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

Ariel Lewis-Ballester,Scott Lew,Farhad Forouhar,Syun-Ru Yeh,Shay Karkashon,Munif Hussain,Liang Tong,Bing-Yu Chiang,Dipanwita Batabyal,Sung-Mi Kim,Jayaraman Seetharaman,Maria Almira Correia,Yongqiang Wang

doi:10.1038/srep35169

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.

Highlights

Trp-O2 ternary complex obtained by mixing of ferrous hTDO-Trp with air-saturated solution in a stopped-flow system. (e) Difference spectrum, (c,d) which likely represents the spectrum of NFK-bound ferrous hTDO
The reaction between O2 and Trp in the active site was monitored by microscopic spectroscopy
A second binding site for L-Trp was observed in XcTDO at the tetramer interface[25] (Supplementary Fig. 3), while the exo site in hTDO is located within each subunit

Summary

Introduction

Trp-O2 ternary complex obtained by mixing of ferrous hTDO-Trp with air-saturated solution in a stopped-flow system (red). (e) Difference spectrum, (c,d) which likely represents the spectrum of NFK-bound ferrous hTDO (blue). No electron density for O2 was observed in crystals mounted directly in the glove box, and the structure of hTDO in this binary complex with L-Trp is mostly the same as that obtained following O2 exposure (Table 1).

Results

Conclusion