Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening

Article abstract of DOI:10.1021/jacs.8b00262

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.

Full text of DOI:10.1021/jacs.8b00262

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Stepwise O-Atom Transfer in Heme-based Tryptophan Dioxygenase:
The Role of Substrate Ammonium in the Epoxide Ring Opening
Inchul Shin, Brett R. Ambler, Daniel Wherritt, Wendell P.
Griffith, Amanda C. Maldonado, Ryan A. Altman, and Aimin Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00262 • Publication Date (Web): 06 Mar 2018
Downloaded from http://pubs.acs.org on March 6, 2018
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Stepwise O-Atom Transfer in Heme-based Tryptophan Dioxygenase:
The Role of Substrate Ammonium in the Epoxide Ring Opening
Inchul Shina, Brett R. Amblerb,†, Daniel Wherritta, Wendell P. Griffitha, Amanda C. Maldonadob,‡, Ryan
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A. Altmanb, and Aimin Liua,*
aDepartment of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249, United States;
bDepartment of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045, United States
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires keywords, provide
significant keywords to aid the reader in literature retrieval.
ABSTRACT: Hemeꢀbased tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomediꢀ
cal interest. Here, we synthesized two mechanistic probes to specifically test if the αꢀamino group of the substrate directly particiꢀ
pates in a critical step of the Oꢀatom transfer during catalysis in human tryptophan 2,3ꢀdioxygenase (TDO). Substitution of the niꢀ
trogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first Oꢀatom transfer
such that transferring the second Oꢀatom becomes less likely to occur, though 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
1
HPLC coupled UVꢀVis spectroscopy, highꢀresolution mass spectrometry, HꢀNMR, 13CꢀNMR, HSQC, HMBC and infrared (IR)
spectroscopies concluded that this monooxygenated product is a furopyrrole compound derived from an unstable epoxyindole inꢀ
termediate. These results prove that small molecules can manipulate the stepwise Oꢀatom transfer reaction of TDO and provide a
showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrateꢀ
based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the
substrate αꢀamino group in the epoxide ring opening step during catalysis. This combined synthetic, biochemical and biophysical
study establishes the catalytic role of the αꢀamino group of the substrate during the Oꢀatom transfer reactions of and thus represents
a
substantial advance to the mechanistic comprehension of the hemeꢀbased tryptophan dioxygenases.
INTRODUCTION
which makes this enzyme a promising drug target for developꢀ
ing cancer immunotherapies.4
Tryptophan is an essential but also the least abundant amino
acid. The vast majority of tryptophan not reserved for protein
synthesis takes a route for degradation through the kynurenine
pathway for energy metabolism and also for providing a pool
of metabolites including quinolinic acid for de novo nicotinaꢀ
mide adenine dinucleotide (NAD) biosynthesis.1 The first and
committed step of the kynurenine pathway is carried out by
two heme dioxygenases, tryptophan 2,3ꢀdioxygenase (TDO,
EC 1.13.11.11) and the homolog enzyme indoleamine 2,3ꢀ
dioxygenase (IDO, EC 1.13.11.17) in two isoforms. TDO is a
homotetrameric protein with high substrate specificity for Lꢀ
Trp (Figure 1). This protein is mostly found in the liver and
also present at low expression levels in other tissues. IDO is a
monomeric enzyme with broader substrate specificity and is
located in nonꢀhepatic tissues. Both enzymes utilize a monoꢀ
nuclear bꢀtype heme catalyzing the same tryptophan oxidation
reaction by inserting molecular dioxygen to cleave the pyrrole
ring, forming Nꢀformylkynurenine (NFK) (Scheme 1).2 Of
note, TDO served as one of the first demonstrations of the
incorporation of both O atoms of molecular oxygen into an
organic substrate by an enzyme.3 Other than mediating tryptoꢀ
phan degradation, IDO and TDO are also the representative
metabolic enzymes used in mammals to maintain proper amꢀ
plitude and duration of immune responses and prevent unreꢀ
strained immune activation in healthy cells. However, maligꢀ
nant tumors recruit this mechanism for immune evasion,
The mechanism of TDO/IDO has been long debated
for either a Criegeeꢀtype rearrangement, implicated dioxetane
pathway, or a novel epoxyindoleꢀbased mechanism (Scheme
S1).5ꢀ12 In the past decade, mechanistic studies have focused
on the differentiation between a stepwise (through an epoxyꢀ
indole) or a concerted (through a dioxetane) oxygen insertion
mechanism. Recently, compelling evidence from multiple
groups support the stepwise oxygen insertion mechanism.8ꢀ9, 11,
13ꢀ14, 15,16ꢀ17
Of note, the model shown in Scheme 1 is a novel mechanism
in heme chemistry. It predicts an alkylperoxo radical intermeꢀ
diate followed by an epoxyindole and a histidineꢀligated comꢀ
pound IIꢀlike species (an oxoferryl heme). Interestingly, the Oꢀ
atom transfer from the compound IIꢀlike species to the epoxyꢀ
indole intermediate predicts the involvement of the αꢀamino
group of the tryptophan substrate. Such a highꢀvalent intermeꢀ
diate has been observed in the catalytic pathway of IDO8, 13
and the enzyme reactivation process of TDO.17 The possible
participation of the ammonium group in catalysis was initially
suggested from studies of dyeꢀsensitized photooxygenation of
tryptophan in model studies.18 This interaction was later echꢀ
oed by EPR/ENDOR and computational studies leading to a
proposal that the ammonium group stabilizes the Fe(III)ꢀ
superoxide in the initial reaction steps of the catalytic cycle.9,
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Scheme 1. Mechanistic model of stepwise oxygen insertion via an epoxyindole intermediate
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14
In particular, quantum mechanics/molecular mechanics
results indicate the crucial role of the ammonium group in
proton transfer assisted epoxyindole ring opening and strongly
supports the stepwise oxygen insertion mechanism via the
(QM/MM) simulations have suggested that the ammonium
epoxyindole intermediate and compound II pathway.
MATERIALS AND METHODS
Organic synthesis of probe 1 and probe 2. Methylꢀ and hyꢀ
droxylꢀcontaining tryptophan analogs probe 1 and probe 2
were prepared by adapting previously reported protocols (see
Supporting Information Scheme S2 and the associated text for
synthetic routes and characterization details).19ꢀ21 Methylꢀ
based analog probe 1 was synthesized from indoleꢀ3ꢀ
carboxaldehyde using a threeꢀstep sequence involving Wittig
olefination, reduction, and hydrolysis.19 Hydroxylꢀcontaining
analog probe 2 was generated from indole using a SnCl4ꢀ
mediated epoxide opening followed by saponification.20 The
detailed chemical analysis and spectroscopic characterization
of these probes are included in Supporting Information.
Figure 1. The interaction of LꢀTrp with the
active site of human TDO with molecular
oxygen based on the crystal structure (PDB
entry: 5TI911)
Enzymatic reaction. The cloning, expression, and purificaꢀ
tion of human TDO has been described in a previous report.22
The enzyme assays were conducted using a modified proceꢀ
dure published previously.16ꢀ17 In a typical reaction for product
analyses and structural identification, 1 mM of each mechanisꢀ
tic probe and Lꢀtryptophan were reacted with 50 ꢁM of puriꢀ
fied human TDO at room temperature for a day. The enzyme
reaction mixtures were filtered using 10 kDa molecular weight
cut off centrifugal filter unit (Millipore). Filtered samples were
analyzed using Thermo Scientific Ultimateꢀ3000SD HPLC
rapid separation system equipped with photodiode array detecꢀ
tor. Samples injected were eluted using gradient mixing solꢀ
vent A, 0.1% FA in 90:10 water: acetonitrile and solvent B,
acetonitrile at the flowrate of 1.2 mL/min. InertSustain C18
column with the particle size of 5 ꢁm and the dimension of 4.6
I.D. × 100 mm (GL Sciences Inc.) was used for HPLC analyꢀ
sis and sample preparation for further MS and NMR analysis.
group in tryptophan is crucial for epoxyindole ring opening.9,
15
However, this possibility has not been explored experimenꢀ
tally. In particular, a substrate lacking the αꢀamino group, i.e.,
7
indole propionic acid , can function as an alternate substrate
of human IDO,13 which casts into question the role of the αꢀ
amino group in catalysis.
We hypothesized that if the proton transfer from the ammoniꢀ
um group of tryptophan is critical for opening the ring of the
epoxyindole intermediate, then replacing the ammonium
moiety with a substituent that is less able to donate a proton
will slow down the TDO enzyme reaction. In this study, we
probed the TDO mechanism by employing two synthesized
mechanistic probes (tryptophan analogs), 1 and 2 (Figure 2).
Here, we report a study of these probes with human TDO. The
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Inhibition mechanism study. The inhibition assays were
conducted in a buffer system containing 50 mM TrisꢀHCl, 200
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mM NaCl, 5% v/v glycerol (pH 8.0) at room temperature. The
reaction was initiated by adding 1 ꢁM enzyme to the mixture
containing 1 mM Lꢀascorbate and LꢀTrp (20 – 3200 ꢁM) in
the absence or presence of probe 1 or 2 at three different conꢀ
centrations. Steadyꢀstate formation of NFK was monitored at
321 nm (extinction coefficient, 3752 Mꢀ1cmꢀ1) using a Perkin
Elmer Lambda 25 UVꢀvisible spectrophotometer.
The data were analyzed using OriginPro (OriginLab, Northꢀ
ampton, Massachusetts, USA). Global fittings were conducted
across the datasets using the following equation:
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1
ꢆꢇꢈ
ꢉ
1
ꢆꢇꢈ
1
ꢂ ꢃꢄ1 ꢅ
ꢋ
ꢌ ꢑ
ꢉ 1 ꢍꢎꢏꢐ ꢆꢒꢈ
ꢅ ꢄ1 ꢅ
ꢋ
ꢀꢁ
ꢉ 2 ꢍꢎꢏꢐ
ꢊ
ꢊ
in which parameters, kcat, Km, Ki1, and Ki2 are shared.
Figure 2. Chemical structure of the synthetic probes and
comparison of reaction rates of human TDO with LꢀTrp
and the probes. With 200 ꢁM of LꢀTrp, the NFK forꢀ
mation by 1.0 ꢁM TDO was complete within 150 s. Conꢀ
versely, at 200 ꢁM, both probes 1 and 2 reacted ca. 500ꢀ
fold slower under the same conditions. The inset shows
the chemical structure of 1 and 2.
NMR spectroscopy. All NMR spectra were recorded on a
Bruker (Billerica, MA) Avance III HD 500 MHz spectrometer
equipped with a 5ꢀmm Prodigy Probe at 303 K running Topꢀ
Spin 3.5 pl 6. Spectra were recorded in D2O. One dimension
1H (pulse sequence: zg30) spectra were recorded with 1 s reꢀ
laxation delays, 64 k data points, and multiplied with an expoꢀ
nential function for a lineꢀbroadening of 0.3 Hz before Fourier
transformation. 13C (pulse sequence: udeft) spectra were recꢀ
orded with 10 s relaxation delays, 21k data points, and multiꢀ
plied with an exponential function for a lineꢀbroadening for 2
Hz before Fourier transformation. Multiplicity edited HSQC
(pulse sequence: hsqcedetgpsisp 2.3) spectra were acquired
with spectral widths of 16 ppm for 1H and 220 ppm for 13C, 2k
x 128 data points, and with a relaxation delay of 2 s. HMBC
(pulse sequence: hmbcetgpl3nd) spectra were acquired with
spectral widths of 13 ppm for H and 220 ppm for 13C, 4k ×
1
128 data points, and with a relaxation delay of 5 s. 1D gradient
selected NOESY (pulse sequence: selnogp) were recorded
with a mixing time of 300 ms, a relaxation delay of 2 s, and
multiplied with an exponential function for a lineꢀbroadening
of 1 Hz before Fourier transformation.
Mass Spectrometry. Mass spectra were collected on a maXis
plus quadrupoleꢀtime of flight mass spectrometer equipped
with an electrospray ionization source (Bruker Daltonics) and
operated in the negative ionization mode. Samples were introꢀ
duced via syringe pump at a constant flow rate of 3 ꢁL/min.
The critical source parameters are summarized as follows:
capillary voltage, 3500 V with a set end plate offset of –500
V; nebulizer gas pressure, 0.4 bar; dry gas flow rate, 4.0
L/min; source temperature, 200 °C. Mass spectra were average
of one minute of scans collected at a rate of 1 scan per second
in the range 50 ≤ m/z ≤ 1500. Compass Data Analysis software
version 4.3 (Bruker Daltonics) was used to process all mass
spectra.
IR spectroscopy. Infrared spectra were measured at a Shiꢀ
madzu FTIRꢀ8400S Fourier Transform Infrared Spectrometer
using KBr pellets.
RESULTS
Figure 3. LineweaverꢀBurk plots from the inhibitory asꢀ
says of (A) 1 and (B) 2
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Figure 4. Separation of reaction mixtures by HPLC. (A) Superimposed HPLC chromatograms obtained from separate singleꢀ
wavelength (black trace 280 nm; red trace; 294 nm; blue trace, 321 nm) detections for (a) LꢀTrp (control), (b) reaction mixꢀ
ture of LꢀTrp, (c) reaction mixture of 1, and (d) reaction mixture of 2. (B) Absorption properties of each of the fractions obꢀ
tained by an onꢀline diode array UVꢀvis spectrometer coupled with the HPLC system.
Mechanistic probes as alternative substrates and inhibi-
tors. At physiological pH, the ammonium group of LꢀTrp may
donate a proton if needed during catalysis. Two mechanistic
probes were designed by changing the αꢀamino group of the
native substrate to either a methyl group (1) or a hydroxyl
group (2) (Figure 2, inset). The methyl αꢀgroup of 1 would be
unable to donate a proton, and the αꢀhydroxyl group of 2 could
be a hydrogen bond donor or reluctant proton source. We synꢀ
thesized the two probes and performed biochemical analyses
with isolated human TDO.
exhibits cooperativity, and its heme centers are inequivalent.21
Additionally, this enzyme has a secondary LꢀTrp binding site
remote from the catalytic heme center for regulating the enꢀ
zyme stability.11, 22 As such, two distinct binding modes could
arise from binding to two inequivalent hemes or the regulating
site.
Product analysis by HPLC coupled to UV-vis spectrosco-
py. We analyzed the enzymatic reaction products by HPLC
with a diode array detector (Figure 4). The product of the naꢀ
tive substrate, NFK eluted 0.5 min earlier than LꢀTrp. The
peak at 7.2 min was identified as a decay product of NFK
(Figure S1, Table S1, Scheme S3 and Figure S2). A similar
spontaneous deamination product can be formed from NFK.23ꢀ
24 The peaks corresponding to LꢀTrp and NFK showed characꢀ
teristic absorption spectra at 280 and 321 nm, respectively.
Figure 4A shows the HPLC separations of three sets of experꢀ
iments on LꢀTrp, probes 1 and 2, monitored at 280, 294, and
321 nm, respectively, to maximize the opportunity to observe
potential products. The absorption spectrum of each identified
eluent was recorded by an onꢀline diodeꢀarray spectrometer
(Figure 4B). The enzyme reaction mixture of probe 1 showed
the unreacted probe 1 peak with the retention time of 8.5 min,
and a new peak (1a) eluted at 7.5 min. The species showed
similar spectral feature to that of NFK, suggesting a dioxygenꢀ
ated product of probe 1 was produced. Likewise, the HPLC
separation of the reaction mixture with probe 2 contained an
NFKꢀlike peak (2a) with the retention time of 6.3 min and
unreacted probe at 7 min. Intriguingly, the reaction mixture
also contained an additional new peak (2b) that was not obꢀ
served in the reaction with either probe 1 or LꢀTrp. This new
The ability for TDO to activate molecular oxygen using 1 and
2 as the substrate in place of LꢀTrp was assessed by following
product formation as we have previously done with 1ꢀmethylꢀ
Lꢀtryptophan with a bacterial TDO.16 After incubating with
TDO, each probe generated a new peak centered near 321 nm
similar to that observed from the reaction with LꢀTrp, suggestꢀ
ing that both probes are alternate substrates. The catalytic rate
using these probes as the substrate in place of LꢀTrp were
compared with LꢀTrp (Figure 2). The data showed that the
reaction with the probes proceeded at a much slower rate than
that with the native substrate.
Both probes were found to be reversible inhibitors of TDO.
The Ki values determined from the global fitting of the double
reciprocal plots were 320 ± 43 and 444 ± 32 ꢁM for 1 and 2,
respectively (Figure 3), indicating these are relatively weak
inhibitors. Although the plots show a mixed inhibition pattern,
these probes are predominately competitive inhibitors. The
secondary Ki values obtained are ca. 3.6 to 30ꢀfold higher than
the primary Ki values, which are higher than the Km values
obtained from the fitting. This inhibition pattern might derive
from the fact that TDO is a homotetrameric enzyme which
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containing the reaction products of probe 2 with the retention
peak eluted 0.3 min earlier than the NFKꢀlike peak and
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showed UVꢀvis absorption spectra at 294 nm, which was disꢀ
tinct from that of LꢀTrp, probe 1, probe 2 and their correꢀ
sponding NFK products (Figure 4B).
time of 6.0 min and 6.3 min showed increases of 16 and 32
Da, respectively, suggesting the monoꢀ (2b) and dioxygenated
(2a) products of probe 2 (Figure 5, C, D, and E). Theoretical
and experimentally measured accurate masses are summarized
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High-resolution MS analysis of the reaction products prov-
ing O-atom transfer to the probes. Each eluent from the
HPLC separation was further analyzed by highꢀresolution MS.
The fraction containing probe 1 reaction product with the reꢀ
tention time of 7.5 min showed an increase of 32 Da (1.8 ppm
mass accuracy) compared to the unreacted compound, indicatꢀ
ing 1a, the dioxygenated product of probe 1 (Figure 5 A and
B, range 190 ≤ m/z ≤ 250 shown), was produced. Fractions
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Figure 6. NMR analysis of 2b. (A) 500 MHz 1H NMR in
D2O, (B) 1Dꢀselective NOESY spectrum, (C) 125 MHz
13C NMR, (D) HSQC spectrum, CH and CH3’s red, CH2’s
blue, and (E) HMBC spectrum
Scheme 2. Summary of reaction products of probe 2
Figure 5. Negative ion ESI mass spectra of the LC fracꢀ
tions containing (A) 1, (B) 1a, (C) 2, (D) 2b, and (E) 2a.
Tandem mass spectra confirming the identity of (F) 1a, (G)
2b, and (H) 2a. Parent ions are indicated with ●.
Table 1. Calculated and experimentally measured accurate
masses for probes 1, 2, and their oxygenated products deꢀ
tected by ESI mass spectrometry*.
in Table 1. Tandem MS analyses were also carried out, and the
chemical structures and the corresponding masses are shown
in Scheme S4. The results were consistent with a dioxygenated
product (1a) from probe 1 and two products, a monoꢀ (2b) and
a dioxygenated (2a) products from probe 2 (Figure 5, F, G,
and H). Together with the HPLCꢀcoupled UVꢀvis data, we
conclude that an NFKꢀlike dioxygenation product was proꢀ
duced on each of the mechanistic probes and a singleꢀoxygen
inserted product from 2.
Molecular m/z
formula
m/z
Mass
theoretical observed accuracy
(ppm)
1
C12H13NO2 202.0874
202.0872 ꢀ1.0
N/A N/A
1b** C12H13NO3 218.0823
1a
2
2b
2a
C12H13NO4 234.0772
C11H11NO3 204.0666
C11H11NO4 220.0615
C11H11NO5 236.0564
234.0767 ꢀ2.1
204.0660 ꢀ2.9
220.0612 ꢀ1.4
236.0564 0.0
*Ion species are [MꢀH+]ꢀ.
**1b is monooxygenated product of probe 1 which was not
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While IDO has a more relaxed substrate specificity than TDO,
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human TDO is more specific to the chemical structure of the
substrate. Thus, the test with human TDO for the tryptophan
analogꢀbased mechanistic probes as alternate substrates may
have a better chance to reveal the mechanism shared by these
hemeꢀdependent dioxygenases. With a dioxygenated product
observed from both probes as a result of multiple turnovers,
we would expect the TDO reaction to proceed through a pathꢀ
way that resembles the catalytic cycle with LꢀTrp.
Determining the chemical structure of the monooxygenat-
ed product by NMR and IR spectroscopies. Next, the
monooxygenated product 2b from the TDO reaction of probe
2 was analyzed by NMR to determine if it would be an epoxyꢀ
indole intermediate or a decayed derivative (Figure 6, addiꢀ
tional NMR data and analyses are included in Supporting In-
formation Table S2 and Figures S3ꢀS7). 1HꢀNMR spectra conꢀ
tained a unique signal at 5.46 ppm (labeled “11” in Figure
6A), which could correlate with an epoxyindole product.
However, the instability of the initial epoxyindole intermediate
might enable rearrangement to two other cyclized forms with
the same exact mass and same signal “11” in 1HꢀNMR
(Scheme 2). 1Dꢀselective NOESY on proton “2” determined
the stereochemistry of the newly formed stereocenter (Figure
6B). The signal of the proton “11” indicates both protons “11”
and “2” should be on the same side. 13CꢀNMR assigned or
listed all the 11 peaks of carbons (Figure 6C). Together with
1HꢀNMR, the edited HSQC and HMBC spectra were obtained.
The HSQC spectrum showed CꢀH bond connectivity except
for the four quaternary carbons at positions 4 (88.2 ppm), 5
(130.1 ppm), 10 (149.4 ppm), and 1 (178.6 ppm) (Figure 6D).
Finally, the HMBC data provides compelling evidence that the
asꢀisolated monooxygenated product, 2b, is a cyclized comꢀ
pound with a fiveꢀmembered ring (Figure 6E). The unique
cross peak between proton “11” and carbon at position 2 (78.6
ppm) proves its connectivity with 3ꢀbond correlation, which
rules out the epoxyindole itself. The lack of the cross peak
between proton “11” and carbon at position 1 (178.6 ppm)
also supports the structure with a new fiveꢀmembered ring
generated on the probe 2 (Scheme 2). A proposed mechanism
of PrnB in the pyrrolnitrin biosynthesis pathway also predicts
the similar fiveꢀmembered ring compound.25 Finally, our
NMR analysis revealed that the monooxygenated product 2b
NMR analysis revealed that monooxygenation of probe 2 genꢀ
erated a furoindoline, which presumably arose from isomerizaꢀ
tion of unstable epoxyindoline.32 The chemical shifts and splitꢀ
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ting patterns in the H and 13C NMR spectra were consistent
with furoindoline 2b and assignment was made with the assisꢀ
tance of HSQC NMR data. The identity of the monooxygenatꢀ
ed species as furoindoline 2b rather than an epoxyindoline was
supported by HSQC NMR data, which revealed a 3ꢀbond corꢀ
relation between the proton at position 11 and the carbon at
position 2. Further, the relative stereochemistry was assigned
based on 1D selective NOESY, which showed that protons at
positions 2 and 11 resided on the same face of the tetrahydroꢀ
furan ring system. The unambiguous detection of a monooxyꢀ
genated product from probe 2 delivers compelling experiꢀ
mental evidence indicating that the Oꢀatom transfer takes
place stepꢀbyꢀstep, ruling out the dioxetane mechanism.
The formation of an epoxyindole species as an intermediate
has been implicated from a resonance Raman study8 and the
LCꢀMS detection of a minor cyclic aminoacetal byproduct.33
Together with the aminoacetal byproduct from LꢀTrp, the
presence of 2b from the monooxygenation of probe 2 through
the proposed mechanism suggested in Scheme 3 strongly supꢀ
port an epoxyindole intermediate predicted in the stepwise
oxygen insertion mechanism.
At physiological pH, tryptophan is zwitterionic. Hence, the
roles of the ammonium group in the mechanism was first proꢀ
posed by Nakagawa and colleagues in a model study in
1977,18 which, unfortunately, received little attention in the
ensuing 30 years. This proposal was reintroduced and further
from probe
2
is (2S,3aS,8aR)ꢀ3aꢀhydroxyꢀ3,3a,8,8aꢀ
tetrahydroꢀ2Hꢀfuro[2,3ꢀb]indoleꢀ2ꢀcarboxylic acid.
The monooxygenated product 2b from probe 2 was further
analyzed by infrared (film) and the spectral features were obꢀ
served at 3352, 2924, 2852, 1611, 1592, 1484, 1430, 1330,
1301, 1257, 1195, 1122, 1108, 1031, 998, 971, 943, 881, 861,
800, 748, 700, and 592 cmꢀ1 (Figure S8). The IR signal at
~1600 is indicative of an acyclic carboxylate, which is conꢀ
sistent with the proposed structure shown in Scheme 2. A proꢀ
posed pathway leading to the monooxygenated product 2b
from the putative epoxide intermediate of probe 2 is shown in
Scheme 3.
developed by two independent computational studies, a moꢀ
15
lecular dynamics and hybrid QM/MM methods7,
and an
ONIOM study,9 in 2010ꢀ2012. However, the proposed role of
the ammonium group from these computational studies is diꢀ
verse, and there is so far no experimental evidence to support
the proposed function directly. The results described in our
synthetic probe study, namely the 500ꢀfold decrease in reacꢀ
tion rate upon substitution of the amino group and the identifiꢀ
cation of the detected furoindole compound 2b, provide strong
experimental support for the participation of the αꢀamino
group of the substrate in catalysis.
DISCUSSION
Key NMR assignments and evidence for the ammonium
involvement in catalysis. TDO is an enzyme discovered some
82 years ago,26 yet it is one of the hottest inhibition targets for
drug development.4, 27ꢀ31 The mechanistic understanding of its
action will potentially help designing intermediateꢀ or transiꢀ
tion stateꢀbased analogous to effectively inhibit this enzyme.
Dioxygneation vs. monooxygenation—the determining
factor. The substitution of the nitrogen atom of the substrate
ammonium group to oxygen sufficiently reduces the efficiency
of the Oꢀatom transfer from the compound IIꢀlike species to
the epoxyindole intermediate, such that it reaches the limit for
Scheme 3. Proposed mechanism of the formation of the observed monooxygenated product 2b from reaction of probe 2
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the acidꢀcatalyzed ringꢀopening of the epoxyindole at the later
Among these prodrugs, only one is a TDO inhibitor (i.e., inꢀ
dole LM10),28 primarily due to its stringent substrate specificiꢀ
ty. A thorough understanding of the catalytic pathway would
help design more synthetic compounds that target both IDO
and TDO.
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part of the catalytic pathway. Thus, both monooxygenated and
dioxygenated products are produced. A significant amount of
the monooxygenated intermediate is released to the solution
and its instability leads to the epoxyindole ringꢀopening prodꢀ
uct. Although the substituted hydroxyl group is less capable of
donating a proton to the epoxyindole, it may stabilize the
epoxyindole by Hꢀbonding interactions, making it longer lived
at the active site. Of note, a TDO homologues protein MarE in
the maremycin biosynthetic pathway in Streptomyces sp.
B9173, is reported to generate a monooxygenated product,
presumably via an epoxyindole intermediate.34 In contrast, the
carbonꢀsubstitution as shown in probe 1 cannot stabilize the
epoxyindole.
The stepwise oxygen insertion model is composed of two parts
(Scheme 1A). The first half starts with molecular dioxygen
becoming a bridging molecule between the heme iron and the
organic substrate. This could be accomplished by either an
electrophilic addition or an ironꢀbound superoxide radical
attack to the LꢀTrp, which requires further investigation deꢀ
spite extensive studies.7, 10, 38 The second half involves a histiꢀ
dineꢀligated ferrylꢀoxo (a compound IIꢀlike species)8, 13 and an
epoxyindole intermediate. The formation of a substrateꢀbased
epoxide intermediate has been found in other systems such as
nonꢀheme iron extradiol dioxygenase and Rieske dioxygenꢀ
ase.39ꢀ40 The formation of an epoxyindole intermediate accomꢀ
panied by a compound IIꢀlike species defines the key characꢀ
teristic of heme dioxygenase mechanism. The highꢀvalent Fe
species is responsible for oxygen insertion and the pyrrole ring
opening to complete the dioxygenation reaction. This part of
the reaction starts with the proton transferꢀassisted epoxyinꢀ
dole ring opening coupled with a nucleophilic ferryl oxygen
addition reaction.
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It is, however, intriguing that methyl substitution yields only a
dioxygenated product but not a detectable monooxygenated
product. The lack of monooxygenation product may arise from
the inability of the αꢀmethyl group to facilitate an acidꢀ
catalyzed regiospecific ringꢀopening of the epoxyindole, so
that a protein component at the distal pocket is eventually reꢀ
cruited for assisting the catalytic cycle. Likewise, the αꢀ
deaminated substrate, IPA, functions as an alternate substrate
of human IDO, though this substrate requires a long lag phase
(ca. 50 s) to form product and, in that case, also monooxygenꢀ
ated product 13. In the active site of TDO, the binding mode of
LꢀTrp is dictated by the interaction between the Lꢀamino acid
moiety and surrounding residues (Figure 1). Arg144 forms salt
bridge with the carboxyl group in a bidentate fashion. The
main chain amino group of Thr342 also involves the Hꢀ
bonding with the substrate carboxyl group. Thr342 and the
carboxyl group of heme interact with the ammonium group of
the substrate through Hꢀbonding. The substrate specificity of
TDO for LꢀTrp is accomplished by these interactions. In the
primary structure level, Arg144 and Thr342 are also conserved
in the IDO system. Recently, the crystal structure of hIDO in
complex with LꢀTrp and cyanide as an oxygen surrogate beꢀ
came available.37 The structure revealed that the binding mode
of LꢀTrp in hIDO is almost identical as hTDO. This is quite
surprising because broader substrate specificity of IDO has
been believed based on a more relaxed binding pocket than
hTDO, lacking the tight nonꢀcovalent interaction that allows
only LꢀTrp in TDO active site.11, 35ꢀ36 Identical substrate bindꢀ
ing mode of LꢀTrp between TDO and IDO suggests similar
oxygen insertion mechanisms but different kinetic details. IPA
and probe 1, which both lack the αꢀamino group, would suffer
from poor binding to TDO active site because they do not
provide a hydrogen bonding partner to Thr342 and the heme
carboxyl group in TDO. Since the insertion of two oxygen
atoms is observed for IPA and probe 1, another proton source
other than ammonium group and its substitution to hydroxyl
group might be involved in the epoxyindole ring opening. The
loss of a functionally important atom could cause a change in
rateꢀlimiting step so that the accumulation of a monooxygenꢀ
ated intermediate is affected.
From a mechanistic perspective, the dioxygenation mechanism
of TDO and IDO does not share the wellꢀestablished strategy
derived from the studies of the cytochrome P450 enzyme suꢀ
perfamily, which invokes a thiolꢀligated ferrylꢀoxo porphyrin
π cation radical (FeIV=O[porphyrin⋅+]) known as compound I.
Two oxidizing equivalents (relative to the initial ferric state of
the enzyme) are placed on the heme cofactor in the compound
I type of oxidant.41 Instead, a compound IIꢀlike species is the
key oxidant and it is responsible for performing the critical Oꢀ
atom transfer to the organic substrate. The compound IIꢀlike
species is oneꢀelectron more reduced than compound I. Thereꢀ
fore, it was once thought that the compound II species might
be incapable of oxygen insertion. However, P450 enzyme
reaction starts from the ferric species and obtains electrons
from both the organic substrate and NAD(P)H, whereas in the
hemeꢀbased dioxygenases the ferrous, not the ferric, form is
the catalytic active form. Thus, the compound IIꢀlike species
in tryptophan dioxygenases indeed carries two oxidizing
equivalents (relative to its catalytic active ferrous state) and is
apparently competent for performing an Oꢀatom transfer. A
surprising example of Compound II reactivity is found in the
hemeꢀthiolate peroxygenase APO.42 The ferryl hydroxide form
of APOꢀCompound II shows high reactivity toward benzylic
CꢀH bond with the bond dissociation energy up to 85
kcal/mol. In the P450 mechanism, the resting enzyme accepts
electrons and protons from NAD(P)H and undergoes an OꢀO
bond cleavage to generate the compound I species. After the
intended oxidation to an organic substrate, the P450 enzyme
returns to the initial ferric, but not ferrous, state. Similarly,
catalases, peroxidases, peroxygenases, and some other hemeꢀ
dependent enzymes employ an adapted compound Iꢀbased
strategy. Hence, from a bioinorganic perspective, the mechaꢀ
nism illustrated in Scheme 1 is distinct and thus deserves more
attention.
Although the present work provides evidence to support only a
specific step of the catalytic cycle, it helps refine the catalytic
pathway of this group of dioxygenases, whose mechanismꢀ
based inhibitors are much needed. After several decades of
considerable efforts of screening and biochemical studies, only
a few compounds have entered preclinical & Phase I trials to
target immune escape of cancers via IDO/TDO inhibition.
Concluding remarks. By using the ammonium ion of the
substrate as an independent variable in a mechanistic pursuit,
we characterized the monoꢀoxygenated product of an NꢀtoꢀO
atom substituted probe in the reaction with human TDO. Diꢀ
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1.
Stone, T. W.; Darlington, L. G., Nat. Rev. Drug. Discov.
rect product analysis and structural determination revealed that
the product is derived from the proposed epoxyindole interꢀ
mediate, thereby proving the catalytic function of the substrate
αꢀamino group and corroborating the stepwise oxygen inserꢀ
tion mechanism for heme dioxygenase. In the TDOꢀcatalyzed
reaction, the enzyme utilizes not only the heme center and
protein residues, but also the substrate itself in the mechanism
of oxidation.
2002, 1, 609ꢀ20.
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ABBREVIATIONS
7.
Capece, L.; LewisꢀBallester, A.; Batabyal, D.; Di Russo,
TDO, tryptophan 2,3ꢀdioxygenase; IDO, indoleamine 2,3ꢀ
dioxygenase; NFK, Nꢀformylkynurenine; IPA, indole propionic
acid.
N.; Yeh, S.ꢀR.; Estrin, D. A.; Marti, M. A., J. Biol. Inorg. Chem.
2010, 15, 811ꢀ823.
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LewisꢀBallester, A.; Batabyal, D.; Egawa, T.; Lu, C.; Lin,
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Acad. Sci. U. S. A. 2009, 106, 17371ꢀ6.
ASSOCIATED CONTENT
9.
K., J. Am. Chem. Soc. 2010, 132, 11993ꢀ2005.
10. Makino, R.; Obayashi, E.; Hori, H.; Iizuka, T.; Mashima,
K.; Shiro, Y.; Ishimura, Y., Biochemistry 2015, 54, 3604ꢀ3616.
11. LewisꢀBallester, A.; Forouhar, F.; Kim, S. M.; Lew, S.;
Chung, L. W.; Li, X.; Sugimoto, H.; Shiro, Y.; Morokuma,
Supporting Information
The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
Scheme S1, providing the mechanistic models of both
stepwise and concerted oxygen insertion. Scheme S2
and associated text, providing the details of chemical
synthesis and characterization of the molecular probes
of 1 and 2. Figure S1, Table S1, Scheme S3 and Figure
S2, providing the identification of the peak at 7.2 min in
Figure 4Ab. Scheme S4, providing the chemical strucꢀ
tures and masses for the tandem mass spectrometry
analysis. Tables S2, providing the key numbers of NMR
shifts; Figures S3−S8, providing the NMR and IR specꢀ
troscopic characterization of the TDO reaction products
from the synthetic probes (PDF).
Wang, Y.; Karkashon, S.; Seetharaman, J.; Batabyal, D.; Chiang, B.
Y.; Hussain, M.; Correia, M. A.; Yeh, S. R.; Tong, L., Sci. Rep. 2016,
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Davydov, R. M.; Chauhan, N.; Thackray, S. J.; Anderson,
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Capece, L.; LewisꢀBallester, A.; Yeh, S. R.; Estrin, D. A.;
AUTHOR INFORMATION
17.
Fu, R.; Gupta, R.; Geng, J.; Dornevil, K.; Wang, S.; Zhang,
Corresponding Author
Y.; Hendrich, M. P.; Liu, A., J. Biol. Chem. 2011, 286, 26541ꢀ26554.
* Feradical@utsa.edu
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Nakagawa, M.; Watanabe, H.; Kodato, S.; Okajima, H.;
Hino, T.; Flippen, J. L.; Witkop, B., Proc. Natl. Acad. Sci. U. S. A.
1977, 74, 4730ꢀ3.
ORCID
Aimin Liu: 0000ꢀ0002ꢀ4182ꢀ8176
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Matsuda, K.; Toyoda, H.; Nishio, H.; Nishida, T.; Dohgo,
M.; Bingo, M.; Matsuda, Y.; Yoshida, S.; Harada, S.; Tanaka, H.;
Komai, K.; Ouchi, S., J. Agr. Food Chem. 1998, 46, 4416ꢀ4419.
Current address:
† Department of Chemistry, University of Texas at Austin;
20.
Deechongkit, S.; You, S. L.; Kelly, J. W., Org. Lett. 2004,
‡
Department of Medicinal Chemistry and Pharmacognosy, Uniꢀ
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Gupta, R.; Fu, R.; Liu, A.; Hendrich, M. P., J. Am. Chem.
versity of Illinois at Chicago
Soc. 2010, 132, 1098ꢀ109.
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Ferreira, P.; Shin, I.; Sosova, I.; Dornevil, K.; Jain, S.;
Notes
Dewey, D.; Liu, F.; Liu, A., Mol. Genet. Metab. 2017, 120, 317ꢀ324.
The authors declare no competing financial interest.
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Invest. Ophthalmol. Vis. Sci. 1999, 40, 3237ꢀ44.
24. Tomek, P.; Palmer, B. D.; Kendall, J. D.; Flanagan, J. U.;
Ching, L. M., Biochim. Biophys. Acta 2015, 1850, 1772ꢀ80.
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Bova, L. M.; Wood, A. M.; Jamie, J. F.; Truscott, R. J.,
ACKNOWLEDGMENT
This work was supported in whole or part by the National Instiꢀ
tutes of Health (NIH) grants GM108988 (to A.L.) and GM124661
(to. R.A.A.), and the Lutcher Brown Distinguished Chair Enꢀ
dowment fund (to A.L.). The mass spectrometry facility was
sponsored by National Institutes of Health Grant G12MD007591.
The NMR spectrometer used at UTSA is a shared instrument
sponsored by the National Science Foundation (NSF) under the
award number #1625963. Support for the NMR instrumentation at
KU was provided by NSF Academic Research Infrastructure
Grant No. S10RR024664 and NSF Major Research Instrumentaꢀ
tion Grant No. 0320648. The contents of this publication are soleꢀ
ly the responsibility of the authors and do not necessarily repreꢀ
sent the official views of National Institutes of Health.
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Kotake, Y.; Masayama, I., Z. Physiol. Chem. 1936, 243,
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