Monooxygenase Activity of Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a heme-dependent enzyme that catalyses the first, rate-limiting step of the kynurenine pathway – the oxidation of L-tryptophan to N-formylkynurenine (NFK). IDO-catalysed 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 a number of other tryptophan analogues. Alongside dioxygenase activity, we identify a concurrent pathway of heme-dependent monooxygenase activity in the reaction of hIDO with LTrp, 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 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 towards 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 gate-keeper for control of tryptophan levels in cells, the findings provide mechanistic information of relevance to therapeutic strategies focused on IDO inhibition.