Formation of Indigo by Recombinant Mammalian Cytochrome P450

Gillam, Elizabeth M. J.; Aguinaldo, Anna Marie A.; Notley, L.; Kim, Donghak; Mundkowski, Ralf G.; Volkov, Alexander N.; Arnold, Frances H.; Souček, Pavel; DeVoss, James J.; Guengerich, F. Peter

doi:10.1006/bbrc.1999.1702

Cited by 129 publications

(98 citation statements)

References 18 publications

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“…Formation of II (an analogue of N-FK) is notable, because this compound is the only product that we have identified that has not been reported to result from P450-catalyzed oxidation of indole (29)(30)(31)(37)(38)(39). In contrast, oxidation of indoles (40) -, below detection limit.…”

Section: •−mentioning

confidence: 87%

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Kuo

Mauk

2012

Proc. Natl. Acad. Sci. U.S.A.

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The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to catalyze the oxidation of indole by H 2 O 2 , with generation of 2-and 3-oxoindole as the major products. This reaction occurred in the absence of O 2 and reducing agents and was not inhibited by superoxide dismutase or hydroxyl radical scavengers, although it was strongly inhibited by L-Trp. The stoichiometry of the reaction indicated a one-to-one correspondence for the consumption of indole and H 2 O 2 . The 18 O-labeling experiments indicated that the oxygen incorporated into the monooxygenated products was derived almost exclusively from H 2 18 O 2 , suggesting that electron transfer was coupled to the transfer of oxygen from a ferryl intermediate of IDO. These results demonstrate that IDO oxidizes indole by means of a previously unrecognized peroxygenase activity. We conclude that IDO inserts oxygen into indole in a reaction that is mechanistically analogous to the "peroxide shunt" pathway of cytochrome P450.monooxygenase | oxygen isotopic labeling T he heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-determining step of L-tryptophan metabolism in nonhepatic mammalian tissues by inserting both atoms of O 2 into the indole ring to form N-formylkynurenine (N-FK) (1). This dioxygenase activity of IDO requires O 2 and the reduced (Fe 2+ ) enzyme or O 2•− and the oxidized (Fe 3+ ) enzyme. In recent years, an increasing number of physiological consequences of IDO activity have been identified, including links to neural, ocular, and immunological disorders (2). Of these roles, the ability of IDO expressed by tumors to suppress the normal response of T lymphocytes and enable tumor survival and growth has attracted the most intense interest, owing to its implications for the development of new therapeutic approaches in cancer treatment (3).Aside from tryptophan (Trp), the dioxygenase activity of IDO extends to oxidation of other indoleamines, such as 5-hydroxy-LTrp, serotonin, and tryptamine, whereas indole, 3-methylindole, and indoleacetic acid are not substrates (4-6). Although indole can apparently bind to the oxygenated enzyme (IDOFe 3+ -O 2•− ) or to the IDOFe 2+ -CO complex (7), autoxidation to IDOFe 3+ is unaffected by the presence of indole, and oxidation of indole does not occur (5). Notably, IDO does not catalyze oxidation of L-Trp by H 2 O 2 (4, 8, 9). Consequently, the reactivity of IDO with H 2 O 2 received little attention for more than 30 years. During that time, IDO was noted to have peroxidase activity (6) and to catalyze the H 2 O 2 -supported N-demethylation of benzphetamine and hydroxylation of aniline (10), but these activities were not studied further.Recently, reactivity of the enzyme with H 2 O 2 has received considerable attention in light of spectroscopic detection of a compound II-like ferryl species (11, 12) and quantum mechanics/ molecular mechanics simulations implicating a role for this intermediate in the catalytic cycle (13). Subsequently, the peroxidase activity of IDO has received renewed atten...

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Section: •−mentioning

confidence: 87%

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Kuo

Mauk

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The upper layer, containing the phenolic product, was transferred to fluorescence microtiter plates, and the 7-hydroxycoumarin was measured using a microtiter plate fluorescence plate reader (43). For the analysis of 3-hydroxy-7-methoxycoumarin, the reaction products were analyzed by HPLC using a Phenomenex Prodigy ODS (C 18 (44,45), were done with bacterial membranes prepared from E. coli DH5␣-expressing P450 2A6 (or a mutant) and NADPH-P450 reductase. Membranes were dialyzed twice versus 25 volumes of 200 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA at 4°C, first for 90 min and then 14 h, to remove glycerol, which contains traces of HCHO and reduces the assay sensitivity.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Coumarin 7-Hydroxylation Activity of Cytochrome P450 2A6 using Random Mutagenesis

Kim

Guengerich

2005

Journal of Biological Chemistry

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Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k cat and K m ) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k cat /K m ) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher K m values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations. Cytochrome P4502 enzymes are the major catalysts involved in the oxidative metabolism of xenobiotic chemicals, a significant focus in the areas of toxicology, drug metabolism, and pharmacology (3-5). Human P450 2A6 was identified as the major coumarin 7-hydroxlylase in humans (6 -8), and this enzyme also plays an important role in the metabolism of many xenobiotics including coumarin, nicotine, and tobacco-specific nitrosamines (8 -12). Genetic polymorphisms have been identified, and their relevance to cancer risk has been proposed because of the variations in nicotine and N-nitrosamine metabolism (13-17). The oxidation of indoles by P450 2A6 has been characterized, and the new kinase inhibitors have been biosynthesized using P450 2A6 mutants (18 -22).Recently x-ray crystal structures have been reported for P450 2A6 bound with coumarin and methoxsalen (23). The active site of P450 2A6 is six times smaller than that of human P450 2C8 (23, 24), even smaller than that of bacterial P450 101A1 (25). The decrease in size of the active site in P450 2A6 relative to P450 2C8 is caused by repositioning of helix BЈ and helices F to H toward the active site. Large aromatic residues in the active site of P450 2A6 also reduce the volume of the substrate binding site.In recent years, random mutagenesis and high-thro...

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“…Some drugs are oxidized by P450 2A6 (7,22). P450 2A6 also catalyzes the oxidation of indoles (23,24), and we have used P450 2A6 mutants to synthesize new indirubins with activity as protein kinase inhibitors (25).…”

Section: P450mentioning

confidence: 99%

“…P450 2A6 was of interest because of the recently reported structure, the useful fluorescence properties and common use of coumarins as P450 substrates, and our inherent interest in the catalytic properties of P450 2A6 (8,(23)(24)(25)32). Rates of several steps were measured (Scheme 1).…”

Section: P450mentioning

confidence: 99%

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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Formation of Indigo by Recombinant Mammalian Cytochrome P450

Cited by 129 publications

References 18 publications

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Analysis of Coumarin 7-Hydroxylation Activity of Cytochrome P450 2A6 using Random Mutagenesis

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

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