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J. Biol. Chem., Vol. 278, Issue 32, 29525-29531, August 8, 2003
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From the
Australian Cataract Research Foundation,
Department of Chemistry and the Department of
Biological Sciences, University of Wollongong, Wollongong, New South Wales
2522, Australia
Received for publication, February 19, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, and can thereby
modulate the levels of tryptophan, which is vital for cell growth. To identify
which amino acid residues are important in substrate or heme binding in IDO,
site-directed mutagenesis of conserved residues in the IDO gene was
undertaken. Because it had been proposed that a histidine residue might be the
proximal heme ligand in IDO, mutation to alanine of the three highly conserved
histidines His16, His303, and His346 was
conducted. Of these, only His346 was shown to be essential for heme
binding, indicating that this histidine residue may be the proximal ligand and
suggesting that neither His303 nor His16 act as the
proximal ligand. Site-directed mutagenesis of Asp274 also
compromised the ability of IDO to bind heme. This observation indicates that
Asp274 may coordinate to heme directly as the distal ligand or is
essential in maintaining the conformation of the heme pocket. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Increased levels of the kynurenine pathway metabolites quinolinic acid and 3-hydroxykynurenine have been observed in a number of neurological disorders. Quinolinic acid, which is an N-methyl-D-aspartate agonist, may be involved in the pathogenesis of AIDS dementia complex (14–16), cerebral malaria, and Huntington's disease (17, 18). 3-Hydroxykynurenine readily oxidizes in air with the formation of H2O2, and the resulting oxidation products bind proteins (19–21). With the induction of IDO being implicated in several disease states, its suppression may be of potential pharmacological importance. To date there are no potent therapeutic inhibitors of IDO; however, an understanding of the structure of IDO will aid in the development of such compounds.
Heme proteins, such as IDO, are the most extensively studied of all metalloproteins. All heme proteins carry iron coordinated to protoporphyrin IX. The four coordination sites provided by the porphyrin ring are not sufficient to satisfy the coordination requirements of the iron. Normally, groups from the heme-binding protein occupy the remaining coordination sites. The common coordinating functional groups are the imidazole nitrogen of histidine, the phenoxide group of tyrosine, the sulfur of methionine and cysteine, and the carboxylate group of aspartic acid and glutamic acid. In both myoglobin and hemoglobin, the heme is attached to the protein through a histidine residue to the 5th coordination position on the iron (the proximal ligand). A second histidine (the distal ligand) occupies space immediately above the 6th coordination position of the iron, and coordination is through an oxygen molecule, not directly to the iron. Although only the primary structure of IDO has been determined (22, 23), there have been extensive equilibrium studies undertaken to investigate the binding properties of both substrate and oxygen with the additional objective of identifying the proximal and distal heme ligands.
Dioxygenases, such as IDO, catalyze the oxidative cleavage of a substrate
and, as such, both oxygen and the substrate need to bind to the enzyme. A
common property of oxygenases is that the binding of the substrate precedes
that of dioxygen, and this has been shown to be the case with tryptophan
2,3-dioxygenase (24). By
contrast, Hirata et al.
(25) found that IDO could bind
oxygen to form a relatively stable complex that has catalytic activity. In
later investigations it was determined that ferrous IDO binds
L-tryptophan, followed by molecular oxygen
(26). The following mechanism
was proposed for the IDO catalyzed conversion of tryptophan to
N'-formylkynurenine. (i) IDO-Fe2+ is
bound by the substrate. (ii) The IDO-Fe2+-tryptophan
complex is then bound by oxygen to form an
complex (ternary complex); and (iii) this complex is converted to
N'-formylkynurenine, releasing
IDO-Fe2+.
Other studies have used EPR, MCD, and CD spectroscopy to determine the properties of the IDO heme pocket and the nature of the proximal and distal heme ligands. MCD spectra of ferric and ferrous IDO closely resembled that of myoglobin and horseradish peroxidase (27–29); however, the environment of the IDO heme pocket is similar to, but the size is considerably larger than, the prototypical binding heme pocket of myoglobin. This observation is supported by data showing that the heme pocket of IDO allows access to the inhibitors norharman and 4-phenylpyridine (30).
On the basis of EPR studies, Shimizu et al. (31) proposed that the 5th (proximal) ligand of the heme was nitrogen, probably from a histidine imidazole ring. The 6th (distal) ligand in substrate-free IDO was proposed to be a histidine imidazole ring nitrogen by Uchida et al. (28, 29) and Sono and Dawson (27) based on MCD spectroscopy. Uchida et al. (28, 29) suggested that, in the substrate-bound ferric IDO, the 6th ligand was also a histidine (28, 29). This contrasted with Sono and Dawson (27), who showed that the observed changes in MCD upon substrate binding were most consistent with a hydroxide ion being the 6th ligand in substrate-bound ferric IDO (27). The findings suggested that both of the proximal and distal heme ligands in substrate free IDO were histidines, as in hemoglobin and myoglobin.
In this study, we report the use of site-directed mutagenesis to identify residues important in heme binding and enzymatic activity. Mutation of the three highly conserved histidines His16, His303, and His346 identified only His346 as essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His303 nor His16 act as the proximal ligand. Site-directed mutagenesis of Asp274 compromised the ability of IDO to bind heme. Thus, both His346 and Asp274 appear essential for the binding of heme and thus for IDO enzymatic activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-galactopyranoside (IPTG), ampicillin,
kanamycin and sodium dithionite were obtained from Sigma-Aldrich. Bovine serum
albumin (Fraction V) was obtained from Amersham Biosciences. All restriction
enzymes and the protease inhibitor mixture were obtained from Roche Applied
Science. The GeneEditorTM reaction kit was obtained from Promega, and the
Big Dye Terminator was obtained from PerkinElmer Life Sciences.
Bacterial Strains, Plasmids, and Culture Conditions—The
Escherichia coli strains EC538 (J. McCarthy) was used for the
expression of the plasmid pQE9-IDO
(32). Proteins were expressed
and purified as described previously
(32). E. coli strains
were routinely grown at 37 °C in Luria Bertani (LB) medium containing
ampicillin (100 µg/ml) and kanamycin (50 µg/ml)
(33). All liquid cultures were
agitated at 200 rpm during incubation in an orbital-shaking incubator.
Cultures (1 liter) were inoculated with a one-tenth volume of starter culture
and grown to a density of 0.6 OD at 600 nm.
Isopropyl-1-thio--D-galactopyranoside (200 mM),
hemin (3.5 mM in 10 mM NaOH), and PMSF (200
mM in isopropanol) were then added at a final concentration of 10
µM, 7 µM and1mM, respectively. The
cultures were then shaken for a further 3 h. EDTA (500 mM, pH 8.0)
was then added to a final concentration of 1 mM, and the cells were
pelleted by centrifugation (3,600 x g, 20 min, 4 °C). The
pellet from a 1 liter culture was resuspended in 10 ml of ice-cold phosphate
buffered saline containing PMSF (1 mM) and EDTA (1 mM)
and centrifuged at 27,000 x g for 15 min at 4 °C. The
pellet was stored at –20 °C for up to 2 months.
Site-directed Mutagenesis of the IDO Expression Plasmid pQE9-IDO—Plasmid DNA was isolated using the Plasmid Midi-Prep kit or the Plasmid Spin-Prep kit (Qiagen) per the manufacturer's instructions. Site-directed mutagenesis of pQE9-IDO was undertaken using the GeneEditorTM in vitro site-directed mutagenesis system (Promega) per the manufacturer's instructions. All primers were synthesized by Sigma-Genosys (Castle Hill, Australia). Primers used for site-directed mutagenesis were as follows: His16 to Ala16, 5'-ATC AGT AAA CAG TAC GCT ATT GAT GAA GAA GTG-3'; His 303 to Ala303, 5'-AGA AGA TAT ATG CCA CCA GCT GCT AGG AAC TTC CTG TGC TCA TTA-3'; His346 to Ala346, 5'-TCC CTG AGG AGC TAC GCT CTG CAA ATC GTG ACT-3'; Val104 to Ala104, 5'-AAT ATT GCT GCT CCT TAC TGC-3'; Asp274 to Ala274, 5'-GTC TTT CAG TGC TTT GCT GTC CTG CTG GGC ATC-3'; and Lys352 to Ala352, 5'-CTG CAA ATG GTG ACT GCG TAC ATC CTG ATT CCT-3'. The mutagenized codon in each primer is underlined. Additionally, the primers used for DNA sequence analysis to confirm site-directed mutagenesis were the following: IDO down, 5' AAG TGT TTC ACC AAA TCC TCG 3'; IDO up, 5' AAG GGC TTT CTC CAA GCA AGA 3'; pQE9 reverse, 5' GTT CTG AGG TCA TTA CTG G 3'; and pQE9 type II, 5' G GTC CAG GAG GAA AAA GGC 3'. Plasmid DNA was routinely digested with the restriction enzyme SalI to confirm the presence of the IDO gene insert. Dye terminator DNA sequence analysis was performed using an ABI-PRISM 377 DNA sequencer (Applied Biosystems) to confirm mutations. Sequences were analyzed using the Auto Assembler and MacVector programs.
Purification of IDO Wild Type and Mutant Proteins—Potassium phosphate buffer containing 1 mM PMSF was used in the following experiments. All procedures were conducted at 0–4 °C unless stated otherwise. The pellet from 2 liters of bacterial culture was suspended in 20 ml of PBS containing CompleteTM inhibitor mixture (Roche Molecular Biochemicals), sonicated on ice with a Branson Sonifier 250 for 4 min at maximum power, and centrifuged (27,000 x g, 15 min).
Phosphocellulose (P11) resin was activated per the manufacturer's instructions. The crude supernatant from lysed cells was applied to a phosphocellulose (P11) column with a specification of 2.5 x 4 cm equilibrated with potassium phosphate buffer (50 mM). After washing with 50 mM buffer (150 ml) and 100 mM buffer (100 ml), proteins were eluted with a linear gradient of buffer (100–500 mM, 100 ml of each) at a flow rate of 30 ml/h. Fractions that had an absorbance peak at 280 nm were pooled. Imidazole and glycerol were then added to a final concentration of 10 mM and 10% (v/v), respectively.
The buffer used for affinity chromatography consisted of potassium phosphate buffer (250 mM, pH 6.5), glycerol (10% v/v), and PMSF (1 mM) with varying concentrations of imidazole (10–250 mM). Ni-NTA agarose resin was washed with MilliQ water and equilibrated with buffer containing 10 mM imidazole. The pooled fractions from the phosphocellulose column were applied to a Ni-NTA agarose column (2.5 x 3 cm). After washing with 10 mM imidazole buffer (100 ml) and 50 mM imidazole buffer (100 ml), IDO wild type and mutant proteins were eluted with a linear imidazole gradient (50–250 mM imidazole, 100 ml each) and monitored by absorbance at 280 and 406 nm. The samples containing high 406- and/or 280-nm absorbances were pooled.
Analysis of IDO Wild Type and Mutant Proteins—Protein concentration was determined from A280 measurement or with Bio-Rad dye reagent using bovine serum albumin (0–1 mg/ml) as the standard. The colored product was measured at 595 nm using a SPECTRAmax plate reader (Molecular Devices, Sunnyvale, CA). Proteins were visualized using SDS-PAGE analysis by the method of Laemmli (34). Western blot analysis was conducted by the method of Burnette (35) using a monoclonal antibody raised against native human IDO (36).
Ultraviolet spectra were recorded using a Shimadzu UV-2401PC UV-Vis spectrophotometer (Shimadzu Corporation, Japan), with 1-cm pathlength quartz cuvettes. CD spectra were recorded on a JASCO J-810 spectropolarimeter (JASCO Corporation, Tokyo, Japan) with 1-cm pathlength quartz cuvettes. Sensitivity was 100 millidegrees, and the scanning speed was 50 nm/min for an accumulation of 5 scans. Further analysis of CD data was undertaken using the K2D program (37, 38).
Kinetic Studies—IDO activity was determined as described by Takikawa et al. (36) with a minor modification. In brief, the standard reaction mixture (0.5 ml) contained potassium phosphate buffer (50 mM, pH 6.5), ascorbic acid (20 mM, neutralized with NaOH), catalase (200 µg/ml), methylene blue (10 µM), L-tryptophan (400 µM), and IDO. The reaction was carried out at 37 °C for 10–60 min and was stopped by the addition of trichloroacetic acid (100 µl of 30%, w/v). After heating at 65 °C for 15 min, the reaction mixtures were centrifuged at 6,000 x g for 5 min. The supernatant (125 µl) was transferred into a well of a 96-well microplate and mixed with 2% (w/v) p-dimethylaminobenzaldehyde (125 µl) in acetic acid. The yellow pigment derived by reaction of p-dimethylaminobenzaldehyde (Ehrlich's reagent) with kynurenine was measured at 480 nm using a SPECTRAmax 250 microplate reader. L-Kynurenine (1–100 µM) was used as the standard.
Using this assay, the kinetic activity of wild type and the IDO mutants was determined against three substrates, i.e. L-tryptophan, D-tryptophan, and 5-hydroxy-L-tryptophan. Apparent Michaelis-Menten constants (Km) were determined using between 5 and 7 varying concentrations of these substrates. The concentration ranges for L-tryptophan, D-tryptophan, and 5-hydroxy-L-tryptophan are 3.6–288 µM, 0.72–10.8 mM, and 0.072–3.6 mM, respectively. The amount of wild type and mutant protein was adjusted to give initial velocity for each substrate where the percentage of conversion of substrate at each concentration was less than 10%. Each reaction was conducted in triplicate. Kinetic parameters (Km, Vmax) were determined from Lineweaver-Burk plots. As the content of heme varied between mutants, the data is presented as a function of the holoenzyme.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Purification and Structural Analysis of Wild Type and Mutant IDO
Species—Following the purification of the wild type and mutant IDO
proteins, the products obtained were subjected to SDS-PAGE
(Fig. 2A) and Western
blot analyses (Fig.
2B). As determined by SDS-PAGE, only one homologous band
was observed for each isolated protein with a molecular mass of 
45 kDa
and confirmed as IDO by a positive reaction with the anti-IDO monoclonal
antibody. CD spectrums were acquired to determine whether the selected
mutations affected the protein secondary structure of IDO. Far UV CD spectra
were obtained (Fig. 3) and
further analyzed using the K2D deconvolution program (data not shown). This
analysis showed that the wild type IDO protein exhibited a structure
consisting of 31% 
-helical, 11% -sheet, and 58% random coil. The
mutant IDO proteins exhibited similar structural compositions (28–31%
-helical, 10–18% -sheet, and 53–59% random coil).
These data indicate that site-directed mutagenesis of individual residues did
not grossly affect protein secondary structure.
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UV and visible spectroscopy was conducted on all purified mutants (Fig. 4). The His346 and Asp274 mutants have very low or no absorbance at 406 nm, indicating that they do not bind heme. The His16, His303, Val109, and Lys352 mutants maintained the ability to bind heme, and all give the same Soret band of heme with a peak at 406 nm, although, in the case of His16, His303, and Lys352, the binding was diminished compared with wild type. This may reflect a slight batch-by-batch difference in incorporation of heme into wild type and mutant IDO but indicates that these residues are not critical for heme coordination or binding.
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Biochemical Analysis of Wild Type and Mutant IDO Species—The
enzymatic activity of wild type protein and each mutant was analyzed using
L-tryptophan, D-tryptophan, and
5-hydroxy-L-tryptophan as substrates. Because mutant samples were a
mixture of active holoenzyme (heme-containing IDO) and inactive apoenzyme
(heme-free IDO), the reaction velocity was determined as a function of
concentration of holoenzyme. The concentration of holoenzyme for each mutant
was determined using the extinction coefficient at 406 nm determined for
rabbit intestinal IDO of 
= 140
mM–1cm–1
(31). Substrate concentration
(S) versus velocity (V) plots were constructed for
the wild type and each mutant protein for each substrate. The number of
substrate concentrations for L-tryptophan, D-tryptophan,
and 5-hydroxy-L-tryptophan were 5, 7, and 6, respectively. Double
reciprocal plots were constructed, and linear trend lines were fitted by the
least squared method from which the constants Km
and Vmax were determined (results not shown). The kinetic
parameters of wild type and mutant IDO for L-tryptophan,
D-tryptophan, and 5-hydroxy-L-tryptophan are given in
Table I. Each of the mutants
was analyzed for its ability to oxygenate L-tryptophan,
D-tryptophan, and 5-hydroxy-L-tryptophan. Only the
Asp274 and His346 mutants were unable to convert each of
these substrates to N'-formyl kynurenine.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Each of the IDO mutant proteins was expressed and purified by the protocol
established for IDO (1). The
yields of the purification for most mutants were comparable with that obtained
for IDO of 500 µg/liter of culture. However, the yield for the
His346 IDO mutant was lower at 125 µg/liter of culture.
Wild type and mutants proteins remained soluble after storage at 4 °C and
retained initial enzymatic activity, suggesting that the folding of each
protein and stability were not affected. Wild type and mutant IDO proteins
exhibited CD spectra with minima at 208 and 225 nm, which is characteristic of
predominantly -helical structure. The CD spectra were further analyzed
using the K2D deconvolution program. This analysis indicated that the wild
type and each of the IDO mutant proteins contained similar levels of
-helical, -sheet, and random coil structure. From this CD
analysis it can be concluded that all of the mutants maintained secondary
structure and that any loss of enzymatic activity can therefore be attributed
to the affect of the residue change and not from a loss of secondary
structure.
UV and visible spectroscopy were conducted on all wild type and mutant IDO proteins. From the ratio of the absorbance maxima at 406:280 it can be deduced that the heme content of mutant proteins was highly variable. His346 and Asp274 have very low or no absorbance at 406 nm, indicating that these mutant IDO proteins do not bind heme. This confirms the observation made during purification that these proteins had no visible red coloration. The wild type 406:280 ratio was 1.7. The 406:280 ratio for the His16, Val109, His303, and Lys352 mutants were 1.0, 1.6, 0.9, and 0.8, respectively. These data indicate that these protein preparations contained varying amounts of apoprotein; nonetheless, each of these IDO mutant proteins retained the ability to bind heme. Furthermore, these mutations did not markedly compromise enzymatic activity of the holoprotein.
Each of the mutants was analyzed for its ability to oxygenate
L-tryptophan, D-tryptophan, and
5-hydroxy-L-tryptophan. Each mutant, excluding Asp274
and His346, was able to convert each of these substrates to
N'-formyl kynurenine. The Km and
Vmax values obtained were compared with those obtained for
wild type IDO and placental human IDO, as reported in Takikawa et al.
(1). For
L-tryptophan, the mutant Km values
were between 6 and 20 µM. These are comparable with those of
wild type (20 µM) and placental human IDO (21 µM).
For D-tryptophan, the Km values ranged
between 2 and 5 mM, and these are comparable with that determined
for wild type (5 mM) but are low in comparison to that reported for
placental IDO (Km = 14 mM). Because
mutagenesis was carried out on the recombinant protein, values of 5
mM were expected. The reported Km
values for wild type (440 µM) and native IDO (400
µM) were approximately double that obtained for the mutants. The
mutants had the highest affinity for L-tryptophan, followed by
D-tryptophan and 5-hydroxy-L-tryptophan. This agrees
with both the wild type IDO result and the previously reported data for the
human placental enzyme (2). The
Vmax/Km values for
L-tryptophan, D-tryptophan, and
5-hydroxy-L-tryptophan exhibited less than a 8-fold variance
between the enzymatically active mutant and wild type proteins. This indicates
that each of the mutant enzymes, except His346 and
Asp274, maintained enzymatic ability with similar enzyme
efficiency.
Conversion of His346 to Ala resulted in a total loss of heme content in IDO, although the secondary structure of the protein was maintained. This IDO mutant therefore provides strong evidence for the proposal that this histidine is the proximal heme ligand in IDO. Site-directed mutagenesis showed that Asp274 is also required for maintaining heme binding in IDO. The role of His346 and Asp274 in the binding of heme in IDO may be direct; i.e. they may act as the proximal and distal heme ligands. It may be, however, that these residues act indirectly by providing support to the structure of the heme pocket or that these residues are important in influencing the coordination of other residues that act as the heme ligands. Crystallization of wild type IDO or these mutants will directly determine their role in heme binding by IDO.
The proximal ligand in hemoproteins is important for maintaining the basic and rigid structure of the heme; however, the distal ligand is more easily replaced by other ligands. It is therefore proposed that the removal of the proximal ligand by site-directed mutagenesis should remove the ability of the enzyme to bind heme, whereas the removal of the distal ligand may not totally prevent heme binding. Previous EPR and magnetic CD studies have indicated that the proximal ligand of IDO is a histidine residue such as is found in a wide range of other hemoproteins. It had been previously proposed that either His346 or His303acts as the proximal residue (3). The mutation of His303 produced a protein that retained significant heme binding ability, which suggest that His303 is not the proximal ligand. It does not, however, indicate whether or not this residue is the distal ligand. This is because the distal ligand is more flexible and can be replaced by other ligands, including other amino acids. For example if His303 is the distal ligand in the native protein, its removal may allow residues such as Met295, Tyr298, or Met299, which are located near His303 in the primary sequence and are able to coordinate to heme, to act as the distal ligand. The His303 replacement mutation does, however, show that this residue is not essential for heme binding or IDO activity.
This is the first study to show the importance of two specific residues, Asp274 and His346, in the enzymatic activity of the human IDO. By contrast, residues His303, His16, Lys352, and Val109 were found to not be essential for heme binding in IDO. This supports the hypothesis that the highly conserved His346 residue may act as the proximal heme ligand in IDO.
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FOOTNOTES |
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¶ Present address: Dept. of Pharmacology, Hokkaido University School of
Medicine, Sapporo 060-8638, Japan.
|| To whom correspondence should be addressed. Tel.: 61-2-42213013; Fax: 61-2-42214135; E-mail: mark_walker{at}uow.edu.au.
1 The abbreviations used are: IDO, indoleamine 2,3-dioxygenase; CD, circular
dichroism; MCD, magnetic circular dichroism; EPR, electron paramagnetic
resonance; Ni-NTA, nickel nitrilotriacetic acid; PMSF, phenylmethylsulfonyl
fluoride.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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