# Monooxygenase Activity of Indoleamine 2,3-Dioxygenase

## TL;DR

This paper shows that the enzyme IDO can perform two types of reactions, which could impact immune responses and drug development.

## Contribution

The discovery of a monooxygenase activity in IDO alongside its known dioxygenase function.

## Key findings

- Human IDO exhibits monooxygenase activity, forming a cyclic HPIC species from l-Trp.
- The dual reactivity of IDO is observed in HeLa cells overexpressing hIDO.
- Substrate plasticity in the active site of hIDO influences the reaction pathway.

## Abstract

Indoleamine 2,3-dioxygenase
(IDO) is a heme-dependent enzyme that
catalyzes the first, rate-limiting step of the kynurenine pathwaythe
oxidation of l-tryptophan to N-formylkynurenine
(NFK). IDO-catalyzed depletion of tryptophan levels and accumulation
of kynurenine pathway metabolites is an important control mechanism
of the immune responses in cells. IDO has been considered as a dioxygenase
because two atoms of oxygen are inserted into the substrate. Here,
we use LC-MS and NMR to examine the reactivity of human IDO (hIDO)
with l-tryptophan (l-Trp) and several other tryptophan
analogues. Alongside dioxygenase activity, we identify a concurrent
pathway of heme-dependent monooxygenase activity in the reaction of
hIDO with l-Trp, leading to the formation of a cyclic 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo­[2,3-b]­indole-2-carboxylic acid (HPIC) species. Reaction profiles
for the reaction of hIDO with other tryptophan analogues are likewise
examined. Formation of HPIC from l-Trp is reproduced in HeLa
cells induced to overexpress hIDO, indicating that this dual dioxygenase/monooxygenase
reactivity also occurs biologically. Notably, the reaction of hIDO
with β-[3-benzo­(b)­thienyl]-l-alanine (S-l-Trp)a
known inhibitor yielded only the cyclic HPIC analogue, suggesting
that IDO activity can be selectively directed toward the monooxygenase
pathway. Molecular dynamics simulations underscore the critical role
of substrate plasticity within the active site of hIDO, while DFT
calculations provide a mechanistic rationalization for the observed
product distributions. Together, the data demonstrate dual dioxygenase/monooxygenase
functionality for human IDO. As the overall gatekeeper for control
of tryptophan levels in cells, the findings provide mechanistic information
on relevance to therapeutic strategies focused on IDO inhibition.