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Stepwise O atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening
Shin, Inchul Ambler, Brett R. Wherritt, Daniel Griffith, Wendell P. Maldonado, Amanda C. Altman, Ryan A. Liu, Aimin
Shin, Inchul
Ambler, Brett R.
Wherritt, Daniel
Griffith, Wendell P.
Maldonado, Amanda C.
Altman, Ryan A.
Liu, Aimin
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Abstract
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 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.
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This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/jacs.8b00262.
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2018-03-28
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American Chemical Society
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Shin, I., Ambler, B. R., Wherritt, D., Griffith, W. P., Maldonado, A. C., Altman, R. A., & Liu, A. (2018). Stepwise O atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening. Journal of the American Chemical Society, 140(12), 4372–4379. http://doi.org/10.1021/jacs.8b00262