Crystal structure of human indoleamine 2,3-dioxygenase: Catalytic mechanism of O
            <sub>2</sub>
            incorporation by a heme-containing dioxygenase

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...

mentioning

confidence: 99%

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Kuo

Mauk

2012

“…In comparison, IDO is monomeric, and shows activity toward a larger collection of substrates, including L-Trp, Dtryptophan (D-Trp), serotonin, and tryptamine (3), although the K m for D-Trp is Ϸ100-fold higher than that for L-Trp (13). The structure of human IDO in the catalytically inactive, ferric [Fe(III)]-heme state in complex with the 4-phenylimidazole inhibitor has recently been reported (14). Although this structure gave information about important active site residues, the inhibitor is coordinated to the heme iron and does not provide any information regarding Trp or oxygen binding.…”

mentioning

confidence: 99%

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

Forouhar

Anderson

Mowat

et al. 2007

169

284

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.cancer ͉ heme enzymes ͉ immunomodulation ͉ indoleamine 2,3-dioxygenase T ryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring, the first and rate-limiting step in L-Trp catabolism through the kynurenine pathway (1-3). In addition, IDO has been implicated in a diverse range of physiological and pathological conditions, including suppression of T cell proliferation, maternal tolerance to allogenic fetus, and immune escape of cancers (4-8), and is an attractive target for drug discovery against cancer and autoimmune and other diseases (2, 9-12).Despite catalyzing identical biochemical reactions (Fig. 1a), the sequence similarity between TDO and IDO is extremely low. An alignment of their sequences is only possible based on their structures, which suggests a sequence identity of 10% between them (Fig. 1b). In comparison, Xanthomonas campestris TDO shares 34% sequence identity with human TDO (Fig. 1b), demonstrating the remarkable evolutionary conservation of this enzyme. TDO is a homotetrameric enzyme and is highly specific for L-Trp and related derivatives such as 6-fluoro-Trp as the substrate. In comparison, IDO is monomeric, and shows activity toward a larger collection of substrates, including L-Trp, Dtryptophan (D-Trp), serotonin, and tryptamine (3), although the K m for D-Trp is Ϸ100-fold higher than that for L-Trp (13). The structure of human IDO in the catalytically inactive, ferric [Fe(III)]-heme state in complex with the 4-phenylimidazole inhibitor has recently been reported (14). Although this structure gave information about important active site residues, the inhibitor is coordinat...

“…The 3-indolenylperoxy intermediate subsequently converts to the product, NFK, via either a Criegee rearrangement or a dioxetane pathway. A major breakthrough in the understanding of the heme-based dioxygenase chemistry was made recently by the unveiling of the crystallographic structures of hIDO and 2 bacterial isoforms of TDO (23)(24)(25). Structure-based sequence alignment shows that the proximal histidine ligand and most of the critical distal residues involved in substrate-protein interactions in TDO are conserved in IDO (24,25).…”

mentioning

confidence: 99%

Evidence for a ferryl intermediate in a heme-based dioxygenase

Lewis-Ballester

Batabyal

Egawa

et al. 2009

118

217

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions. indoleamine 2,3-dioxygenase ͉ reasonance Raman spectroscopy ͉ tryptophan dioxygenase