ja8b00262_si_001.pdf (1.76 MB)
Stepwise O‑Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening
journal contribution
posted on 2018-03-06, 00:00 authored by Inchul Shin, Brett R. Ambler, Daniel Wherritt, Wendell P. Griffith, Amanda C. Maldonado, Ryan A. Altman, Aimin LiuHeme-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 1) or oxygen (probe 2) 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 2 in a significant quantity.
Analysis of this new product by HPLC coupled UV–vis spectroscopy,
high-resolution mass spectrometry, 1H NMR, 13C 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.