Roles of NADPH-P450 Reductase and Apo- and Holo-Cytochrome b5 on Xenobiotic Oxidations Catalyzed by 12 Recombinant Human Cytochrome P450s Expressed in Membranes of Escherichia coli

Yamazaki, Hiroshi; Nakamura, Mami; Komatsu, Takao; Ohyama, Katsuhiro; Hatanaka, Naoya; Asahi, Satoru; Shimada, Noriaki; Guengerich, F. Peter; Shimada, Tsutomu; Nakajima, Miki; Yokoi, Tsuyoshi

doi:10.1006/prep.2001.1578

Cited by 230 publications

(221 citation statements)

References 30 publications

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“…As reported previously (46,63,85), the presence of b 5 enhanced the steady-state k cat for coumarin 7-hydroxylation (Table I). One general hypothesis for the enhancement of P450 catalytic activities by b 5 is transfer of an electron from ferrous b 5 to the P450 FeO 2ϩ -substrate complex (70,71).…”

supporting

confidence: 88%

“…The b 5 experiments are complex and difficult to interpret, but our overall conclusion is that some electron transfer from ferrous b 5 to the P450 2A6 FeO 2 2ϩ -coumarin complex occurs but that this does not seem to be a particularly efficient process. In other work we have shown that P450 2A6-catalyzed coumarin 7-hydroxylation can be enhanced by apo-b 5 devoid of heme and precluding electron transfer (85). We conclude that part of the enhancement may be attributable to the electron transfer (Figs.…”

supporting

confidence: 48%

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Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

115

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Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

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supporting

confidence: 88%

supporting

confidence: 48%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

115

View full text Add to dashboard Cite

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“…Because of the considerable molar excess of reductase over CYP3A4, the latter is anticipated to be completely complexed with the flavoprotein and the functional properties of the monooxygenase are expected to be largely insensitive to possible variations in CPR content between the batches. This conclusion is supported by the observation of Yamazaki et al that the incorporation of exogenous CPR into CYP3A4 Baculosomes does not affect the rate of testosterone 6β-hydroxylation [53]. It should be noted also that all experiments on BFC debenzylation with intact Baculosomes were done with the first batch (39195B), while BQ experiments and the experiments with BFC and enriched baculosomes were done with the second batch.…”

Section: Activity Measurementssupporting

confidence: 56%

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Davydov

Davydova

Tsalkova

et al. 2008

Archives of Biochemistry and Biophysics

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Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl) coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.

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“…Individual porcine CYP450 enzymes have been studied to determine how metabolic capabilities correlate with those found in humans and for studies in veterinary pharmacology (Juskevich, 1987;Anzenbacherová et al, 2005). In addition, the microsomal haemoprotein cytochrome b5A (CYB5A) has been shown to augment the activity of human CYP2E1 (hCYP2E1) towards chlorzoxazone (CLZ) hydroxylation by apparently acting as an electron donor (Yamazaki et al, 1996;Yamazaki et al, 2002;Wiercinska and Squires, 2010). CYB5 also interacts with a number of other CYP450 isoforms including CYP17A1 (Billen and Squires, 2009); a CYB5 knockout mouse model has a dramatically altered expression of drug metabolizing enzymes and low levels of testicular androgens (McLaughlin et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Wiercinska

Lou

Squires

2012

Animal

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Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.Keywords: boar taint, skatole, 3-methylindole, cytochrome b5A, cytochrome P450 ImplicationsStudies on the metabolism and clearance of skatole, a major component of boar taint, have been hampered by the lack of specific tools to measure the activity of the cytochrome P450 (CYP450) enzymes involved. Here we have used a defined cellbased system to individually express six different isoforms of porcine CYP450 and cytochrome b5A, and measure their role in the metabolism of skatole. Our results show that CYP2A19 is the primary isoform responsible for the formation of 6-hydroxyskatole, although its importance to in vivo clearance will also be affected by its level of expression in vivo.

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Roles of NADPH-P450 Reductase and Apo- and Holo-Cytochrome b5 on Xenobiotic Oxidations Catalyzed by 12 Recombinant Human Cytochrome P450s Expressed in Membranes of Escherichia coli

Cited by 230 publications

References 30 publications

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

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