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

ABSTRACT: 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 O2 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 O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases.

SUBMITTER: Lewis-Ballester A

PROVIDER: S-EPMC5071832 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Lewis-Ballester Ariel A Forouhar Farhad F Kim Sung-Mi SM Lew Scott S Wang YongQiang Y Karkashon Shay S Seetharaman Jayaraman J Batabyal Dipanwita D Chiang Bing-Yu BY Hussain Munif M Correia Maria Almira MA Yeh Syun-Ru SR Tong Liang L

Scientific reports 20161020

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<sub>2</sub> 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 dioxygen ...[more]

PMID: 27762317

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Project description:Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.

Dataset Information

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

Publications

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

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