Histidine 55 of Tryptophan 2,3-Dioxygenase Is Not an Active Site Base but Regulates Catalysis by Controlling Substrate Binding

Thackray, Sarah J.; Bruckmann, Chiara; Anderson, J. L. Ross; Campbell, L.P.; Xiao, Ran; Zhao, Lili; Mowat, Christopher G.; Forouhar, F.; Tong, Liang; Chapman, Stephen K

doi:10.1021/bi801202a

Cited by 41 publications

(78 citation statements)

References 27 publications

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“…44 The first water molecule was found in the ligand binding site ~3.5A away from the heme iron, similar to that observed in the wild type protein. 21 This water molecule, like that in the wild type protein (see Figure 4), forms H-bonds with the ammonium and indole NH groups of the L-Trp, as well as the backbone NH group of the G125.…”

Section: Resultssupporting

confidence: 68%

Substrate−Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76

Batabyal

Yeh

2009

J. Am. Chem. Soc.

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The initial and rate-limiting step of the kynurenine pathway in humans involves the oxidation of tryptophan to N-formyl kynurenine catalyzed by two hemeproteins, tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). In hTDO, the conserved H76 residue is believed to act as an active site base to deprotonate the indole NH group of Trp, the initial step of the Trp oxidation reaction. In hIDO, this histidine is replaced by a serine. To investigate the role of the H76, we have studied the H76S and H76A mutants of hTDO. Activity assays show that the mutations cause a decrease in kcat and an increase in KM for both mutants. The decrease in the kcat is accounted for by the replacement of the active site base catalyst, H76, with a weaker base, possibly a water, whereas the increase in KM is attributed to the loss of the specific interactions between the H76 and the substrate as well as the protein matrix. Resonance Raman studies with various Trp analogs indicate that the substrate is positioned in the active site by the ammonium, carboxylate, and indole groups, via intricate H-bonding and hydrophobic interactions. This scenario is consistent with the observation that L-Trp binding significantly perturbs the electronic properties of the O2-adduct of hTDO. The important structural and functional roles of H76 in hTDO is underscored by the observation that the electronic configuration of the active ternary complex, L-Trp-O2-hTDO, is sensitive to the H76 mutations.

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Section: Resultssupporting

confidence: 68%

Substrate−Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76

Batabyal

Yeh

2009

J. Am. Chem. Soc.

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“…3a). It is noteworthy that although the deprotonation of the indoleamine group of the substrate does not appear to be essential for the dioxygenase reaction, the hydrogen bond between the indoleamine group of the substrate and H55 in xcTDO (or H76 in hTDO) could still be a critical factor in controlling substrate binding as well as catalysis, on the basis of two observations: (1) the substitution of the indoleamine with N -methyl in the substrate significantly diminishes dioxygenase activity in TDO [49] and (2) the mutation of H55 (in xcTDO) [50, 51] or H76 (in hTDO) [42] to Ala or Ser caused a significant increase in the K m value and a decrease in the k cat value.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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Tryptophan dioxygenase (TDO) and indole-amine 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. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C2=C3 bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371–17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states.

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“…20 The crystal structures of H55A and H55S variants from xcTDO in binary complexes with L-Trp were reported in a subsequent paper, which show that the aforementioned H-bonding interaction is abolished in the mutant proteins. 25 Since both His55 mutants exhibited detectable activity, the distal histidine was proposed to function as a transition-state stabilizer, rather than an essential base. 25 A detailed model for such a stabilization role still remains to be explored.…”

Section: ■ Introductionmentioning

confidence: 99%

Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

2012

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Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

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Histidine 55 of Tryptophan 2,3-Dioxygenase Is Not an Active Site Base but Regulates Catalysis by Controlling Substrate Binding

Cited by 41 publications

References 27 publications

Substrate−Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76

Substrate−Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76

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

Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

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