The mechanism of formation of N-formylkynurenine by heme dioxygenases

Abstract

Heme dioxygenases catalyze the oxidation of L-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O(2)-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with (18)O(2) confirm the origin of the oxygen atom as arising from O(2)-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed.

Scheme 1. Previous Proposals for Reaction Mechanism in the Heme Dioxygenases

(A) The reaction catalyzed by the heme dioxygenases. X = H for tryptophan and X = Me for 1-methyl-tryptophan. (B) Left: Previous proposal^, for the first step of the reaction mechanism, involving base-catalyzed abstraction. Right: A more recent proposal.^, (C) Proposed(1) Criegee (top, solid line) and dioxetane (bottom, dashed line) reaction mechanisms for formation of N-formylkynurenine.

Scheme 2. Proposed Mechanism for Formation of the Cyclic Amino Acetal (3a-Hydroxypyrroloindole-2-carboxylic acid, Species Y), Showing the Proposed Ring-Opening and Cyclization Mechanism

The species labeled X and Y have also been suggested(40) as intermediates in the PrnB mechanism (see Discussion).

Figure 1

Formation of NFK formed during O₂-dependent turnover by hTDO. LC–MS and LC–MS/MS analyses of the products obtained from the O₂-dependent reaction of hTDO with l-Trp in the steady state. (A) Elution profile for selected ion chromatogram with m/z = 221. The peak eluting at t = 8.58 min is kynurenine. l-Trp elutes at t = 13 min. (B) Corresponding positive ESI mass spectrum for the product eluted at 5.72 min, showing a peak at m/z = 221. (C) Elution profile for the same peak (at 5.60 min in this elution profile) and (D) corresponding MS/MS spectrum, showing the peak at 221 and a fragmentation pattern corresponding to the structure proposed in Scheme 2. (E) Positive ESI mass spectrum for the product eluted at 7.02 min, showing a peak at m/z = 237 corresponding to NFK formation. For all enzymes examined, the intensity of the 221 peak is invariably lower than that for the 237 peak, but we have been unable to extract quantitative time profiles from the mass spectrometric analyses.

Figure 2

Formation of NFK during dioxygenase-catalyzed turnover using ¹⁸O₂. LC–MS/MS analyses of the products obtained on reaction of hTDO with l-Trp and ¹⁸O₂ ((A) fragmentation pattern for ion with m/z = 223, (B) fragmentation pattern for ion with m/z = 241) and on reaction of hIDO with Me-Trp and ¹⁸O₂ ((C) fragmentation pattern for ion with m/z = 237, (D) fragmentation pattern for ion with m/z = 255).

Figure 3

Evidence for mixed incorporation of ¹⁸O and ¹⁶O in NFK in reactions with ¹⁸O₂. LC–MS/MS analyses of NFK (m/z = 239) carrying one atom of ¹⁶O and one atom of ¹⁸O from reaction of (A) hTDO with l-Trp and ¹⁸O₂ (¹⁶O¹⁸O-NFK) and (B) hIDO with Me-Trp and ¹⁸O₂ (¹⁶O¹⁸O-Me-NFK).

Figure 4

Analysis of oxygen exchange in NFK. LC–MS/MS analyses of the NFK product peak (m/z = 239) in reactions of (A) hTDO, (B) hIDO, and (C) xTDO with l-Trp and ¹⁸O₂ in H₂¹⁶O buffer; and LC–MS/MS analyses of the NFK product peak (m/z = 239) in reactions of hTDO (D) with l-Trp and ¹⁶O₂ in H₂¹⁸O buffer. For clarity of discussion in the Results, the two different carbonyl groups are differentiated using color (see text).

Scheme 3. Mechanistic Proposal for NFK Formation in the Heme Dioxygenases

Epoxide formation is envisaged by two possible routes, an electrophilic mechanism (step 1a) or by radical addition (step 1b): as we highlight in the Discussion, both routes can lead to formation of an epoxide, but neither mechanism has been definitely proven. Ring-opening is indicated in step 3 (leading to the species labeled as X in Scheme 2), but could be synchronous with step 4. Cleavage of the C²–C³ bond and formation of NFK is subsequently straightforward. For further details see text. R = CH₂CH(NH₃⁺)CO₂^–. The amine group of the side chain could potentially form a hydrogen bond to the O^– of the singly oxygenated intermediate.

Scheme 4. Comparisons of the Reactivity of the Dioxygenase and PrnB Enzymes

(A) The reaction catalyzed by PrnB.(40) (B) Comparison of possible reaction mechanisms in PrnB (right) and the dioxygenases (left). In both, formation of the species labeled X is implicated, but after that the mechanisms branch (the species labeled X and Y are the same as also shown in Scheme 2). In PrnB this leads to formation of the tricyclic (amino acetal) intermediate, without insertion of oxygen, whereas in the dioxygenases C²–C³ bond cleavage and further O atom insertion is the preferred route.