Enzymatic Oxidation of Tryptophan and Its Analogues by Heme-Containing Dioxygenases

Abstract

The study of the mechanism of tryptophan oxidation by heme dioxygenase enzymes to produce
N-formylkynurenine (NFK) has been of wide interest because the reaction is the first ratelimiting step of the kynurenine pathway. In this work, oxidation of L-tryptophan and other
related substrates by human indoleamine-2,3-dioxygenases (hIDO) and X. campestris
tryptophan-2,3-dioxygenases (XcTDO) enzyme was examined to understand the distribution
of products generated by different enzymes and different substrates. The study began with
optimizing the concentration of ascorbic acid as a reducing agent for subsequent enzymatic
assays. Enzymatically catalyzed product formation was analyzed by UV-visible
spectrophotometry, LC-MS, 1H-NMR and HSQC. Additionally, the crystal structure of XcTDO
in complex with (β-[3-benzo(b)thienyl]-L-alanine (S-L-Trp) was resolved using X-ray
crystallography.
We successfully proved NFK formation on hIDO- and XcTDO-catalysed reactions with Ltryptophan, fully labelled 13C L-trytophan and L-trytophan (indole ring-2
13C). We also
improved NFK production using an oxygenated buffer, which resulted in a threefold increase
in NFK yield compared to the standard buffer. Beyond NFK, the hIDO-catalysed oxidation of
L-tryptophan produced cyclic products of hydroxypyrrolo indole carboxylic acid (HPIC).
Analogous cyclic products were observed in reactions with S-L-Trp and L-tryptophanol,
yielding 3a-hydroxy-2,3,3a,8a-tetrahydro-1H-benzo[4,5]thieno[2,3-b]pyrrole-2-carboxylic
acid (an HPIC analogue) and tetrahydropyranoindolol (ATPI) respectively. The substrate
flexibility within the active site of hIDO influenced the formation of these cyclic products.
Furthermore, the production of HPIC in the hIDO-catalysed reaction with L-tryptophan
suggests the potential of HPIC formation through hIDO activity in the human body. On the
other hand, the crystal structure of XcTDO in complex with S-L-Trp showed that S-L-Trp binds
favourably to the active site of XcTDO. However, despite this favorable binding, no enzymatic
products were detected in the reaction with S-L-Trp.
These findings, particularly the observation that hIDO can catalyse the formation of NFK and
cyclic products, provide an up-to-date understanding of the mechanism underlying hemecontaining dioxygenases catalysis.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Emma Raven (Supervisor) & Jonathan Clayden (Supervisor)