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The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily. 相似文献
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Kapoor N Pant AB Dhawan A Dwievedi UN Gupta YK Seth PK Parmar D 《Life sciences》2006,79(16):1514-1522
The expression of the cytochrome P450s (CYPs) may vary in the different brain cells depending on their specialization and the presence of different endogenous factors. The present study was initiated to investigate the expression and catalytic activity of the constitutive and inducible forms of CYP2E1, the major ethanol inducible CYP, in cultured rat brain neuronal and glial cells. These cells exhibited relatively two-fold higher activity of N-nitrosodimethylamine demethylase (NDMA-d) when compared with the liver enzyme. Pretreatment with ethanol revealed a significant time and concentration dependent induction in NDMA-d activity in both cell types. Western blot, immunocytochemistry and RT-PCR also indicated significant induction of CYP2E1 in the cultured brain cells. Interestingly, the neuronal cells exhibited greater magnitude of induction than the glial cells. The relatively higher degree of induction in cultures of neurons has indicated enhanced sensitivity of neurons to the inductive effects of ethanol. This enhanced induction of CYP2E1 in neuronal cells has indicated that like regional specificity, cell specificity also exists in the induction of CYP2E1 and other CYPs. 相似文献
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Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C10:0 to C18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, Km, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined. 相似文献
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Yelena V. Grinkova 《Biochemical and biophysical research communications》2010,398(2):194-539
Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 °C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug-drug interaction studies. 相似文献
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Immunohistochemical localization of cytochrome P450 CYP1B1 in breast cancer with monoclonal antibodies specific for CYP1B1. 总被引:3,自引:0,他引:3
M C McFadyen S Breeman S Payne C Stirk I D Miller W T Melvin G I Murray 《The journal of histochemistry and cytochemistry》1999,47(11):1457-1464
Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase. (J Histochem Cytochem 47:1457-1464, 1999) 相似文献
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根据细胞色素P450家族4(CYP4)的氨基酸保守序列设计1对简并引物,从椰心叶甲Brontispa longissima成虫总RNA中扩增得到5个cDNA片段(GenBank登录号: DQ238840-DQ238844)。以3′-RACE法获得片段BLWH4的3′端序列,推导的氨基酸序列表明其结构中含有CYP家族的特征性保守序列: 螺旋K区的ETLR和血红素结合区的F××G×××C×G。以18S 为对照的RT-PCR分析表明,BLWH4在成虫的mRNA表达量远大于幼虫。绿僵菌Metarhizium anisopliae菌株MA-3和MA-4侵染椰心叶甲成虫及5龄幼虫后,BLWH4的mRNA表达增强,提示BLWH4可能具有增强椰心叶甲抵抗绿僵菌侵染的作用。 相似文献
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Karlgren M Gomez A Stark K Svärd J Rodriguez-Antona C Oliw E Bernal ML Ramón y Cajal S Johansson I Ingelman-Sundberg M 《Biochemical and biophysical research communications》2006,341(2):451-458
A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues, CYP2W1 mRNA was expressed preferentially in fetal but also in adult colon. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1-intron 1 region which was methylated in cell lines lacking CYP2W1 expression, but unmethylated in cells expressing CYP2W1. Re-expression of CYP2W1 was seen following demethylation by 5-Aza-2'-deoxycytidine. Transfection of HEK293 cells with CYP2W1 caused the formation of a properly folded enzyme, which was catalytically active with arachidonic acid as a substrate. It is concluded that CYP2W1 represents a tumor-specific P450 isoform with potential importance as a drug target in cancer therapy. 相似文献
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Barbosa-Sicard E Markovic M Honeck H Christ B Muller DN Schunck WH 《Biochemical and biophysical research communications》2005,329(4):1275-1281
CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites. 相似文献
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Enantioselectivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketoconazole: Testosterone and methadone as substrates 总被引:4,自引:0,他引:4
Racemic ketoconazole (KTZ) was the first orally active azole antifungal agent used in clinical practice and has become widely used in the treatment of mucosal fungal infections associated with AIDS immunosuppression and cancer chemotherapy. However, the use of KTZ has been limited because of adverse drug-drug interactions. KTZ blocks ergosterol biosynthesis by inhibiting the fungal cytochrome P450 (CYP51). KTZ is also a potent inhibitor of human cytochrome P450 3A4 (CYP3A4) enzyme, the major drug-metabolizing CYP isozyme in the human liver. We examined the enantioselective differences of KTZ in the inhibition of human CYP3A4 and in antifungal action. Dextro- and levo-KTZ exhibited modest enantioselective differences with respect to CYP3A4 inhibition of testosterone and methadone metabolism. For both substrates levo-KTZ was approximately a 2-fold more potent inhibitor. We examined the enantioselective differences in the in vitro activity of KTZ against medically relevant species of Candida and Aspergillus, as well as Cryptococcus neoformans. Overall, levo-KTZ was 2-4-fold more active than dextro-KTZ. Therefore, levo-KTZ is a more potent inhibitor of CYP3A4 and has stronger in vitro antifungal activity. Chirality 16:79-85, 2004. 相似文献
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Shabana Vohra Maria Musgaard Luet‐Lok Wong Weihong Zhou Philip C. Biggin 《Protein science : a publication of the Protein Society》2013,22(9):1218-1229
The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit. 相似文献
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Regev-Shoshani G Shoseyov O Kerem Z 《Biochemical and biophysical research communications》2004,323(2):668-673
Resveratrol, a polyphenol found in red wine, was recently suggested to act as an irreversible, mechanism-based inactivator of cytochrome P450 3A4 (CYP3A4). We found a significant inhibition of human CYP3A4-dependent transformation of cyclosporine by resveratrol, with IC50 = 4.5 microM. We studied the kinetics parameters of CYP3A4 transformation of resveratrol and structurally related, naturally occurring stilbenes. Resveratrol, piceid, resveratroloside, 5,4'-dihydroxy-3-O-methoxystilbene, and 5,3-dihydroxy-4'-O-methoxystilbene were all shown to inhibit hydroxylation of testosterone by CYP3A4. Both methoxy-stilbenes had lower IC50 values, ranging from 0.43 to 0.47 microM, suggesting that lipophilicity rather than number or positions of free hydroxyls (3,5 or 5,4') determines the CYP3A4 inhibition capacity of polyphenols. In line with these findings, both glucosyl-stilbenes were found to be weak inhibitors of CYP3A4. The affinity of the enzyme towards methoxy-stilbenes, expressed as apparent Km, was indeed higher than those for the parent resveratrol and its glucosides, in CYP3A4 reaction mixtures. Vmax values were similar, except for piceid. These results support the role of lipophilicity in the interaction of polyphenols with CYP3A4. It is suggested that selective structural modifications of substrates add significantly to knowledge acquired through molecular modifications of the enzyme. 相似文献
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Sandra Irmisch Sybille B Unsicker Jonathan Gershenzon Tobias G K?llner 《Plant signaling & behavior》2013,8(12)
After herbivore feeding, poplar trees produce complex volatile blends containing terpenes, green leaf volatiles, aromatics, and nitrogen-containing compounds such as aldoximes and nitriles. It has been shown recently that volatile aldoximes released from gypsy moth (Lymantria dispar) caterpillar-damaged black poplar (Populus nigra) trees attract parasitoids that are caterpillar enemies. In western balsam poplar (P. trichocarpa), volatile aldoximes are produced by 2 P450 monooxygenases, CYP79D6v3 and CYP79D7v2. A gene fragment with high similarity to CYP79D6/7 was recently shown to be upregulated in herbivore-damaged leaves of P. nigra. In the present study we report the cloning and characterization of this gene, designated as CYP79D6v4. Recombinant CYP79D6v4 was able to convert different amino acids into the corresponding aldoximes, which were also found in the volatile blend of P. nigra. Thus, CYP79D6v4 is most likely involved in herbivore-induced aldoxime formation in black poplar. 相似文献
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Rupasinghe S Schuler MA Kagawa N Yuan H Lei L Zhao B Kelly SL Waterman MR Lamb DC 《FEBS letters》2006,580(27):6338-6342
In this work, we have spectroscopically characterised CYP157C1 from Streptomyces coelicolor A3(2) which has the motif E(297)QSLW(301) rather than the invariant EXXR motif in the P450 K-helix. Site-directed mutagenesis of native E(297)QSLW(301) in CYP157C1 to E(297)ESLR(301) or E(297)QSRW(301) both containing standard EXXR motifs produced cytochrome P420 proteins thought to be inactive forms of P450 even though wild type CYP157C1 has the spectral properties of a normal P450. These results indicate that the EXXR motif is not required in all CYP tertiary architectures and only a single cysteine residue, which coordinates as the fifth thiolate ligand to the P450 haem iron, is invariant in all CYPs structures. 相似文献
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