Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis<sup>,</sup>

Zhang, Yang; Kang, Seong A.; Mukherjee, Tathagata; Bale, Shridhar; Crane, Brian R.; Begley, Tadhg P.; Ealick, S.E.

doi:10.1021/bi0620095

Cited by 109 publications

(144 citation statements)

References 30 publications

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“…Expression and Purification of Ferric TDO-The construction of the plasmid encoding full-length Cupriavidus metallidurans TDO (CmTDO) has been described elsewhere (13). The protein was purified by using a 100-ml HiLoad nickel-affinity column and a Superdex 200 size-exclusion column on an Ä KTA FPLC system as described in an earlier spectroscopic study of the enzyme (24).…”

Section: Reagents-l-trpmentioning

confidence: 99%

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Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Gupta

Geng

et al. 2011

Journal of Biological Chemistry

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An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (␦ ‫؍‬ 0.055 mm/s and ⌬E Q ‫؍‬ 1.755 mm/s) and a protein-based free radical (g ‫؍‬ 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation byproducts by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.Hemoproteins perform a wide range of biological functions, including oxygen transport, storage, electron transfer, monooxygenation, and reduction of dioxygen. However, they rarely express dioxygenase activity as their native biological function. Tryptophan 2,3-dioxygenase (TDO) 3 is the first described exception (1-3). This enzyme employs a b-type ferrous heme prosthetic group to catalyze the oxidative cleavage of the indole ring of L-Trp, converting it to N-formylkynurenine (NFK) (Scheme 1). This is the first and rate-limiting step of the kynurenine pathway of L-Trp metabolism, which oxidizes over 99% of L-Trp in mammalian intracellular and extracellular pools (2, 4 -9). The kynurenine pathway constitutes the major steps in biosynthesis of NAD, an essential redox cofactor in all living systems (5).TDO is a hepatic enzyme first discovered in rat liver extracts in 1936 (1). An analogous enzyme, indoleamine 2,3-dioxygenase (IDO), was isolated 31 years later from tissues other than the liver (10). Although both enzymes catalyze the same reaction, TDO is highly substrate-specific with L-Trp, whereas IDO presents a more relaxed specificity. TDO is a homotetramer with a total mass of ϳ134 kDa, whereas IDO is a monomeric protein. The two enzymes share only 14% sequence identity but conserve similar active site architectures (11-13). In addition to humans, TDO has also been found in other mammals, such as rats and mice, as well as in mosquitoes and bacteria (2, 5, 14 -18). Recently, a potential heme-dependent dioxygenase enzyme superfamily has been proposed (19). Moreover, several other heme-based proteins, such as my...

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Section: Reagents-l-trpmentioning

confidence: 99%

“…TDO is a homotetramer with a total mass of ϳ134 kDa, whereas IDO is a monomeric protein. The two enzymes share only 14% sequence identity but conserve similar active site architectures (11)(12)(13). In addition to humans, TDO has also been found in other mammals, such as rats and mice, as well as in mosquitoes and bacteria (2, 5, 14 -18).…”

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confidence: 99%

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Gupta

Geng

et al. 2011

Journal of Biological Chemistry

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“…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).…”

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confidence: 99%

“…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). Spectroscopic studies suggested that the active site base for hTDO is an evolutionarily conserved residue, H76, while that in hIDO is the heme-bound dioxygen (see SI Text and Fig.…”

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confidence: 99%

Evidence for a ferryl intermediate in a heme-based dioxygenase

Lewis-Ballester

Batabyal

Egawa

et al. 2009

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

120

217

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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

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“…Apart from covering the top of the heme-binding site, the role of the noncatalytic small domain is unclear. Interestingly, TDO has a homotetrameric structure, in which each monomer, when aligned to IDO, appears to contain the large catalytic but not the small domain (31). Although not crystallized yet, the protein sequence of IDO2 suggests that it also possesses the small domain (29).…”

Section: Idos Itims and Evolutionmentioning

confidence: 99%

Different Partners, Opposite Outcomes: A New Perspective of the Immunobiology of Indoleamine 2,3-Dioxygenase

Orabona

Pallotta

Grohmann

2012

Mol Med

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Indoleamine 2,3-dioxygenase (IDO), a metabolic enzyme that catalyzes tryptophan conversion into kynurenines, is a crucial regulator of immunity. Altered IDO activity is often associated with pathology, including neoplasia and autoimmunity. IDO is highly expressed in dendritic cells (DCs) that exploit the enzyme's activity and the production of tryptophan catabolites to regulate immune responses by acting on several cell types, including T lymphocytes, of which they promote a regulatory phenotype. IDO also contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that, once bound by distinct molecular partners, will either promote degradation or initiate signaling activity and self-maintenance of the enzyme. We here discuss how ITIM-dependent molecular events can affect the functional plasticity of IDO by modifying the protein half-life and its enzymic and non enzymic functions.

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Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis^,

Cited by 109 publications

References 30 publications

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Evidence for a ferryl intermediate in a heme-based dioxygenase

Different Partners, Opposite Outcomes: A New Perspective of the Immunobiology of Indoleamine 2,3-Dioxygenase

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Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis,

Cited by 109 publications

References 30 publications

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Evidence for a ferryl intermediate in a heme-based dioxygenase

Different Partners, Opposite Outcomes: A New Perspective of the Immunobiology of Indoleamine 2,3-Dioxygenase

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Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis^,