Inhibitory Substrate Binding Site of Human Indoleamine 2,3-Dioxygenase

Lu, Chong‐Dao; Lin, Yu; Yeh, Syun Ru

doi:10.1021/ja9029768

Cited by 79 publications

(163 citation statements)

References 20 publications

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“…Similarly, no appreciable electron transfer from Trp bound near the heme iron to the presumed IDO compound I intermediate occurs to initiate substrate oxidation in the peroxygenase reaction. Without more detailed structural or spectroscopic information, it seems likely that the proposed ability of Trp to bind to more than one location at the active site of IDO is relevant to these observations (7,56). Specifically, differences in the binding of Trp and indole to secondary sites at the active center of IDO may influence the binding orientations of both substrates in proximity to the heme iron and contribute significantly to the catalytic selectivity and the product distribution that we have observed.…”

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 NFK Green assay is a homogeneous mix-and-measure assay that avoids heating and addition of very acidic or toxic reagents, common for other IDO/TDO assays. [19][20][21][22][23] We Table S1.…”

Section: Discussionmentioning

confidence: 99%

“…One often-used approach is based on deformylation of NFK to kynurenine, followed by reaction with 4-(dimethylamino) benzaldehyde (pDMAB). [19][20][21] Unfortunately, pDMAB reacts with all compounds containing primary amines. 21 In one recent alternative assay format, kynurenine was linked to a chemosensor, such as sensor-1, to produce a new fluorophore.…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

et al. 2014

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Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384-or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

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“…This is because the racemization of d-Trp to l-Trp is weak (35,36) and the turnover number (kcat) of tryptophan 2,3-dioxygenase (TDO) is tenfold lower in d-Trp than in l-Trp in vitro (34,37). Conversely, the kcat of indoleamine 2,3-dioxygenase (IDO) for d-Trp and l-Trp is almost identical (38,39).…”

Section: Discussionmentioning

confidence: 99%

The Urinary Ratio of 3-Hydroxykynurenine/3-Hydroxyanthranilic Acid Is an Index to Predicting the Adverse Effects of D-Tryptophan in Rats

Shibata

Ohno

Sano

et al. 2014

J Nutr Sci Vitaminol

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SummaryThe adverse effects of d-tryptophan and the possibility of it being a surrogate index for predicting adverse effects in rats were investigated. Male rats were fed one of several test diets (20% casein diets with 0% (control), 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% d-tryptophan) for 21 d, and 24-h urine samples on the final day of the experiment were collected. Analyses of food intake and body-weight changes revealed adverse effects to be observed in the group fed the 0.3% d-tryptophan diet. We propose urinary levels of 3-hydroxykynurenine/3-hydroxyanthranilic acid to be surrogate indicators for predicting the adverse effects of d-tryptophan from the break point of body-weight gains and urinary levels of d-tryptophan metabolites. The reaction 3-hydroxykynurenine→3-hydroxyanthranilic acid is catalyzed by the pyridoxal phosphate-dependent enzyme kynureninase. Increasing urinary 3-hydrokykynurenine indicates kynureninase deficiency. Intake of d-tryptophan in rats fed the 0.3% d-tryptophan diet was 0.21 g/kg body weight and feeding of the 0.3% d-tryptophan diet did not elicit adverse effects. Thus, the safe level of d-tryptophan was less than 0.2% in the diet, 0.15 g/kg body weight, in rats.

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Inhibitory Substrate Binding Site of Human Indoleamine 2,3-Dioxygenase

Cited by 79 publications

References 20 publications

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

The Urinary Ratio of 3-Hydroxykynurenine/3-Hydroxyanthranilic Acid Is an Index to Predicting the Adverse Effects of D-Tryptophan in Rats

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