Functional Characterization of Human Cytochrome P450 2S1 Using a Synthetic Gene-Expressed Protein in<i>Escherichia coli</i>

Bui, Peter; Hankinson, Oliver

doi:10.1124/mol.109.057752

Cited by 45 publications

(55 citation statements)

References 58 publications

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“…This result is in contrast to the absence of functional interactions between P450 reductase and CYP2S1 described by Bui and Hankinson (2009). The basis for this discrepancy might be due to the fact that Bui and Hankinson, as well as ourselves, have used aerobic conditions, whereas Nishida et al (2010) conducted experiments under strictly anaerobic conditions when studying the electron transfer to CYP2S1 and CYP2W1.…”

Section: Glycosylation Of Cyp2w1 Protein 1009contrasting

confidence: 49%

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Gomez¹,

Nekvindová²,

Travica³

et al. 2010

Mol Pharmacol

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Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.

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Section: Glycosylation Of Cyp2w1 Protein 1009contrasting

confidence: 49%

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Gomez¹,

Nekvindová²,

Travica³

et al. 2010

Mol Pharmacol

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“…CYP1B1, including P450 reductase, was purchased from BD Gentest (Woburn, MA). Purified synthetically encoded CYP2S1 was prepared as described in the accompanying article (Bui and Hankinson, 2009). r7,t8,t9,r7,t8,t9, were purchased from the National Cancer Institute's Chemical Carcinogen Repository (Kansas City, MO).…”

Section: Methodsmentioning

confidence: 99%

“…All assays were conducted in 1.5-ml Eppendorf tubes in duplicate, except for those with the c33 cell lysate, which were performed in replicates of four. The purified CYP2S1 used in all enzyme assays was the synthetically encoded protein (Bui and Hankinson, 2009). In experiments designed to determine whether lipid hydroperoxides can support CYP2S1-, CYP1A1-, CYP1A2-, CYP1B1-, or CYP3A4-mediated oxidation of action mixtures consisted of 100 mM potassium phosphate buffer, pH 7.5; 0.1 M P450; 50 M 5-HpETE, 12-HpETE, 15-HpETE, or 13-HpODE; and 100 M BaP-7,8-diol.…”

Section: Methodsmentioning

confidence: 99%

“…In the accompanying report (Bui and Hankinson, 2009), we report that CYP2S1 cannot use NADPH for oxidative metabolism because of its inability to accept electrons from NADPH. However, CYP2S1 can use cumene hydroperoxide and hydrogen peroxide to oxidize several carcinogens, including BaP-7,8-diol.…”

mentioning

confidence: 99%

“…Although cumene hydroperoxide supported CYP2S1-catalyzed oxidation, it is not present endogenously and therefore does not serve as a physiological cofactor. Although hydrogen peroxide is found endogenously, and could potentially be a relevant physiological cofactor for CYP2S1, the concentration necessary for effective oxidation in vivo is probably considerably greater than endogenous levels under most circumstances (Bui and Hankinson, 2009). On the other hand, lipid hydroperoxides have been shown in many cases to support cytochrome P450-catalyzed oxidations both in vivo and in vitro and have been suggested to serve as physiological cofactors for cytochrome P450-mediated oxidations (Dix and Marnett, 1983;Muindi and Young, 1993;Wang and Liehr, 1994;Smith et al, 1995;McCallum et al, 1996;Anari et al, 1997;Tsai et al, 2001;Caro and Cederbaum, 2006).…”

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confidence: 99%

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Fatty Acid Hydroperoxides Support Cytochrome P450 2S1-Mediated Bioactivation of Benzo[a]pyrene-7,8-dihydrodiol

Bui¹,

Hsu²,

Hankinson³

2009

Mol Pharmacol

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In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo into the highly mutagenic and carcinogenic benzo [a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O 2 . Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of . In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K m , 3.4 Ϯ 0.8 M; turnover, 4.51 Ϯ 0.13 min Ϫ1 ), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K m , 2.8 Ϯ 0.7 M; turnover, 3.7 Ϯ 0.1 min Ϫ1 ), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (K m , 2.7 Ϯ 0.8 M; turnover, 3.69 Ϯ 0.09 min Ϫ1 ), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K m , 11.6 Ϯ 0.3 M; turnover, 0.578 Ϯ 0.030 min Ϫ1 ). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis.Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant produced during combustion, including the burning of cigarettes. It has been identified in ambient air, surface water, drinking water, waste water, and char-broiled foods and is classified as a human carcinogen by the International Agency for Research on Cancer (1983). Exposure can occur by ingestion, inhalation, or dermal absorption (Agency for Toxic Substances and Disease Registry, 1990). For BaP to be carcinogenic, it must be converted by oxidative metabolism to mutagenic and carcinogenic derivatives (Wislocki et al., 1976;Wood et al., 1976;Gelboin, 1980) (see Scheme 1). The first step in this process is the formation of BaP-trans-7,8-epoxide, followed by hydrolysis to the . The latter metabolite is further epoxidized to the mutagenic BaP-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), which is extremely reactive with DNA and protein. BaP-diol-t-epoxide is very unstable, because it rapidly undergoes hydrolysis to t8,t9, and t8,t9,, whose detection is indicative of BaP-diol-t-epoxide formation...

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Oxidative Drug Metabolism by Mammalian CytochromeP450Enzymes Using Their Monooxygenase and Peroxygenase Functions

Hrycay

Bandiera

2017

Encyclopedia of Drug Metabolism and Interactions

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The cytochrome P450 (CYP) enzyme system represents the predominant biotransformation pathway in the human body for lipophilic exogenous and endogenous compounds. The monooxygenase reaction catalyzed by CYP enzymes involves the addition of one oxygen atom from molecular oxygen to a lipophilic substrate, while incorporating the other oxygen atom into a molecule of water, thereby leading to the production of more polar and easily excreted metabolites. Thus, CYP enzymes are major determinants of duration of action and clearance of drugs. In some cases, CYP enzymes are involved in the formation of chemically reactive and potentially toxic metabolites, as well as in clinically significant drug–drug interactions, which are a widely recognized cause of adverse drug reactions in humans. This chapter focuses on the oxidative biotransformation of therapeutic drugs catalyzed by human CYP enzymes. The topics covered include the identification of human hepatic and extrahepatic CYP enzymes involved in drug biotransformation, CYP bioactivation of prodrugs to pharmacologically active compounds, human CYP drug–drug interactions, CYP monooxygenase and peroxygenase reactions involving drug substrates, and mechanisms involved in CYP oxidative drug reactions.

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Functional Characterization of Human Cytochrome P450 2S1 Using a Synthetic Gene-Expressed Protein inEscherichia coli

Cited by 45 publications

References 58 publications

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Fatty Acid Hydroperoxides Support Cytochrome P450 2S1-Mediated Bioactivation of Benzo[a]pyrene-7,8-dihydrodiol

Oxidative Drug Metabolism by Mammalian CytochromeP450Enzymes Using Their Monooxygenase and Peroxygenase Functions

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