Structural Insights into Protein-Inhibitor Interactions in Human Tryptophan Dioxygenase.

Human tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two important targets in cancer immunotherapy. Extensive research has led to a large number of potent IDO inhibitors; in addition, 52 structures of IDO in complex with inhibitors with a wide array of chemical scaffolds have been documented. In contrast, progress in the development of TDO inhibitors has been limited.

Characterization of the structural determinants of the ubiquitin-dependent proteasomal degradation of human hepatic tryptophan 2,3-dioxygenase.

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ∼2.3 h, relative to an average t1/2 of ∼2-3 days for total liver protein.

Conformational Plasticity in Human Heme-Based Dioxygenases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, l-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures.

Structural Basis of Inhibitor Selectivity in Human Indoleamine 2,3-Dioxygenase 1 and Tryptophan Dioxygenase.

Indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are two of the only three heme-based dioxygenases in humans. They have recently been identified as key cancer immunotherapeutic drug targets. While structures of hIDO1 in complex with inhibitors have been documented, so far there are no structures of hTDO-inhibitor complexes available.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome oxidase.

Cytochrome oxidase (CO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CO. It is assigned to the P-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation.

Diaryl hydroxylamines as pan or dual inhibitors of indoleamine 2,3-dioxygenase-1, indoleamine 2,3-dioxygenase-2 and tryptophan dioxygenase.

Tryptophan (Trp) catabolizing enzymes play an important and complex role in the development of cancer. Significant evidence implicates them in a range of inflammatory and immunosuppressive activities. Whereas inhibitors of indoleamine 2,3-dioxygenase-1 (IDO1) have been reported and analyzed in the clinic, fewer inhibitors have been described for tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase-2 (IDO2) which also have been implicated more recently in cancer, inflammation and immune control.

Inhibition Mechanisms of Human Indoleamine 2,3 Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE).

Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae.

HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified.

A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s.

The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis.

Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O on the C atom of the L-Trp indole ring.

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure.

O-alkylhydroxylamines as rationally-designed mechanism-based inhibitors of indoleamine 2,3-dioxygenase-1.

Indoleamine 2,3-dioxygenase-1 (IDO1) is a promising therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. Recently important advances have been made in understanding IDO1's catalytic mechanism. Although much remains to be discovered, there is strong evidence that the mechanism proceeds through a heme-iron bound alkylperoxy transition or intermediate state.

Complete reaction mechanism of indoleamine 2,3-dioxygenase as revealed by QM/MM simulations.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been recognized as a potent anticancer drug target, a fact that triggered intense research on the reaction and inhibition mechanisms of hIDO. Our recent studies revealed that the dioxygenase reaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C(2)═C(3) bond of Trp to form a ferryl and Trp-epoxide intermediate, via a 2-indolenylperoxo radical transition state.

Molecular basis for the substrate stereoselectivity in tryptophan dioxygenase.

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine. While human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. In this work, we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO.

Structural determinants for the formation of sulfhemeprotein complexes.

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H(2)O(2) or O(2) in the presence of H(2)S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H(2)S molecule. This moiety precedes and triggers the sulfheme formation.

The first step of the dioxygenation reaction carried out by tryptophan dioxygenase and indoleamine 2,3-dioxygenase as revealed by quantum mechanical/molecular mechanical studies.

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of L: -tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO.

Evidence for a ferryl intermediate in a heme-based dioxygenase.

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.