Residue 285 in Cytochrome P450 2B4 Lacking the NH2-Terminal Hydrophobic Sequence Has a Role in the Functional Association of NADPH–Cytochrome P450 Reductase

Schulze, Johannes; Tschöp, Katharina; Lehnerer, Michael; Hlavica, Peter

doi:10.1006/bbrc.2000.2495

Cited by 10 publications

(6 citation statements)

References 27 publications

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“…Previous reported studies have tested for substrates in the presence of NADPH and oxygen. The inability to consistently demonstrate an enzymatic activity for the protein in these studies is due to its inability to accept electrons from NADPH via P450 reductase (Bui and Hankinson, 2009), probably due, at least in part, to the fact that CYP2S1 lacks certain amino acids known to be important for the interaction of other P450s with the P450 reductase (Crespi and Miller, 1997;Schulze et al, 2000;Nikfarjam et al, 2006). However, we showed that CYP2S1 can oxidize several substrates using cumene hydroperoxide and hydrogen peroxide.…”

Section: Discussionmentioning

confidence: 72%

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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Section: Discussionmentioning

confidence: 72%

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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“…Even when we attempted to reconstitute the purified CYP2S1 and P450 reductase using L-␣-1,2-dilauroyl-sn-glycero-3-phosphocholine according to a method described previously (Brian et al, 1990), we failed to demonstrate any interaction between the two proteins or NADPH-dependent activity (data not shown). The lack of CYP2S1 and P450 reductase interaction can be explained, at least in part, by the fact that CYP2S1 does not contain certain lysine and arginine amino acids at the important positions 147, 326, and 388, which have been proposed to be important for the interactions of various P450s with P450 reductase (Crespi and Miller, 1997;Schulze et al, 2000;Nikfarjam et al, 2006). Although it is possible that N-terminal modification of the enzyme negates its interaction with the P450 reductase, this is unlikely, because many mammalian P450s have been expressed in E. coli and none of their activities have been reported to be affected by the N-terminal modifications that are always included (Barnes et al, 1991;Guengerich et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

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

Bui¹,

Hankinson²

2009

Mol Pharmacol

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Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H 2 O 2 , such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a] pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K m ϭ 12.4 Ϯ 2 M, turnover ϭ 2.3 min Ϫ1

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“…4A shows the CD spectra in the far UV region (197-250 nm). They are typical for P450 (25,26) and superimposable. The ␣-helical content estimated from mean residue ellipticity ([] R ) at 223 nm is close to 50%, which is in a good agreement with 44% determined from the MTCYP51 structure.…”

Section: Characterization Of Mtcyp51 Mutantsmentioning

confidence: 93%

Folding Requirements Are Different between Sterol 14α-Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs

Lepesheva

Podust

Bellamine

et al. 2001

Journal of Biological Chemistry

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Upon sequence alignment of CYP51 sterol 14␣-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting ␤-strand 3-2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways.

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Residue 285 in Cytochrome P450 2B4 Lacking the NH2-Terminal Hydrophobic Sequence Has a Role in the Functional Association of NADPH–Cytochrome P450 Reductase

Cited by 10 publications

References 27 publications

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

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

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

Folding Requirements Are Different between Sterol 14α-Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs

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