Shin, Inchul Ambler, Brett R. Wherritt, Daniel Griffith, Wendell P. Maldonado, Amanda C. Altman, Ryan A. Liu, Aimin Stepwise O‑Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe <b>1</b>) or oxygen (probe <b>2</b>) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe <b>2</b> in a significant quantity. Analysis of this new product by HPLC coupled UV–vis spectroscopy, high-resolution mass spectrometry, <sup>1</sup>H NMR, <sup>13</sup>C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases. O atom transfer reactions;O atom transfer;HPLC;TDO;substrate;monooxygenated product;product analysis results;1 H NMR;heme-based tryptophan dioxygenases;O atom transfer reaction;HSQC;Heme-based tryptophan dioxygenases;UV;probe 2;HMBC;catalysi;epoxide ring-opening step;Heme-Based Tryptophan Dioxygenase;13 C NMR;IR;α- 2018-03-06
    https://acs.figshare.com/articles/journal_contribution/Stepwise_O_Atom_Transfer_in_Heme-Based_Tryptophan_Dioxygenase_Role_of_Substrate_Ammonium_in_Epoxide_Ring_Opening/5987128
10.1021/jacs.8b00262.s001