19. Optimization of a Cytochrome-P450-Monooxygenase-1A-Mediated EROD Assay in the Cape Hake Species Merluccius capensis and Merluccius paradoxus (Pisces)

    PubMed Central

    De Almeida, Louise; Froneman, William; Pletschke, Brett

    2011-01-01

    Cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds, for example, polychlorinated biphenyls (PCBs) and the induction of this protein is often measured in terms of CYP1A-mediated 7-ethoxyresorufin-O-deethylase (EROD) activity. This study was aimed at developing this assay in the Cape hake species Merluccius capensis and Merluccius paradoxus (considered one stock). Microsomal fractions were obtained from frozen fish liver samples by differential centrifugation. Fluorimetric and spectrophotometric analysis of the EROD assay resulted in the spectrophotometric (at 572 nm) detection method being selected, as this method resulted in a lower degree of variability and demonstrated higher reproducibility. The activity in the EROD assay was enhanced in the presence of NADPH, and the addition of dicumarol (phase II enzyme inhibitor) to the reaction mixtures prevented the underestimation of this assay by the inhibition of DT-diaphorase. In summary, an EROD assay was established for use in Cape hake species. PMID:22145075

  20. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  1. Quantum Chemical Studies of Methane Monooxygenase: Comparison with P450

    SciTech Connect

    Guallar, Victor; Gherman, Benjamin F.; Lippard, Stephen J.; Friesner, Richard A.

    2002-04-01

    The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron-containing enzymes, are described and compared. Recent extensive density functional ab initio electronic structure calculations have revealed many similarities in a number of the key catalytic steps, as well as some important differences. A particularly interesting and significant contrast is the role played by the protein in each system. For sMMO, the protein stabilizes various species in the catalytic cycle through a series of carboxylate shifts. This process is adequately described by a relatively compact model of the active site (similar to100 atoms), providing a reasonable description of the energetics of hydrogen atom abstraction. For P450CAM, in contrast, the inclusion of the full protein is necessary for an accurate description of the hydrogen atom abstraction.

  2. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

  3. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

  4. Stereoselectivity in S- and N-oxygenation by the mammalian flavin-containing and cytochrome P-450 monooxygenases.

    PubMed

    Cashman, J R

    1998-11-01

    In general, the use of stereoselectivity studies in examining the contribution of monooxygenases or other catalysts to the N- and S-oxidation of drugs, xenobiotics and endogenous substrates provides a useful method to distinguish enzymatic from nonenzymatic processes. Recent developments in this active area of research have been rapid, presumably due to advances in bioanalytical chemistry, chiral stationary-phase HPLC, and attendant breakthroughs in the instruments to measure centers of chirality. This research area has also been aided by the availability of enzymes and other catalysts. In light of the ever-increasing necessity for new single-isomer drugs, metabolites, and other chiral drug market materials, the demand for stereoselectivity information in the drug development business should continue to expand. In the future, demand for enantiomeric intermediates and metabolites to be studied in their own right for pharmacological activity will undoubtedly increase. Finally, technologies related to the creation or characterization of enantiomerically pure drugs or their metabolites presumably will grow because of the increased number of compounds entering the drug development pipeline due to combinatorial chemistry. PMID:9844806

  5. Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism.

    PubMed

    Bostick, Chris D; Hickey, Katherine M; Wollenberg, Lance A; Flora, Darcy R; Tracy, Timothy S; Gannett, Peter M

    2016-05-01

    Cytochrome P450 (P450) protein-protein interactions have been shown to alter their catalytic activity. Furthermore, these interactions are isoform specific and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show that these effects are also dependent on the protein partner cytochrome P450 reductase (CPR) and the order of protein addition to purified reconstituted enzyme systems. In this study, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homomeric/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real time utilizing surface plasmon resonance. Finally, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4, in rank order.KDvalues obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowestKD, followed by CYP2C9-CYP2C9, CYP2C9-CPR, and CYP2C9-CYP3A4. Metabolic incubations show that immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 at saturating CPR concentrations in solution but was inhibited when CYP2C9 was immobilized. The order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced the magnitude of inhibition for CYP3A4 and CYP2D6. These results indicate isoform-specific P450 interactions and effects on P450-mediated metabolism. PMID:26961240

  6. Spectroelectrochemistry of cytochrome P450cam.

    PubMed

    Bistolas, Nikitas; Christenson, Andreas; Ruzgas, Tautgirdas; Jung, Christiane; Scheller, Frieder W; Wollenberger, Ulla

    2004-02-13

    The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis. PMID:14741708

  7. Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs.

    PubMed

    Higashi, K; Ikeuchi, K; Karasaki, Y; Obara, M

    1983-08-30

    A highly purified cytochrome P-450 was obtained from the microsomes of tulip bulbs (Tulipa gesneriana L.). The molecular weight (Mr = 52,500) and amino acid composition of this plant cytochrome P-450 are similar to those reported for rat livers. On the contrary, Ouchterlony double diffusion analyses indicated that cytochrome P-450 isolated from tulip bulbs shares no common antigenic determinants with those of 9 other plants, in spite of the presence of comparable contents of cytochrome P-450 and/or trans-cinnamate 4-monooxygenase with tulip bulbs. PMID:6412714

  8. Intronic polymorphisms of cytochromes P450

    PubMed Central

    2010-01-01

    The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2) gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41, which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5*3 and CYP3A5*5, common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism. PMID:20846929

  9. Cytochrome P450 and Non-Cytochrome P450 Oxidative Metabolism: Contributions to the Pharmacokinetics, Safety, and Efficacy of Xenobiotics.

    PubMed

    Foti, Robert S; Dalvie, Deepak K

    2016-08-01

    The drug-metabolizing enzymes that contribute to the metabolism or bioactivation of a drug play a crucial role in defining the absorption, distribution, metabolism, and excretion properties of that drug. Although the overall effect of the cytochrome P450 (P450) family of drug-metabolizing enzymes in this capacity cannot be understated, advancements in the field of non-P450-mediated metabolism have garnered increasing attention in recent years. This is perhaps a direct result of our ability to systematically avoid P450 liabilities by introducing chemical moieties that are not susceptible to P450 metabolism but, as a result, may introduce key pharmacophores for other drug-metabolizing enzymes. Furthermore, the effects of both P450 and non-P450 metabolism at a drug's site of therapeutic action have also been subject to increased scrutiny. To this end, this Special Section on Emerging Novel Enzyme Pathways in Drug Metabolism will highlight a number of advancements that have recently been reported. The included articles support the important role of non-P450 enzymes in the clearance pathways of U.S. Food and Drug Administration-approved drugs over the past 10 years. Specific examples will detail recent reports of aldehyde oxidase, flavin-containing monooxygenase, and other non-P450 pathways that contribute to the metabolic, pharmacokinetic, or pharmacodynamic properties of xenobiotic compounds. Collectively, this series of articles provides additional support for the role of non-P450-mediated metabolic pathways that contribute to the absorption, distribution, metabolism, and excretion properties of current xenobiotics. PMID:27298339

  10. Unusual properties of the cytochrome P450 superfamily

    PubMed Central

    Lamb, David C.; Waterman, Michael R.

    2013-01-01

    During the early years of cytochrome P450 research, a picture of conserved properties arose from studies of mammalian forms of these monooxygenases. They included the protohaem prosthetic group, the cysteine residue that coordinates to the haem iron and the reduced CO difference spectrum. Alternatively, the most variable feature of P450s was the enzymatic activities, which led to the conclusion that there are a large number of these enzymes, most of which have yet to be discovered. More recently, studies of these enzymes in other eukaryotes and in prokaryotes have led to the discovery of unexpected P450 properties. Many are variations of the original properties, whereas others are difficult to explain because of their unique nature relative to the rest of the known members of the superfamily. These novel properties expand our appreciation of the broad view of P450 structure and function, and generate curiosity concerning the evolution of P450s. In some cases, structural properties, previously not found in P450s, can lead to enzymatic activities impacting the biological function of organisms containing these enzymes; whereas, in other cases, the biological reason for the variations are not easily understood. Herein, we present particularly interesting examples in detail rather than cataloguing them all. PMID:23297356

  11. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes

    EPA Science Inventory

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was fo...

  12. The Mycobacterium tuberculosis Cytochrome P450 System

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2009-01-01

    Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M. tuberculosis genome revealed the existence of twenty cytochrome P450 enzymes, some of which are potential candidates for drug targeting. The recent burst of studies reporting microarray-based gene essentiality and transcriptome analyses under in vitro, ex vivo and in vivo conditions highlight the importance of selected P450 isoforms for M. tuberculosis viability and pathogenicity. Current knowledge of the structural and biochemical properties of the M. tuberculosis P450 enzymes and their putative redox partners is reviewed, with an emphasis on findings related to their physiological function(s) as well as their potential as drug targets. PMID:19635450

  13. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    SciTech Connect

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs.

  14. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

    PubMed

    Cook, D J; Finnigan, J D; Cook, K; Black, G W; Charnock, S J

    2016-01-01

    Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s. PMID:27567486

  15. From the Cover: Structural Determinants of the Position of 2,3',4,4',5-Pentachlorobiphenyl (CB118) Hydroxylation by Mammalian Cytochrome P450 Monooxygenases.

    PubMed

    Mise, Shintaro; Haga, Yuki; Itoh, Toshimasa; Kato, Akira; Fukuda, Itsuko; Goto, Erika; Yamamoto, Keiko; Yabu, Miku; Matsumura, Chisato; Nakano, Takeshi; Sakaki, Toshiyuki; Inui, Hideyuki

    2016-08-01

    Polychlorinated biphenyls (PCBs) accumulate in mammals via the food chain because of their characteristics such as slow degradation and high hydrophobicity. One of the 209 PCB congeners, 2,3',4,4',5-pentachlorobiphenyl (CB118), is abundantly found in the environment and in mammals. Understanding the metabolic fate of CB118 can provide important information toward evaluating its toxicity. In vitro studies on the metabolism of CB118 by cytochrome P450 enzymes (P450 or CYP) revealed that human CYP2B6 and rat CYP2B1 primarily metabolized it to 3-hydroxy (OH)-CB118, whereas rat CYP1A1 metabolized CB118 to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). Docking models of CYP2Bs with CB118 revealed a short distance between the 3-position of CB118 and the heme iron caused by polarization of the substrate-binding cavity, and maintenance of this pose through interaction with the peripheral amino acids determines the activity and position of hydroxylation. 4-Hydroxylation by rat CYP1A1 occurs owing to the longitudinal shape of the substrate-binding cavity toward the heme of CYP1A1. The metabolites 3-OH-CB118 and 4-OH-CB107 decreased potential for activating the aryl hydrocarbon receptor compared with that of CB118, thereby leading to a decrease in dioxin-like toxicity; however, the neurodevelopmental toxicity of 4-OH-CB107 has been previously reported. The results suggest that these 3 P450 isoforms play an important role in determining the extent of CB118 toxicity. This study will contribute to understanding of the metabolic fates and toxicity of CB118 in vivo. PMID:27208082

  16. Spectroscopic features of cytochrome P450 reaction intermediates

    PubMed Central

    Luthra, Abhinav; Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Preface Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2-3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV-Vis absorption spectra of the reduced CO-saturated state [4-5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle. PMID:21167809

  17. Novel extrahepatic cytochrome P450s

    SciTech Connect

    Karlgren, Maria . E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-09-01

    The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.

  18. Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations.

    PubMed

    Yang, Yi; Li, Zhi

    2015-01-01

    P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations. PMID:26507217

  19. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology.

    PubMed

    Girvan, Hazel M; Munro, Andrew W

    2016-04-01

    Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by random mutagenesis/directed evolution approaches. This highlights the robustness and malleability of the P450 fold, and the capacity of these biocatalysts to oxidise a wide range of chemical scaffolds. This article reviews recent research on the application of wild-type and engineered P450s in the production of important chemicals, including pharmaceuticals and drug metabolites, steroids and antibiotics. In addition, the properties of unusual members of the P450 superfamily that do not follow the canonical P450 catalytic pathway are described. PMID:27015292

  20. Aldehyde Reduction by Cytochrome P450

    PubMed Central

    Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  1. Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling.

    PubMed

    Eichler, Anja; Gricman, Łukasz; Herter, Susanne; Kelly, Paul P; Turner, Nicholas J; Pleiss, Jürgen; Flitsch, Sabine L

    2016-03-01

    Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions. PMID:26698167

  2. Pharmacophore modeling of cytochromes P450.

    PubMed

    de Groot, Marcel J; Ekins, Sean

    2002-03-31

    Understanding the binding of ligands in the active site of a membrane-bound protein is difficult in the absence of a crystal structure. When these proteins are the enzymes involved in drug metabolism, it leaves little option but to use site-directed mutagenesis and in vitro studies to provide critical information relating to determinants of binding affinity. Pharmacophore models and three-dimensional quantitative structure-activity relationships have been used either alone or in combination with protein homology models to provide this information for cytochrome P450s. At present, their application has been directed to the major enzymes but this may escalate in future as more in vitro data are generated for other P450s. The following review outlines the methodologies and models as well as future prospects for applying these technologies to P450s in the hope that future drugs will be selected with increased metabolic stability and fewer incidences of undesirable drug-drug interactions. PMID:11922953

  3. Evaluating cytochrome p450 in lesser scaup (Aythya affinis) and tree swallow (Tachycineta bicolor) by monooxygenase activity and immunohistochemistry: Possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2006-01-01

    Six-month-old lesser scaup (Aythya affinis) and nestling tree swallows (Tachycineta bicolor) were injected intraperitoneally with beta-naphthoflavone (BNF) in corn oil or in vehicle alone. Liver samples were taken and stored at -80 degrees C until microsome preparation and monooxygenase assay. Skin samples were placed in buffered formalin for subsequent immunohistochemical (IHC) analysis for cytochrome P4501A (CYP1A). Lesser scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 6- to 18-fold increases in four monooxygenases (benzyloxyresorufin-O-dealkylase, ethoxyresorufin-O-dealkylase, methoxyresorufin-O-dealkylase, and pentoxyresorufin-O-dealkylase). No IHC response was observed for CYP1A in the skin of vehicle-injected ducks, whereas in the skin from BNF-treated ducks, the positive IHC response was of similar magnitude for both dose levels of BNF. Tree swallows injected with BNF at 100 mg/kg, but not at. 20 mg/kg, showed significant increases (approximately fivefold) in hepatic microsomal O-dealkylase activities. Cytochrome P4501A was undetectable by IHC response in skin from corn oil-treated swallows, but positive IHC responses were observed in the skin of one of five swallows at 20 mg/kg and four of five swallows at 100 mg/kg. Although these data do not allow construction of significant dose-response curves, the IHC responses for CYP1A in skin support the possible use of this nonlethal approach for biomonitoring contaminant exposure of birds. In addition, the CYP1A signal observed at the bases of emerging feathers suggest that these might provide less invasive sampling sites for IHC analysis of CYP1A.

  4. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    SciTech Connect

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W. . Patuxent Wildlife Research Center); Woodin, B.R.; Stegeman, J.J. )

    1993-09-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene or phenobarbital. Compared to controls, 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p[prime]-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP1B) were significantly associated with total PCB burdens.

  5. Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution▿ †

    PubMed Central

    Trefzer, Axel; Jungmann, Volker; Molnár, István; Botejue, Ajit; Buckel, Dagmar; Frey, Gerhard; Hill, D. Steven; Jörg, Mario; Ligon, James M.; Mason, Dylan; Moore, David; Pachlatko, J. Paul; Richardson, Toby H.; Spangenberg, Petra; Wall, Mark A.; Zirkle, Ross; Stege, Justin T.

    2007-01-01

    Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. PMID:17483257

  6. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation

    PubMed Central

    Jin, Shuangxia; Singh, Nameirakpam D.; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-01-01

    Summary In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. PMID:25782349

  7. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation.

    PubMed

    Jin, Shuangxia; Singh, Nameirakpam D; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-04-01

    In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. PMID:25782349

  8. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... P450 oxidoreductase deficiency is a disorder of hormone production. This condition specifically affects steroid hormones, which are ... activity of cytochrome P450 oxidoreductase, which disrupts the production of steroid hormones. Changes in sex hormones such ...

  9. Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

    PubMed

    Yu, J; Chang, P K; Ehrlich, K C; Cary, J W; Montalbano, B; Dyer, J M; Bhatnagar, D; Cleveland, T E

    1998-12-01

    The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee. PMID:9835571

  10. Cytochrome P450 expression in oesophageal cancer.

    PubMed Central

    Murray, G I; Shaw, D; Weaver, R J; McKay, J A; Ewen, S W; Melvin, W T; Burke, M D

    1994-01-01

    The cytochrome P450 superfamily of enzymes play a central part in the metabolism of carcinogens and anti-cancer drugs. The expression, cellular localisation, and distribution of different forms of P450 and the functionally associated enzymes epoxide hydrolase and glutathione S-transferases have been investigated in oesophageal cancer and non-neoplastic oesophageal tissue using immunohistochemistry. Expression of the different enzymes was confined to epithelial cells in both non-neoplastic samples and tumour samples except the CYP3A was also identified in mast cells and glutathione S-transferase pi was present in chronic inflammatory cells. CYP1A was present in a small percentage of non-neoplastic samples but both CYP2C and CYP3A were absent. Epoxide hydrolase was present in half of the non-neoplastic samples and the different classes of glutathione S-transferase were present in a low number of samples. In carcinomas CYP1A, CYP3A, epoxide hydrolase, and glutathione S-transferase pi were expressed in at least 60% of samples. The expression of glutathione S-transferases alpha and mu were significantly less in adenocarcinoma compared with squamous carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8200549

  11. Mechanisms that Regulate Production of Reactive Oxygen Species by Cytochrome P450

    SciTech Connect

    Zangar, Richard C.; Davydov, Dmitri R.; Verma, Seema

    2004-09-15

    Mammalian cytochromes P450 (P450) are a family of heme-thiolate enzymes involved in the oxidative metabolism of a variety of endogenous and exogenous lipophilic compounds. Poor coupling of the P450 catalytic cycle results in continuous production of reactive oxygen species (ROS), which affect signaling pathways and other cellular functions. P450 generation of ROS is tightly controlled by regulation of gene transcription, as well as by modulation of interactions between protein constituents of the monooxygenase that affects its activity, coupling and stability. Malfunction of these mechanisms may result in a burst of ROS production, which can cause lipid peroxidation and oxidative stress. In turn, oxidative stress downregulates P450 levels by a variety of feedback mechanisms. This review provides an overview of recent advances in our understanding of these feedback mechanisms that serve to limit P450 production of ROS. Some of the more likely physiological and cellular effects of P450 generation of ROS are also discussed.

  12. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    NASA Astrophysics Data System (ADS)

    Roccatano, Danilo

    2015-07-01

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

  13. Flower colour and cytochromes P450

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  14. Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784.

    PubMed

    Nodate, Miho; Kubota, Mitsutoshi; Misawa, Norihiko

    2006-07-01

    Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450. PMID:16195793

  15. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  16. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  17. Purification of cytochrome P-450 enzymes.

    PubMed

    Bell-Parikh, L C; Hosea, N A; Martin, M V; Guengerich, F P

    2002-01-01

    Among the liver P-450 xenobiotic-metabolizing enzymes, P450-2E1 is of interest because of its activation of potent carcinogens, and P-450 1A2 is of interest because of its role in oxidation of drugs and carcinogens. This unit describes column chromatography protocols for purification of recombinant forms of these enzymes expressed in a bacterial expression system. PMID:23045082

  18. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  19. Monoclonal antibody-directed radioimmunoassay of specific cytochromes P-450

    SciTech Connect

    Song, B.J.; Fujino, T.; Park, S.S.; Friedman, F.K.; Gelboin, H.V.

    1984-02-10

    A rapid solid phase radioimmunoassay (RIA) for cytochromes P-450 has been developed utilizing specific monoclonal antibodies to major forms of rat liver cytochrome P-450 that are induced by 3-methylcholanthrene (MC-P-450) and phenobarbital (PB-P-450). Monoclonal antibodies (MAbs) that were endogenously labeled with (/sup 35/S)methionine were used to detect MAb-specific cytochromes P-450 in liver microsomes from untreated rats and rats pretreated with 3-methylcholanthrene (MC) or phenobarbital. The competitive binding assays are rapid and can detect cytochrome P-450 in less than 100 ng of microsomal protein. Tthe RIA was used to examine the distribution of MAb-specific cytochromes P-450 in extrahepatic tissues of MC-treated rats; an approximately 30- to 50-fold greater amount of MC-P-450 in liver relative to lung and kidney was observed, which corresponds well with aryl hydrocarbon hydroxylase activity in these tissues. The inducibility of MAb-specific cytochromes P-450 were observed in MC-treated rats, guinea pigs, and C57BL/6 mice, all highly inducible for aryl hydrocarbon hydroxylase; little increase was observed for the relatively noninducible DBA/2 mouse strain.

  20. Cloning and expression of an atrazine inducible cytochrome P450 from Chironomus tentans (Diptera: Chironomidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies performed in our lab have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a ...

  1. Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.

    ERIC Educational Resources Information Center

    Groves, John T.

    1985-01-01

    Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

  2. Recent Structural Insights into Cytochrome P450 Function.

    PubMed

    Guengerich, F Peter; Waterman, Michael R; Egli, Martin

    2016-08-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past 2 years (2014-2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about 'drug-metabolizing' P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction. PMID:27267697

  3. Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy*

    PubMed Central

    Zhang, Meng; Huang, Rui; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-01-01

    Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners. PMID:25795780

  4. Canine cytochrome P450 (CYP) pharmacogenetics

    PubMed Central

    Court, Michael H.

    2013-01-01

    Synopsis The cytochrome P450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences. Canine CYP1A2, which metabolizes phenacetin, caffeine, and theophylline, is the most widely studied polymorphic canine CYP. A single nucleotide polymorphism resulting in a CYP1A2 premature stop codon (c.1117C>T; R383X) with a complete lack of enzyme is highly prevalent in certain dog breeds including Beagle and Irish wolfhound. This polymorphism was shown to substantially affect the pharmacokinetics of several experimental compounds in Beagles during preclinical drug development. However, the impact on the pharmacokinetics of phenacetin (a substrate specific for human CYP1A2) was quite modest probably because other canine CYPs are capable of metabolizing phenacetin. Other canine CYPs with known genetic polymorphisms include CYP2C41 (gene deletion), as well as CYP2D15, CYP2E1, and CYP3A12 (coding SNPs). However the impact of these variants on drug metabolism in vitro or on drug pharmacokinetics is unknown. Future systematic investigations are needed to comprehensively identify CYP genetic polymorphisms that are predictive of drug effects in canine patients. PMID:23890236

  5. Rearrangement Reactions Catalyzed by Cytochrome P450s

    PubMed Central

    Ortiz de Montellano, Paul R.; Nelson, Sidney D.

    2010-01-01

    Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants. PMID:20971058

  6. Cytochrome P-450 epitope typing in animals and humans with monoclonal antibodies to ethanol induced rat liver microsomal cytochrome P-450 (P-450et)

    SciTech Connect

    Park, S.S.; Ko, I.Y.; Yang, C.; Guengerich, F.G.; Schenkman, J.B.; Coon, M.J.; Gelboin, H.V.

    1986-05-01

    Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice that had been immunized with P-450et. The monoclonal antibody (MAb)-producing hybridomas were screened by RIA. Thirty one independent hybrid clones were isolated with each producing an MAb of a single immunoglobulin subclass. All of these MAbs had high affinities for P-450et but only one MAb had a strong inhibitory effect on aniline rho-hydroxylase and N-nitrosodimethylamine demethylase. Western blots and RIAs based on ten MAbs (C1-C10) were used to determine the epitope homology of purified cytochromes P-450 from rats, rabbits, and humans. All ten MAbs had high affinity for both P-450et and a rat P-450 which is induced by acetone (P-450ac). Classes of these MAbs were identified which crossreacted toward different forms of rat P-450. In addition, several MAbs (C3, C6, C9) recognized a P-450 form of human liver, while other MAbs (C7, C9) recognized P-450/sub LM2/ of rabbits. Three MAbs (C4, C5, C8) were specific for only P-450et and P-450ac. These results demonstrate the different degrees of epitope relatedness among the multiple forms of cytochrome P-450.

  7. Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

    SciTech Connect

    Estabrook, Ronald W. . E-mail: Ronald.estabrook@utsouthwestern.edu

    2005-12-09

    The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11{beta}-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O{sup 18} studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17{alpha}-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17{alpha}-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11{beta}-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction.

  8. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    SciTech Connect

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K.

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  9. Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

    PubMed Central

    O'Keefe, Daniel P.; Leto, Kenneth J.

    1989-01-01

    The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450. Images Figure 3 PMID:16666677

  10. Cytochrome P450 Initiates Degradation of cis-Dichloroethene by Polaromonas sp. Strain JS666

    PubMed Central

    Nishino, Shirley F.; Shin, Kwanghee A.; Gossett, James M.

    2013-01-01

    Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes. PMID:23354711

  11. Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

    PubMed Central

    Benveniste, I; Lesot, A; Hasenfratz, M P; Durst, F

    1989-01-01

    Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals. Images Fig. 5. PMID:2499315

  12. Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids

    PubMed Central

    Hallahan, David L.; Nugent, Jonathan H. A.; Hallahan, Beverly J.; Dawson, Glenn W.; Smiley, Diane W.; West, Jevon M.; Wallsgrove, Roger M.

    1992-01-01

    The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity. PMID:16668790

  13. TERATOGEN METABOLISM: THALIDOMIDE ACTIVATION IS MEDIATED BY CYTOCHROME P-450

    EPA Science Inventory

    A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a...

  14. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2015-12-09

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  15. Hot-spot residues in the cytochrome P450cam-putidaredoxin binding interface.

    PubMed

    Hiruma, Yoshitaka; Gupta, Ankur; Kloosterman, Alexander; Olijve, Caroline; Olmez, Betül; Hass, Mathias A S; Ubbink, Marcellus

    2014-01-01

    Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers. PMID:24302683

  16. Cytochrome P450-based cancer gene therapy: current status.

    PubMed

    Kan, On; Kingsman, Susan; Naylor, Stuart

    2002-12-01

    Results from a number of preclinical studies have demonstrated that a P450-based gene-directed enzyme prodrug therapy (GDEPT) strategy for the treatment of cancer is both safe and efficacious. This strategy has now moved forward into the clinic. At least two different approaches using different delivery methods (retroviral vector MetXia [Oxford BioMedica] and encapsulated P450 expressing cells), different cytochrome P450 isoforms (human CYP2B6 versus rat CYP2B1) and different prodrugs (cyclophosphamide [CPA] versus ifosfamide [IFA]) have concluded Phase I/II clinical trial with encouraging results. In the future, P450-based GDEPT can potentially be further enhanced by improved vectors for P450 gene delivery and disease-targeted promoters for focused gene expression at the target site. In addition, there is scope for developing synthetic P450s and their respective prodrugs to improve both enzyme kinetics and the profile of the active moiety. PMID:12517265

  17. The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

    PubMed Central

    Nelson, David R.; Goldstone, Jared V.; Stegeman, John J.

    2013-01-01

    The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution. PMID:23297357

  18. Purification of a soluble cytochrome P450 from Trichosporon montevideense.

    PubMed

    Stündl, U M; Patzak, D; Schauer, F

    2000-01-01

    The yeast Trichosporon montevideense CBS 6721 expressed large amounts of cytochrome P450 after cultivation in a glucose-peptone medium. The P450, which could be detected in the cytosolic fraction after cell breakage and ultracentrifugation, was purified to electrophoretic homogeneity and migrated in SDS-PAGE with a M(r) of 43,000. As indicated by IEF, the preparation consisted of two different P450 isoforms with pI-values of 5.9 and 6.2, which were named P450MS1 and P450MS2 respectively. Both isoforms had a characteristic maximum at 446 nm in the reduced carbon monoxide difference spectra. Partial N-terminal sequencing of P450MS1 and P450MS2 demonstrated a high degree of sequence homology between the soluble P450 enzymes of T. montevideense CBS 6721 and their close relationship to the soluble P450 forms of Trichosporon spec. SBUG 752, T. cutaneum ATCC 58094 and to the P450s of the CYP55 family of Fusarium oxysporum and Cylindrocarpon tonkinense. PMID:10986675

  19. Lateral diffusion of cytochrome P-450 in phospholipid bilayers.

    PubMed

    Wu, E S; Yang, C S

    1984-01-01

    The lateral diffusion coefficient (D) of cytochrome P-450 (P-450) has been measured in lipid multibilayers with the method of fluorescence recovery after photobleaching. In the liquid-crystal phase of egg phosphatidylcholine (EPC) and dimyristoylphosphatidylcholine (DMPC), the diffusion of P-450 is fast with D about 2 X 10(-8) cm2/s. In DMPC multibilayers, P-450 diffusion dropped by a factor of 20 near the liquid crystal to gel phase transition region, and D is about 5 X 10(-10) cm2/s in the gel phase. A value of 50 mol % of cholesterol reduced the diffusion of P-450 in the liquid-crystal phase only slightly but enhanced the diffusion of P-450 in the gel phase significantly. In EPC membranes, P-450 diffusion underwent a stepwise drop as the cholesterol contents increased from 20 to 30 mol %. With the assumption of a lateral diffusion mediated electron transfer between P-450 and NADPH-P-450 reductase and with D = 2.5 X 10(-8) cm2/s for both enzymes, the reduction rate for P-450 in liposomes was calculated and compared with the reported experimental value. PMID:6691964

  20. A novel cytochrome P450 CYP6AB14 gene in Spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th...

  1. Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench 1

    PubMed Central

    Halkier, Barbara Ann; Møller, Birger Lindberg

    1991-01-01

    The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. Images

  2. Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding ethB Gene for Elucidation of Methyl tert-Butyl Ether Biodegradation in Aerated Treatment Pond Systems▿ †

    PubMed Central

    Jechalke, Sven; Rosell, Mònica; Martínez-Lavanchy, Paula M.; Pérez-Leiva, Paola; Rohwerder, Thore; Vogt, Carsten; Richnow, Hans H.

    2011-01-01

    Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ɛC] of −0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ɛH]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ɛC of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ɛH of −5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem. PMID:21148686

  3. Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice.

    PubMed

    Wang, Cuiting; Yang, Yang; Wang, Haihai; Ran, Xiaojuan; Li, Bei; Zhang, Jiantao; Zhang, Hongxia

    2016-09-01

    In Arabidopsis thaliana and Oryza sativa, the cytochrome P450 (CYP) 714 protein family represents a unique group of CYP monooxygenase, which functions as a shoot-specific regulator in plant development through gibberellin deactivation. Here, we report the functional characterizations of PtCYP714A3, an OsCYP714D1/Eui homologue from Populus trichocarpa. PtCYP714A3 was ubiquitously expressed with the highest transcript level in cambium-phloem tissues, and was greatly induced by salt and osmotic stress in poplar. Subcellular localization analyses indicated that PtCYP714A3-YFP fusion protein was targeted to endoplasmic reticulum (ER). Expression of PtCYP714A3 in the rice eui mutant could rescue its excessive-shoot-growth phenotype. Ectopic expression of PtCYP714A3 in rice led to semi-dwarfed phenotype with promoted tillering and reduced seed size. Transgenic lines which showed significant expression of PtCYP714A3 also accumulated lower GA level than did the wild-type (WT) plants. The expression of some GA biosynthesis genes was significantly suppressed in these transgenic plants. Furthermore, transgenic rice plants exhibited enhanced tolerance to salt and maintained more Na(+) in both shoot and root tissues under salinity stress. All these results not only suggest a crucial role of PtCYP714A3 in shoot responses to salt toxicity in rice, but also provide a molecular basis for genetic engineering of salt-tolerant crops. PMID:26970512

  4. Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system.

    PubMed

    Sevrioukova, Irina F; Poulos, Thomas L

    2011-03-01

    The P450cam monooxygenase system consists of three separate proteins: the FAD-containing, NADH-dependent oxidoreductase (putidaredoxin reductase or Pdr), cytochrome P450cam and the 2Fe2S ferredoxin (putidaredoxin or Pdx), which transfers electrons from Pdr to P450cam. Over the past few years our lab has focused on the interaction between these redox components. It has been known for some time that Pdx can serve as an effector in addition to its electron shuttle role. The binding of Pdx to P450cam is thought to induce structural changes in the P450cam active site that couple electron transfer to substrate hydroxylation. The nature of these structural changes has remained unclear until a particular mutant of P450cam (Leu358Pro) was found to exhibit spectral perturbations similar to those observed in wild type P450cam bound to Pdx. The crystal structure of the L358P variant has provided some important insights on what might be happening when Pdx docks. In addition to these studies, many Pdx mutants have been analyzed to identify regions important for electron transfer. Somewhat surprisingly, we found that Pdx residues predicted to be at the P450cam-Pdx interface play different roles in the reduction of ferric P450cam and the ferrous P450-O(2) complex. More recently we have succeeded in obtaining the structure of a chemically cross-linked Pdr-Pdx complex. This fusion protein represents a valid model for the noncovalent Pdr-Pdx complex as it retains the redox activities of native Pdr and Pdx and supports monooxygenase reactions catalyzed by P450cam. The insights gained from these studies will be summarized in this review. PMID:20816746

  5. Oxidation of nonionic detergents by cytochrome P450 enzymes.

    PubMed

    Hosea, N A; Guengerich, F P

    1998-05-15

    Nonionic phenolic detergents are commonly used in the purification of membrane-associated proteins. Triton N-101 was shown to be oxidized by NADPH-fortified human liver microsomes and recombinant human cytochromes P450 (P450). Oxidation was monitored using HPLC and the fluorescence properties of Triton N-101 and other alkylphenol ethoxylate detergents, which are similar to those of anisole. Human liver microsomes and recombinantly expressed reconstituted P450 3A4-oxidized Triton N-101 in a concentration-dependent manner which could be inhibited by ketoconazole, a P450 3A4-selective inhibitor. Triton N-101 inhibition of testosterone oxidation by human liver microsomes was of a mixed nature but mainly non-competitive. Electrospray ionization mass spectrometry and tandem mass spectrometry indicated that the major product formed was hydroxylated on the alkyl moiety. Human liver microsomes also oxidized other Tritons (X-100 and X-114), Emulgens 911 and 913, and Tergitol NP-10 to a similar extent. P450s 1A1, 1A2, and 2C9 also oxidized Triton N-101 but to a lesser extent than P450 3A4. We conclude that Triton N-101 and similar nonionic detergents are oxidized by P450 3A4 and some other P450s. PMID:9606971

  6. Enhanced expression of cytochrome P450 in stomach cancer.

    PubMed Central

    Murray, G. I.; Taylor, M. C.; Burke, M. D.; Melvin, W. T.

    1998-01-01

    The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9569036

  7. [Cytochrome P-450-dependent reactions during intensified biosynthesis of coenzyme A in hepatocytes].

    PubMed

    Sushko, L I; Sheĭbak, V M; Abakumov, G Z; Moĭseenok, A K

    1986-01-01

    After subcutaneous administration into male rats of 4-phosphopantothenic acid and pantethine during 10 days at a dose equivalent to 30 mg/kg of calcium pantothenate total content of CoA was increased in liver tissue. Both these preparations activated the liver endoplasmic reticulum monooxygenase system mainly at the step of substrate hydroxylation. The phenomenon observed appears to occur due to activation of cytochrome P-450 biosynthesis and/or to alterations in phospholipid composition of microsomal membranes. PMID:3765494

  8. Comparison of basal and induced cytochromes P450 in 6 species of waterfowl

    USGS Publications Warehouse

    Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

    1999-01-01

    Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

  9. Role of Cytochrome P450s in Inflammation.

    PubMed

    Christmas, Peter

    2015-01-01

    Cytochrome P450 epoxygenases and hydroxylases play a regulatory role in the activation and suppression of inflammation by generating or metabolizing bioactive mediators. CYP2C and CYP2J epoxygenases convert arachidonic acid to anti-inflammatory epoxyeicosatrienoic acids, which have protective effects in a variety of disorders including cardiovascular disease and metabolic syndrome. CYP4A and CYP4F hydroxylases have the ability to metabolize multiple substrates related to the regulation of inflammation and lipid homeostasis, and it is a challenge to determine which substrates are physiologically relevant for each enzyme; the best-characterized activities include generation of 20-hydroxyeicosatetraenoic acid and inactivation of leukotriene B4. The expression of hepatic drug-metabolizing cytochrome P450s is modulated by cytokines during inflammation, resulting in changes to the pharmacokinetics of prescribed medications. Cytochrome P450s are therefore the focus of intersecting challenges in the pharmacology of inflammation: not only do they represent targets for development of new anti-inflammatory drugs but they also contribute to variability in drug efficacy or toxicity in inflammatory disease. Animal models and primary hepatocytes have been used extensively to study the effects of cytokines on cytochrome P450 expression and activity. However, it is difficult to predict changes in drug exposure in patients because the response to inflammation varies depending on the disease state, its time course, and the cytochrome P450 involved. In these circumstances, the development of endogenous markers of cytochrome P450 metabolism might provide a useful tool to reevaluate drug dosage and choice of therapy. PMID:26233907

  10. Pulmonary oxygen toxicity in rats treated with cytochrome P-450 inducers

    SciTech Connect

    Ebel, R.E.; Barlow, R.L.; Gregory, E.M.

    1987-05-01

    Pulmonary oxygen toxicity is assumed to result from damage caused by superoxide (O/sub 2//sup -/) hydrogen peroxide (H/sub 2/O/sub 2/) and/or hydroxyl radical (OH) produced by the partial reduction of molecular oxygen (O/sub 2/). The microsomal cytochrome P-450 (P-450) monooxygenase system is known to produce O/sub 2//sup -/ and H/sub 2/O/sub 2/. They have studied the influence of monooxygenase induction using phenobarbital (PB) and ..beta..-naphthoflavone (..beta..-NF) on O/sub 2/ toxicity in the rat. PB- or ..beta..-NF induce hepatic P-450 but only ..beta..-NF induces pulmonary P-450. Pulmonary microsomes produced O/sub 2//sup -/ and H/sub 2/O/sub 2/ at rates (expressed per mg microsomal protein) which did not vary as a function of pretreatment. Rats were exposed to 100% O/sub 2/ for up to 3 days. After 3 days of O/sub 2/, lung weights were about 50% above controls regardless of pretreatment. The microsomal monooxygenase enzymes (P-450, b/sub 5/ and NADPH P-450 reductase) were quantified in liver and lung. Lung microsomal P-450 was reduced after 3 days of O/sub 2/ exposure regardless of pretreatment. The protective enzymes (catalase, superoxide dismutase (SOD) and glutathione (GSH) peroxidase) and non-protein sulfhydryl groups (NPSH) were also quantified in lung and liver samples. Lung NPSH and GSH peroxidase were increased after 3 days of O/sub 2/ exposure regardless of pretreatment while SOD was increased in controls and PB- but not ..beta..-NF-treated rats. Three of 14 ..beta..-NF-treated rats died during O/sub 2/ exposure while no animals in the control or PB-treated groups died.

  11. CROSS-SPECIES COMPARISON OF CONAZOLE FUNGICIDE METABOLITES USING RAT AND RAINBOW TROUT (ONCHORHYNCHUS MYKISS) HEPATIC MICROSOMES AND PURIFIED HUMAN CYTOCHROME P450 3A4

    EPA Science Inventory

    Conazoles represent a unique class of azole-containing fungicides that are widely used in both pharmaceutical and agriculture applications. The antifungal property of conazoles occurs via complexation with cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell...

  12. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  13. Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor).

    PubMed

    McQuarters, Ashley B; Wirgau, Nathaniel E; Lehnert, Nicolai

    2014-04-01

    Denitrifying bacteria and fungi efficiently detoxify the toxic metabolite nitric oxide (NO) through reduction to nitrous oxide (N2O) using nitric oxide reductase (NOR) enzymes. In fungi, for example Fusarium oxysporum, NO is reduced by a Cytochrome P450 NOR (P450nor). This enzyme contains a heme b center coordinated to a proximal cysteinate ligand in the active site. In the proposed mechanism of P450nor, the ferric heme binds NO first to form a ferric heme-nitrosyl complex, which is subsequently reduced by NAD(P)H to generate a ferrous HNO species as the next key intermediate. Recently, key progress has been made in our understanding of the electronic structures and fundamental reactivity of these important intermediates, using suitable model complexes. In this review, model complexes of ferric heme-nitrosyls with varied axial anionic ligands (such as N-donors, O-donors, and S-donors) are discussed first. Then, the generation and reactivity of ferrous heme-HNO complexes is summarized and related back to the mechanism of P450nor. PMID:24658055

  14. Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

    PubMed Central

    Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

  15. Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

    PubMed Central

    Huang, Wei-Cheng; Ellis, Jacqueline; Moody, Peter C.E.; Raven, Emma L.; Roberts, Gordon C.K.

    2013-01-01

    Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. PMID:23911089

  16. Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

    PubMed Central

    Poupin, P.; Truffaut, N.; Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.

    1998-01-01

    A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring. PMID:9435074

  17. Degradation of morpholine by an environmental Mycobacterium strain involves a cytochrome P-450.

    PubMed

    Poupin, P; Truffaut, N; Combourieu, B; Besse, P; Sancelme, M; Veschambre, H; Delort, A M

    1998-01-01

    A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C-N bond of the morpholine ring. PMID:9435074

  18. Interindividual Variability in Cytochrome P450-Mediated Drug Metabolism.

    PubMed

    Tracy, Timothy S; Chaudhry, Amarjit S; Prasad, Bhagwat; Thummel, Kenneth E; Schuetz, Erin G; Zhong, Xiao-Bo; Tien, Yun-Chen; Jeong, Hyunyoung; Pan, Xian; Shireman, Laura M; Tay-Sontheimer, Jessica; Lin, Yvonne S

    2016-03-01

    The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals. PMID:26681736

  19. Cytochrome P450 Inhibitors Reduce Creeping Bentgrass (Agrostis stolonifera) Tolerance to Topramezone

    PubMed Central

    Elmore, Matthew T.; Brosnan, James T.; Armel, Gregory R.; Kopsell, Dean A.; Best, Michael D.; Mueller, Thomas C.; Sorochan, John C.

    2015-01-01

    Creeping bentgrass (Agrostis stolonifera L.) is moderately tolerant to the p-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide topramezone. However, the contribution of plant metabolism of topramezone to this tolerance is unknown. Experiments were conducted to determine if known cytochrome P450 monooxygenase inhibitors 1-aminobenzotriazole (ABT) and malathion alone or in combination with the herbicide safener cloquintocet-mexyl influence creeping bentgrass tolerance to topramezone. Creeping bentgrass in hydroponic culture was treated with ABT (70 μM), malathion (70 μm and 1000 g ha-1), or cloquintocet-mexyl (70 μM and 1000 g ha-1) prior to topramezone (8 g ha-1) application. Topramezone-induced injury to creeping bentgrass increased from 22% when applied alone to 79 and 41% when applied with malathion or ABT, respectively. Cloquintocet-mexyl (70 μM and 1000 g ha-1) reduced topramezone injury to 1% and increased creeping bentgrass biomass and PSII quantum yield. Cloquintocet-mexyl mitigated the synergistic effects of ABT more than those of malathion. The effects of malathion on topramezone injury were supported by creeping bentgrass biomass responses. Responses to ABT and malathion suggest that creeping bentgrass tolerance to topramezone is influenced by cytochrome P450-catalyzed metabolism. Future research should elucidate primary topramezone metabolites and determine the contribution of cytochrome P450 monooxygenases and glutathione S-transferases to metabolite formation in safened and non-safened creeping bentgrass. PMID:26186714

  20. Cytochrome P450 Inhibitors Reduce Creeping Bentgrass (Agrostis stolonifera) Tolerance to Topramezone.

    PubMed

    Elmore, Matthew T; Brosnan, James T; Armel, Gregory R; Kopsell, Dean A; Best, Michael D; Mueller, Thomas C; Sorochan, John C

    2015-01-01

    Creeping bentgrass (Agrostis stolonifera L.) is moderately tolerant to the p-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide topramezone. However, the contribution of plant metabolism of topramezone to this tolerance is unknown. Experiments were conducted to determine if known cytochrome P450 monooxygenase inhibitors 1-aminobenzotriazole (ABT) and malathion alone or in combination with the herbicide safener cloquintocet-mexyl influence creeping bentgrass tolerance to topramezone. Creeping bentgrass in hydroponic culture was treated with ABT (70 μM), malathion (70 μm and 1000 g ha(-1)), or cloquintocet-mexyl (70 μM and 1000 g ha(-1)) prior to topramezone (8 g ha(-1)) application. Topramezone-induced injury to creeping bentgrass increased from 22% when applied alone to 79 and 41% when applied with malathion or ABT, respectively. Cloquintocet-mexyl (70 μM and 1000 g ha(-1)) reduced topramezone injury to 1% and increased creeping bentgrass biomass and PSII quantum yield. Cloquintocet-mexyl mitigated the synergistic effects of ABT more than those of malathion. The effects of malathion on topramezone injury were supported by creeping bentgrass biomass responses. Responses to ABT and malathion suggest that creeping bentgrass tolerance to topramezone is influenced by cytochrome P450-catalyzed metabolism. Future research should elucidate primary topramezone metabolites and determine the contribution of cytochrome P450 monooxygenases and glutathione S-transferases to metabolite formation in safened and non-safened creeping bentgrass. PMID:26186714

  1. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    PubMed Central

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B.

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

  2. Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings

    USGS Publications Warehouse

    Rattner, B.; Melancon, M.; Custer, T.; Hothem, R.

    1995-01-01

    Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of arylhydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) weremodestly elevated (monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBs, arylhydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

  3. Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings

    SciTech Connect

    Rattner, B.; Melancon, M.; Custer, T.; Hothem, R. ||

    1995-12-31

    Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of aryl hydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) were modestly elevated ({<=} three-fold) in nestlings from polluted sites. Concentrations of p,p{prime}DDE, other organochlorine pesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos, At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBS, aryl hydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

  4. Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. PMID:22819650

  5. Regioselective hydroxylation of steroid hormones by human cytochromes P450.

    PubMed

    Niwa, Toshiro; Murayama, Norie; Imagawa, Yurie; Yamazaki, Hiroshi

    2015-05-01

    This article reviews in vitro metabolic activities [including Michaelis constants (Km), maximal velocities (Vmax) and Vmax/Km] and drug-steroid interactions [such as induction and cooperativity (activation)] of cytochromes P450 (P450 or CYP) in human tissues, including liver and adrenal gland, for 14 kinds of endogenous steroid compounds, including allopregnanolone, cholesterol, cortisol, cortisone, dehydroepiandrosterone, estradiol, estrone, pregnenolone, progesterone, testosterone and bile acids (cholic acid). First, we considered the drug-metabolizing P450s. 6β-Hydroxylation of many steroids, including cortisol, cortisone, progesterone and testosterone, was catalyzed primarily by CYP3A4. CYP1A2 and CYP3A4, respectively, are likely the major hepatic enzymes responsible for 2-/4-hydroxylation and 16α-hydroxylation of estradiol and estrone, steroids that can contribute to breast cancer risk. In contrast, CYP1A1 and CYP1B1 predominantly metabolized estrone and estradiol to 2- and 4-catechol estrogens, which are endogenous ultimate carcinogens if formed in the breast. Some metabolic activities of CYP3A4, including dehydroepiandrosterone 7β-/16α-hydroxylation, estrone 2-hydroxylation and testosterone 6β-hydroxylation, were higher than those for polymorphically expressed CYP3A5. Next, we considered typical steroidogenic P450s. CYP17A1, CYP19A1 and CYP27A1 catalyzed steroid synthesis, including hydroxylation at 17α, 19 and 27 positions, respectively. However, it was difficult to predict which hepatic drug-metabolizing P450 or steroidogenic P450 will be mainly responsible for metabolizing each steroid hormone in vivo based on these results. Further research is required on the metabolism of steroid hormones by various P450s and on prediction of their relative contributions to in vivo metabolism. The findings collected here provide fundamental and useful information on the metabolism of steroid compounds. PMID:25678418

  6. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  7. ISOLATION OF THE ALKANE INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a gtll library. solation of the gene has been identified on the basis of its inducibility and partial DNA sequence. ranscripts of this gene were indu...

  8. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

    PubMed Central

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing

    2016-01-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  9. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver.

    PubMed

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing; Qiao, Hai-Ling

    2016-08-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  10. Demethylation of Veratrole by Cytochrome P-450 in Streptomyces setonii

    PubMed Central

    Sutherland, John B.

    1986-01-01

    The actinomycete Streptomyces setonii 75Vi2 demethylates vanillic acid and guaiacol to protocatechuic acid and catechol, respectively, and then metabolizes the products by the β-ketoadipate pathway. UV spectroscopy showed that this strain could also metabolize veratrole (1,2-dimethoxybenzene). When grown in veratrole-containing media supplemented with 2,2′-dipyridyl to inhibit cleavage of the aromatic ring, S. setonii accumulated catechol, which was detected by both liquid chromatography and gas chromatography. Reduced cell extracts from veratrole-grown cultures, but not sodium succinate-grown cultures, produced a carbon monoxide difference spectrum with a peak at 450 nm that indicated the presence of soluble cytochrome P-450. Addition of veratrole or guaiacol to oxidized cell extracts from veratrole-grown cultures produced difference spectra that indicated that these compounds were substrates for cytochrome P-450. My results suggest that S. setonii produces a cytochrome P-450 that is involved in the demethylation of veratrole and guaiacol to catechol, which is then catabolized by the β-ketoadipate pathway. PMID:16347120

  11. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  12. Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

    PubMed Central

    Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

    1997-01-01

    One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

  13. Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

    PubMed

    Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

    1997-08-01

    One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

  14. Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.

    PubMed

    Zernia, Sarah; Ott, Florian; Bellmann-Sickert, Kathrin; Frank, Ronny; Klenner, Marcus; Jahnke, Heinz-Georg; Prager, Andrea; Abel, Bernd; Robitzki, Andrea; Beck-Sickinger, Annette G

    2016-04-20

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants. PMID:26967204

  15. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval)

    PubMed Central

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it’s much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

  16. Highly reactive electrophilic oxidants in cytochrome P450 catalysis

    SciTech Connect

    Newcomb, Martin . E-mail: men@uic.edu; Chandrasena, R. Esala P.

    2005-12-09

    The cytochrome P450 enzymes effect a wide range of oxidations in nature including difficult hydroxylation reactions of unactivated C-H. Most of the high energy reactions of these catalysts appear to involve highly electrophilic active species. Attempts to detect the reactive transients in the enzymes have met with limited success, but evidence has accumulated that two distinct electrophilic oxidants are produced in the P450 enzymes. The consensus electrophilic oxidant termed 'iron-oxo' is usually thought to be an analogue of Compound I, an iron(IV)-oxo porphyrin radical cation species, but it is possible that a higher energy electronic isomer of Compound I is required to account for the facility of the C-H oxidation reactions. The second electrophilic oxidant of P450 is speculative; circumstantial evidence suggests that this species is iron-complexed hydrogen peroxide, but this oxidant might be a second spin state of iron-oxo. This overview discusses recent studies directed at detection of the electrophilic oxidants in P450 enzymes and the accumulated evidence for two distinct species.

  17. Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5.

    PubMed

    Shaw, P M; Hosea, N A; Thompson, D V; Lenius, J M; Guengerich, F P

    1997-12-01

    The development of enzyme and buffer premixes for in vitro biotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochrome b5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at -80 degrees C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different Vmax and K(m) values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that Vmax and K(m) could be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays. PMID:9390180

  18. Epoxidation Activities of Human Cytochromes P450c17 and P450c21

    PubMed Central

    2015-01-01

    Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon–carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes. PMID:25386927

  19. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

  20. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  1. Cytochrome P450 responses and PCB congeners in pipping heron embryos from Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Tillett, D.E.; Woodin, Bruce R.; Stegeman, John J.

    1992-01-01

    Pipping black-crowned night-heron (Nvcticorax nvcticorax) embryos were collected from undisturbed (Chincoteague National Wildlife Refuge VA; CNWR) and industrialized (Cat Island, Green Bay WI and San Francisco Bay, CA; SFB) locations. Hepatic monooxygenases (AHH, EROD, BROD, ECOD) were induced up to 100-fold, and were correlated (r=0.50 to 0.72) with total PCB burdens (N =61 embryos). A subset of 30 embryos have now been analyzed by GC/MS for 12 AHH-active PCB congeners and by Western blot for cytochromes P450lA and P450llB. At Cat Island, concentrations of 8 congeners were greater (P <0.05) than at CNWR. P450lA and P450llB were detected in 44% and 100% of the Cat Island embryos compared to 8% and 33% of the CNWR + SFB embryos. Cytochrome P450 parameters were correlated with the total PCBs (r =0.44 to 0.67) and with at least 9 PCB congeners (r =0.39 to 0.77). Since P450 responses might be affected by other contaminants, sample extract potency in the H411E rat hepatoma bioassay is being determined to study relationships among dioxin equivalents and cytochrome P450 parameters.

  2. FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

    PubMed

    Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

    2007-03-01

    Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex. PMID:17014964

  3. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  4. Evaluation of hydroxyimine as cytochrome P450-selective prodrug structure.

    PubMed

    Kumpulainen, Hanna; Mähönen, Niina; Laitinen, Marja-Leena; Jaurakkajärvi, Marja; Raunio, Hannu; Juvonen, Risto O; Vepsäläinen, Jouko; Järvinen, Tomi; Rautio, Jarkko

    2006-02-01

    Hydroxyimine derivatives of ketoprofen (1) and nabumetone (2) were synthesized and evaluated in vitro and in vivo as cytochrome P450-selective intermediate prodrug structures of ketones. 2 released nabumetone in vitro in the presence of isolated rat and human liver microsomes and in different recombinant human CYP isoforms. Bioconversion of 2 to both nabumetone and its active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), was further confirmed in rats in vivo. Results indicate that hydroxyimine is a useful intermediate prodrug structure for ketone drugs. PMID:16451086

  5. Regulation of cytochrome P450 expression in Drosophila: Genomic insights.

    PubMed

    Giraudo, Maeva; Unnithan, G Chandran; Le Goff, Gaëlle; Feyereisen, René

    2010-06-01

    Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the "phenobarbital-type" induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from "detox" microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action. PMID:20582327

  6. Regulation of cytochrome P450 expression in Drosophila: Genomic insights

    PubMed Central

    Giraudo, Maeva; Unnithan, G. Chandran; Le Goff, Gaëlle; Feyereisen, René

    2009-01-01

    Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action. PMID:20582327

  7. Human cytochromes P450 in health and disease

    PubMed Central

    Nebert, Daniel W.; Wikvall, Kjell; Miller, Walter L.

    2013-01-01

    There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

  8. Differential induction of cytochrome P450-mediated triasulfuron metabolism by naphthalic anhydride and triasulfuron.

    PubMed Central

    Persans, M W; Schuler, M A

    1995-01-01

    Cytochrome P450 monooxygenases play paramount roles in the detoxification of herbicides as well as in the synthesis of lignins, flavonoids, and phenolic acids. Biochemical analysis of triasulfuron metabolism in maize (Zea mays) seedlings has demonstrated that the P450(s) responsible for detoxification of this herbicide is induced by naphthalic anhydride (NA), a plant safener, and by triasulfuron, the herbicide itself. Induction studies conducted with seedlings of different ages suggest that two separate response pathways modulate this P-450 activity. Induction by NA is independent of the developmental age of the seedlings up to 6.5 d; induction by triasulfuron is tightly modulated with respect to developmental age in that triasulfuron metabolism can be induced by triasulfuron in young (2.5 d) but not older (6.5 d) seedlings. Induction by NA administered in combination with triasulfuron synergistically enhances triasulfuron metabolism in younger seedlings to levels substantially above that obtained with either herbicide or safener treatment alone. In older seedlings, NA plus triasulfuron treatment induces triasulfuron metabolism to only the level of NA treatment alone, indicating again that the induction cascade responding to triasulfuron is nonfunctional in later development. MnCl2 studies indicate that the triasulfuron insensitivity of older seedlings does not result from a general limitation in the inducibility of this P-450 detoxification system but rather from specific limitations in the triasulfuron-response pathway. PMID:8539299

  9. Water oxidation by a cytochrome p450: mechanism and function of the reaction.

    PubMed

    Prasad, Brinda; Mah, Derrick J; Lewis, Andrew R; Plettner, Erika

    2013-01-01

    P450(cam) (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450(cam) catalysis is controlled by oxygen levels: at high O2 concentration, P450(cam) catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using (17)O and (2)H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450(cam) , and we present a plausible mechanism that accounts for the 1:1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450(cam) and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce. PMID:23634216

  10. Molecular genetic analysis of the cytochrome P450-debrisoquine hydroxylase locus and association with cancer susceptibility.

    PubMed Central

    Smith, C A; Moss, J E; Gough, A C; Spurr, N K; Wolf, C R

    1992-01-01

    The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including beta-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population. PMID:1486838

  11. A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

    PubMed

    Winter, J; Eckerskorn, C; Waditschatka, R; Kayser, H

    2001-11-01

    A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450. PMID:11767943

  12. Cytochrome P450-like substrate oxidation catalyzed by cytochrome c and immobilized cytochrome c.

    PubMed

    Akasaka, R; Mashino, T; Hirobe, M

    1993-03-01

    Cytochrome c (cyt.c) was shown to catalyze cytochrome P450 (P450)-like oxidative reactions, such as N-, and O-demethylation, S-oxidation, and epoxidation of olefins. A more detailed examination showed that (i) N-methylcarbazole and thioanisole oxidation with H2(18)O2 catalyzed by cyt.c resulted in introduction of 18O into the product, and (ii) during the epoxidation of cis-stilbene catalyzed by cyt.c, the stereochemistry of the substrate was retained and 18O was introduced when H2(18)O2 was used as an oxidant. These results show that cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as P450. To utilize these P450-like reactivities effectively, cyt.c was immobilized on poly-gamma-methyl-L-glutamate. Up to 99% of the cyt.c used was immobilized. This immobilized cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as both P450 and free cyt.c, and the activities of these reactions were increased by the immobilization. In N-demethylation of N,N-dimethylaniline with cumene hydroperoxide (CHP) catalyzed by cyt.c, the Vmax for CHP was increased by 4.4-fold by the immobilization of the enzyme, while the Km remained unchanged. Since P450 is involved in the metabolism of many xenobiotics, the above results suggest that immobilized cyt.c may be useful in drug metabolism research. PMID:7681661

  13. Induction of cytochrome P-450, cytochrome b-5, NADPH-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment.

    PubMed

    Kawamoto, T; Hobara, T; Nakamura, K; Imamura, A; Ogino, K; Kobayashi, H; Iwamoto, S; Sakai, T

    1988-12-30

    Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB. PMID:3145630

  14. Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.

    PubMed

    Zhao, Fanglong; Bai, Peng; Liu, Ting; Li, Dashuai; Zhang, Xiangmei; Lu, Wenyu; Yuan, Yingjin

    2016-08-01

    Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc. PMID:26757342

  15. Steady-state kinetic investigation of cytochrome P450cam: interaction with redox partners and reaction with molecular oxygen.

    PubMed

    Purdy, Matthew M; Koo, Laura S; Ortiz de Montellano, Paul R; Klinman, Judith P

    2004-01-13

    Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex. PMID:14705955

  16. Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3.

    PubMed

    Sowden, Rebecca J; Yasmin, Samina; Rees, Nicholas H; Bell, Stephen G; Wong, Luet-Lok

    2005-01-01

    The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3. PMID:15602599

  17. Induction of cytochrome P-450 BM-3 (CYP 102) by non-steroidal anti-inflammatory drugs in Bacillus megaterium.

    PubMed Central

    English, N; Hughes, V; Wolf, C R

    1996-01-01

    Bacillus megaterium contains a cytochrome P-450 fatty acid mono-oxygenase which is inducible with barbiturate drugs. We have demonstrated that this enzyme system is inducible with peroxisome proliferators. In mammals, peroxisome proliferators also induce mono-oxygenases in the CYP4A gene family. In this paper we demonstrate that the non-steroidal anti-inflammatory drugs ibuprofen, ketoprofen and indomethacin are potent inducers of fatty acid mono-oxygenase activity as well as of P-450BM-3 protein in B. megaterium. The levels of induction of P-450 protein were 11.8-, 3.9- and 3.0-fold respectively. In addition, we demonstrate that these inducing agents interact with a transcriptional repressor, Bm3R1, which leads to its dissociation from its operator sequence. This provides a rational mechanism for the induction process. This is the first report which demonstrates that non-steroidal anti-inflammatory drugs can interact directly with a transcription factor to initiate gene expression, and further substantiates the structure-activity relationships that identify inducers of cytochrome P-450BM-3 and compounds that have the potential to act as peroxisome proliferators and induce CYP4A expression in mammals. PMID:8645218

  18. Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation.

    PubMed

    Park, Sun-Ha; Kim, Dong-Hyun; Kim, Dooil; Kim, Dae-Hwan; Jung, Heung-Chae; Pan, Jae-Gu; Ahn, Taeho; Kim, Donghak; Yun, Chul-Ho

    2010-05-01

    Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency. PMID:20100815

  19. Development of cytochromes P450 in avian species as a biomarker for environmental contaminant exposure and effect: Procedures and baseline values

    USGS Publications Warehouse

    Melancon, M.J.

    1996-01-01

    As in mammals and fish, birds respond to many environmental contaminants with induction of hepatic cytochromes P450. In order to monitor cytchromes P450 in specific avian species, for assessing the status of that species or the habitat it utilizes, it is necessary to have background information on the appropriate assay conditions and the responsiveness of cytochrome P450 induction in that species. Assay of four monooxygenases which give resorufin as product using a fluorescence microwell plate scanner has proven to be an effective approach. Information is provided on the incubation conditions and baseline activity for twenty avian species at ages ranging from pipping embryo to adult. Induction responsiveness is presented for sixteen of them. This information can serve as a guide for those who wish to utilize cytochrome P450 as a biomarker for contaminant exposure and effect to aid in selection of appropriate species, age, and monooxygenase assay(s).

  20. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    PubMed Central

    Elenewski, Justin E.; Hackett, John C

    2015-01-01

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis. PMID:25681906

  1. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

    PubMed Central

    Moskaleva, Natalia; Moysa, Alexander; Novikova, Svetlana; Tikhonova, Olga; Zgoda, Victor; Archakov, Alexander

    2015-01-01

    Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine. PMID:26561010

  2. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-01-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

  3. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    NASA Astrophysics Data System (ADS)

    Elenewski, Justin E.; Hackett, John C.

    2015-02-01

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

  4. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

    Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

    2004-08-01

    Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

  5. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

    NASA Astrophysics Data System (ADS)

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-04-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport (ETp) depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of ETp processes in the enzyme, in addition to occupying the active site.

  6. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    SciTech Connect

    Elenewski, Justin E.; Hackett, John C

    2015-02-14

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

  7. Mechanisms underlying the inhibition of the cytochrome P450 system by copper ions.

    PubMed

    Letelier, M E; Faúndez, M; Jara-Sandoval, J; Molina-Berríos, A; Cortés-Troncoso, J; Aracena-Parks, P; Marín-Catalán, R

    2009-11-01

    Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu2+-binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu2+ alone and Cu2+/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro-oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p-nitroanisole O-demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu2+ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu2+ and Cu2+/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed. PMID:19629952

  8. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    SciTech Connect

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

  9. Cholesterol-metabolizing cytochromes P450: implications for cholesterol lowering

    PubMed Central

    Pikuleva, Irina A.

    2010-01-01

    Cardiovascular disease (CVD) continues to be a leading cause of death worldwide. Elevated serum cholesterol is one of the classical risk factors for CVD which also include age, hypertension, smoking, diabetes mellitus, obesity and family history. A number of therapeutic drug classes have been developed to treat hypercholesterolemia, yet, an important percentage of patients do not reach their treatment goals. Therefore, new cholesterol-lowering medications, having a site of action different from that of currently available drugs need to be developed. This review summarizes new information about cytochrome P450 enzymes 7A1, 27A1, and 46A1, that play key roles in cholesterol elimination and that have potential to serve as targets for cholesterol-lowering. PMID:18950282

  10. Novel Bioactivation Pathway of Benzbromarone Mediated by Cytochrome P450.

    PubMed

    Kitagawara, Yumina; Ohe, Tomoyuki; Tachibana, Kumiko; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2015-09-01

    Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity. PMID:26106235

  11. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping. PMID:26233905

  12. Cytochrome P450 epoxygenase pathway of polyunsaturated fatty acid metabolism

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2014-01-01

    Polyunsaturated fatty acids (PUFA) are oxidized by cytochrome P450 epoxygenases to PUFA epoxides which function as potent lipid mediators. The major metabolic pathways of PUFA epoxides are incorporation into phospholipids and hydrolysis to the corresponding PUFA diols by soluble epoxide hydrolase. Inhibitors of soluble epoxide hydrolase stabilize PUFA epoxides and potentiate their functional effects. The epoxyeicosatrienoic acids (EETs) synthesized from arachidonic acid produce vasodilation, stimulate angiogenesis, have anti-inflammatory actions, and protect the heart against ischemia-reperfusion injury. EETs produce these functional effects by activating receptor-mediated signaling pathways and ion channels. The epoxyeicosatetraenoic acids synthesized from eicosapentaenoic acid and epoxydocosapentaenoic acids synthesized from docosahexaenoic acid are potent inhibitors of cardiac arrhythmias. Epoxydocosapentaenoic acids also inhibit angiogenesis, decrease inflammatory and neuropathic pain, and reduce tumor metastasis. These findings indicate that a number of the beneficial functions of PUFA may be due to their conversion to PUFA epoxides. PMID:25093613

  13. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  14. Ipriflavone as an inhibitor of human cytochrome P450 enzymes

    PubMed Central

    Monostory, Katalin; Vereczkey, László; Lévai, Ferenc; Szatmári, István

    1998-01-01

    Reduction of theophylline metabolism and elimination were observed in a theophylline-treated patient during ipriflavone administration. After withdrawal of ipriflavone, the serum theophylline level decreased to an extent similar to that found before administration of ipriflavone. The effects of ipriflavone and its major metabolites 7-hydroxy-isoflavone and 7-(1-carboxy-ethoxy)-isoflavone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors. Ipriflavone and 7-hydroxy-isoflavone competitively inhibited phenacetin O-deethylase and tolbutamide hydroxylase activity. The parent compound and its dealkylated metabolite were strong inhibitors exhibiting Ki values around 10–20 μM, while 7-(1-carboxy-ethoxy)-isoflavone had no effect on the cytochrome P450 activities investigated. 7-Hydroxy-isoflavone is the only one that influenced nifedipine oxidase activity. It competitively inhibited this activity with a Ki value of 129.5 μM. The steady state concentrations of ipriflavone and 7-hydroxy-isoflavone in plasma of patients receiving 3×200 mg daily doses of ipriflavone for 48 weeks were found to be 0.33±0.32 μM and 1.44±0.77 μM, respectively. The results indicate that the decrease in theophylline metabolism observed in a patient treated with ipriflavone may be due to a competitive interaction of ipriflavone or its metabolite, 7-hydroxy-isoflavone with CYP1A2. On the other hand, our in vitro findings predict some more interaction with CYP2C9. PMID:9517377

  15. Potential Biological Functions of Cytochrome P450 Reductase-dependent Enzymes in Small Intestine

    PubMed Central

    D'Agostino, Jaime; Ding, Xinxin; Zhang, Peng; Jia, Kunzhi; Fang, Cheng; Zhu, Yi; Spink, David C.; Zhang, Qing-Yu

    2012-01-01

    NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine. PMID:22453923

  16. Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450

    PubMed Central

    2015-01-01

    Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms. PMID:26299431

  17. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black- crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P450-associated monooxygenases and cytochrome P450 proteins, induced up to 85- fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r super(2) often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah- active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  18. Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  19. Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors.

    PubMed

    Hara, M; Iazvovskaia, S; Ohkawa, H; Asada, Y; Miyake, J

    1999-01-01

    P450 monooxygenases exhibit great potential for application to bioreactors for the decomposition of various hydrophobic chemicals including pollutant compounds. P450-containing microsomes were immobilized in spinach chloroplasts for use in light-driven bioreactors. We tested three methods (entrapment, adsorption and cross-linking) to immobilize chloroplasts and yeast microsomes containing a genetically engineered fusion enzyme between rat P450 1A1 and yeast P450 reductase. Entrapment in agarose gave the best activity for the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin under illumination of 6200 lx. We then tested three light-driven bioreactors (two column-type and one batch-type reactors developed) using the immobilized gels. A two-phase column-type reactor with separately immobilized microsomes and chloroplasts showed a higher conversion rate than a reactor with co-immobilization of both components. The reactor showed a turnover rate of 6.32 mol product/mol P450/min after a 40-min run, and 2.49 after a 180-min run. These turnover rates are higher than the values reported by others using immobilized microsomal P450s. PMID:16232556

  20. Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).

    PubMed Central

    Funk, C.; Croteau, R.

    1993-01-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. PMID:12231778

  1. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    SciTech Connect

    Funk, C.; Croteau, R. )

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  2. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

    Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

    1988-01-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  3. Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

    PubMed Central

    Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu

    2013-01-01

    Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM−1 s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

  4. Ethynyl and Propynylpyrene Inhibitors of Cytochrome P450.

    PubMed

    Zhu, Naijue; Lightsey, Danielle; Liu, Jiawang; Foroozesh, Maryam; Morgan, Kathleen M; Stevens, Edwin D; Klein Stevens, Cheryl L

    2010-04-01

    The single-crystal X-ray structures and in vivo activities of three aryl acetylenic inhibitors of cytochromes P450 1A1, 1A2, 2A6, and 2B1 have been determined and are reported herein. These are 1-ethynylpyrene, 1-propy-nylpyrene, and 4-propynylpyrene. To investigate electronic influences on the mechanism of enzyme inhibition, the experimental electron density distribution of 1-ethynylpy-rene has been determined using low-temperature X-ray diffraction measurements, and the resulting net atomic charges compared with various theoretical calculations. A total of 82,390 reflections were measured with Mo Kα radiation to a (sinθ/λ)(max) = 0.985 Å(-1). Averaging symmetry equivalent reflections yielded 8,889 unique reflections. A least squares refinement procedure was used in which multipole parameters were added to describe the distortions of the atomic electron distributions from spherical symmetry. A map of the model electron density distribution of 1-ethynylpyrene was obtained. Net atomic charges calculated from refined monopole population parameters yielded charges that showed that the terminal acetylenic carbon atom (C18) is more negative than the internal carbon (C17). Net atomic charges calculated by ab initio, density functional theory, and semi-empirical methods are consistent with this trend suggesting that the terminal acetylenic carbon atom is more likely to be the site of oxidation. This is consistent with the inhibition mechanism pathway that results in the formation of a reactive ketene intermediate. This is also consistent with assay results that determined that 1-ethynylpyrene acts as a mechanism-based inhibitor of P450s 1A1 and 1A2 and as a reversible inhibitor of P450 2B1. Crystallographic data: 1-ethynylpyrene, C(18)H(10), P2(1)/c, a = 14.571(2) Å, b = 3.9094(5) Å, c = 20.242(3) Å, β = 105.042(2)°, V = 1,113.5(2) Å(3); 1-propynylpyrene, C(19)H(12), P2(1)/n, a = 8.970(2) Å, b = 10.136(1) Å, c = 14.080(3) Å, β = 99.77(2)°, V = 1,261.5(4)

  5. A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

    PubMed Central

    Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A.; Halberg, Kenneth A.; Dow, Julian A.T.; Davies, Shireen-A.

    2015-01-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. PMID:26073628

  6. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  7. Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes.

    PubMed

    Kotewong, Rattanawadee; Pouyfung, Phisit; Duangkaew, Panida; Prasopthum, Aruna; Rongnoparut, Pornpimol

    2015-07-01

    The cytochrome P450 monooxygenases play a major role in insecticide detoxification and become a target for development of insecticide synergists. In this study, a collection of rhinacanthins (rhinacanthin-D, -E, -G, -N, -Q, and -H/I) purified from Rhinacanthus nasutus, in addition to previously purified rhinacanthin-B and -C, were isolated. These compounds displayed various degrees of inhibition against benzyloxyresorufin-O-debenzylation mediated by CYP6AA3 and CYP6P7 which were implicated in pyrethroid resistance in Anopheles minimus malaria vector. Inhibition modes and kinetics were determined for each of rhinacanthins. Cell-based inhibition assays by rhinacanthins employing 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity test were explored their synergistic effects with cypermethrin toxicity on CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells. Rhinacanthin-B, -D, -E, -G, and -N exhibited mechanism-based inhibition against CYP6AA3, an indication of irreversible inhibition, while rhinacanthin-B, -D, -G, and -N were mechanism-based inhibitors of CYP6P7. There was structure-function relationship of these rhinacanthins in inhibition effects against both enzymes. In vitro enzymatic inhibition assays revealed that there were synergistic interactions among rhinacanthins, except rhinacanthin-B and -Q, in inhibition against both enzymes. These rhinacanthins exerted synergism with cypermethrin toxicity on Sf9 cells expressing each of the two P450 enzymes via P450 inhibition and in addition could interact in synergy to further increase cypermethrin toxicity. The inhibition potentials, synergy among rhinacanthins in inhibition against the P450 detoxification enzymes, and synergism with cypermethrin toxicity of the R. nasutus constituents of reported herein could be beneficial to implement effective resistance management of mosquito vector control. PMID:25869958

  8. Structure and function of cytochrome P450S in insect adaptation to natural and synthetic toxins: insights gained from molecular modeling.

    PubMed

    Schuler, Mary A; Berenbaum, May R

    2013-09-01

    Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management. PMID:24036972

  9. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

    PubMed Central

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L.; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  10. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

    PubMed

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and -300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  11. Redox-dependent dynamics in cytochrome P450cam.

    PubMed

    Pochapsky, Susan Sondej; Dang, Marina; OuYang, Bo; Simorellis, Alana K; Pochapsky, Thomas C

    2009-05-26

    Local protein backbone dynamics of the camphor hydroxylase cytochrome P450(cam) (CYP101) depend upon the oxidation and ligation state of the heme iron. (1)H-(15)N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H-D exchange) and (15)N relaxation measurements, and these results are compared with previously published results obtained by H-D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and beta3 and beta5 sheets) and binding of putidaredoxin (C and L helices), the iron-sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed. PMID:19366254

  12. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  13. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  14. Functional coupling of ATP-binding cassette transporter Abcb6 to cytochrome P450 expression and activity in liver.

    PubMed

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-03-20

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

  15. Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver*

    PubMed Central

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-01-01

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

  16. Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria.

    PubMed

    Guo, Yanqiong; Zhang, Jianzhen; Yu, Rongrong; Zhu, Kun Yan; Guo, Yaping; Ma, Enbo

    2012-05-01

    Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust. PMID:22300555

  17. Hepatic metabolism of cyclodiene insecticides by constitutive forms of cytochrome P-450 from lower vertebrates.

    PubMed

    Ronis, M J; Walker, C H; Peakall, D

    1987-01-01

    1. Multiple forms of cytochrome P-450 were separated from the hepatic microsomes of untreated male rats, pigeons (Columbia livia), razorbills (Alca torda), puffins (Fratercula arctica), and rainbow trout (Salmo gairdnerii), using anion exchange chromatography and DEAE-cellulose. 2. In some cases cytochrome P-450 forms were further purified on hydroxylapatite and carboxymethyl-sephadex columns. 3. Considerable differences in the distribution of forms between these five species were evident from elution profiles on DEAE cellulose, and on analysis of the cytochrome P-450 containing pools by SDS-PAGE. 4. The metabolism of two organochlorine compounds, aldrin and the dieldrin analogue HCE, were studied in (a) intact microsomes and (b) reconstituted systems containing cytochrome P-450, from each of the five species. 5. In spite of their close structural similarity, significant differences were found between the two substrates in the distribution of catalytic activity between the cytochrome P-450 isozymes of each species. PMID:2888582

  18. Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system.

    PubMed

    Kuznetsov, Vadim Yu; Ivanov, Yuri D; Archakov, Alexander I

    2004-08-01

    The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm. PMID:15274134

  19. Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man.

    PubMed Central

    Sesardic, D; Boobis, A R; Murray, B P; Murray, S; Segura, J; de la Torre, R; Davies, D S

    1990-01-01

    1. Furafylline (1,8-dimethyl-3-(2'-furfuryl)methylxanthine) is a methylxanthine derivative that was introduced as a long-acting replacement for theophylline in the treatment of asthma. Administration of furafylline was associated with an elevation in plasma levels of caffeine, due to inhibition of caffeine oxidation, a reaction catalysed by one or more hydrocarbon-inducible isoenzymes of P450. We have now investigated the selectivity of inhibition of human monooxygenase activities by furafylline. 2. Furafylline was a potent, non-competitive inhibitor of high affinity phenacetin O-deethylase activity of microsomal fractions of human liver, a reaction catalysed by P450IA2, with an IC50 value of 0.07 microM. 3. Furafylline had either very little or no effect on human monooxygenase activities catalysed by other isoenzymes of P450, including P450IID1, P450IIC, P450IIA. Of particular interest, furafylline did not inhibit P450IA1, assessed from aryl hydrocarbon hydroxylase activity of placental samples from women who smoked cigarettes. 4. It is concluded that furafylline is a highly selective inhibitor of P450IA2 in man. 5. Furafylline was a potent inhibitor of the N3-demethylation of caffeine and of a component of the N1- and N7-demethylation. This confirms earlier suggestions that caffeine is a selective substrate of a hydrocarbon-inducible isoenzyme of P450 in man, and identifies this as P450IA2. Thus, caffeine N3-demethylation should provide a good measure of the activity of P450IA in vivo in man. 6. Although furafylline selectively inhibited P450IA2, relative to P450IA1, in the rat, this was at 1000-times the concentration required to inhibit the human isoenzyme, suggesting a major difference in the active site geometry between the human and the rat orthologues of P50IA2. PMID:2378786

  20. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

    SciTech Connect

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.

  1. Induction of drug-metabolizing enzymes by tricyclic antidepressants in human liver: characterization and partial resolution of cytochromes P-450.

    PubMed Central

    Cresteil, T; Célier, C; Kremers, P; Flinois, J P; Beaune, P; Leroux, J P

    1983-01-01

    Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine. PMID:6661349

  2. Characterization of human cytochrome P450 induction by pesticides.

    PubMed

    Abass, Khaled; Lämsä, Virpi; Reponen, Petri; Küblbeck, Jenni; Honkakoski, Paavo; Mattila, Sampo; Pelkonen, Olavi; Hakkola, Jukka

    2012-03-29

    Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR. PMID:22310298

  3. Cytochrome P450-Dependent Metabolism of Caffeine in Drosophila melanogaster

    PubMed Central

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie

    2015-01-01

    Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone—an inhibitor of CYP enzymes—showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects. PMID:25671424

  4. Differential Cytochrome P450 2D Metabolism Alters Tafenoquine Pharmacokinetics

    PubMed Central

    Vuong, Chau; Xie, Lisa H.; Potter, Brittney M. J.; Zhang, Jing; Zhang, Ping; Duan, Dehui; Nolan, Christina K.; Sciotti, Richard J.; Zottig, Victor E.; Nanayakkara, N. P. Dhammika; Tekwani, Babu L.; Walker, Larry A.; Smith, Philip L.; Paris, Robert M.; Read, Lisa T.; Li, Qigui; Pybus, Brandon S.; Sousa, Jason C.; Reichard, Gregory A.; Smith, Bryan

    2015-01-01

    Cytochrome P450 (CYP) 2D metabolism is required for the liver-stage antimalarial efficacy of the 8-aminoquinoline molecule tafenoquine in mice. This could be problematic for Plasmodium vivax radical cure, as the human CYP 2D ortholog (2D6) is highly polymorphic. Diminished CYP 2D6 enzyme activity, as in the poor-metabolizer phenotype, could compromise radical curative efficacy in humans. Despite the importance of CYP 2D metabolism for tafenoquine liver-stage efficacy, the exact role that CYP 2D metabolism plays in the metabolism and pharmacokinetics of tafenoquine and other 8-aminoquinoline molecules has not been extensively studied. In this study, a series of tafenoquine pharmacokinetic experiments were conducted in mice with different CYP 2D metabolism statuses, including wild-type (WT) (reflecting extensive metabolizers for CYP 2D6 substrates) and CYPmouse 2D knockout (KO) (reflecting poor metabolizers for CYP 2D6 substrates) mice. Plasma and liver pharmacokinetic profiles from a single 20-mg/kg of body weight dose of tafenoquine differed between the strains; however, the differences were less striking than previous results obtained for primaquine in the same model. Additionally, the presence of a 5,6-ortho-quinone tafenoquine metabolite was examined in both mouse strains. The 5,6-ortho-quinone species of tafenoquine was observed, and concentrations of the metabolite were highest in the WT extensive-metabolizer phenotype. Altogether, this study indicates that CYP 2D metabolism in mice affects tafenoquine pharmacokinetics and could have implications for human tafenoquine pharmacokinetics in polymorphic CYP 2D6 human populations. PMID:25870069

  5. The cytochrome P450 (CYP) gene superfamily in Daphnia pulex

    PubMed Central

    Baldwin, William S; Marko, Peter B; Nelson, David R

    2009-01-01

    Background Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome genomes. Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone (Nematostella vectensis) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex, whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans. Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex. Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (aromatase). Conclusion Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan. PMID:19383150

  6. Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.

    PubMed

    Rothwell, C E; Khazaeli, M B; Bernstein, I A

    1985-08-15

    A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b. PMID:3935002

  7. Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics.

    PubMed

    Purdy, Matthew M; Koo, Laura S; de Montellano, Paul R Ortiz; Klinman, Judith P

    2006-12-26

    The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured. PMID:17176102

  8. Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

    SciTech Connect

    Hamdane, Djemel; Xia, Chuanwu; Im, Sang-Choul; Zhang, Haoming; Kim, Jung-Ja P.; Waskell, Lucy

    2010-01-20

    NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.

  9. Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm moth (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura (Lepidoptera, Noctuidae) has been shown to be resistant to a wide range of insecticides. In this stu...

  10. Clofibrate-induced cytochrome P450-lauric acid omega hydroxylase(P450LA omega):purification, cDNA cloning, sequence and regulation

    SciTech Connect

    Hardwick, J.P.; Song, B.J.; Gonzalez, F.J.

    1986-05-01

    A cytochrome P450 that hydroxylates lauric acid at the 12 position (P450LA omega) was isolated from liver microsomes of clofibrate treated rats. P450LA omega was immunologically distinct from P450s a,b,c,d,e,f,g,h,j,PB1, and PCN1. Polyclonal antibody against P450LA omega was utilized to screen a gt11 cDNA library. A clone (pP450LA omega), was isolated and its sequence determined. The P450LA omega mRNA is a minimum 2387 nts in length and codes for a P450 of Mr.58,222 daltons. This protein shares less than 35% amino acid similarity with P450s b,c,d,e,f,PB1, and PCN1; however, it does contain a hydrophobic amino terminal peptide and a conserved sequence surrounding the Cys residue at position 456, which is similar to other microsomal P450s. P450LA omega is present at high levels in untreated rat kidney and is induced by clofibrate in both kidney and liver. This induction is the result of an accumulation of mRNA through a rapid transcriptional activation of the P450LA gene. Southern blotting data suggest the presence of 2 or 3 genes in the P450LA omega family. This P450 gene family may be associated with arachidonic acid and prostraglandin metabolism in kidney and other tissues.