proteins STRUCTURE O FUNCTION O BIOINFORMATICS
STRUCTURE NOTE
Structural and functional analyses of human tryptophan 2,3-dioxygenase Bing Meng,1,2 Dong Wu,3* Jianhua Gu,4 Songying Ouyang,1 Wei Ding,1 and Zhi-Jie Liu1,5* 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 iHuman Institute, ShanghaiTech University, Shanghai 201210, China 4 Department of General Surgery, Tianjin First Center Hospital, Tianjin 300192, China 5 Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China
ABSTRACT Tryptophan 2,3-dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2-C3 bond of L-tryptophan. This is a rate-limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 A˚ resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in L-tryptophan oxidation. Proteins 2014; 82:3210–3216. C 2014 Wiley Periodicals, Inc. V
Key words: L-tryptophan metabolism; dioxygenase; crystal structure; enzymatic activity; TDO.
INTRODUCTION Tryptophan 2,3-dioxygenase (TDO, EC 1.13.11.11) and indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.52) are the only two heme-containing enzymes that catalyze the first and rate-limiting step of L-tryptophan (L-Trp) catabolism in the kynurenine pathway. This step converts LTrp to N-formyl kynurenine (NFK) by cleaving the C2– C3 bond in the indole moiety of L-Trp and incorporating one oxygen molecule.1 In addition to its role in protein synthesis, 95% of L-Trp in the human body is processed by the kynurenine pathway, leading to the production of nicotinamide adenine dinucleotide. A small amount of LTrp (1%) is used to synthesize the neurotransmitter serotonin.2 As key enzymes in this pathway, both TDO and IDO play important roles in biological processes and are associated with some diseases. IDO is involved in the
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regulation of immune system, including inhibition of T cell proliferation, suppression of adaptive immunity, and immune escape of tumors.3 TDO was found to be Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 31330019 and 31200559; Grant sponsor: Ministry of Science and Technology of China; Grant numbers: 2014CB910400 and 2013CB911103; Grant sponsor: National Key Technology Research and Development Program of the Ministry of Science and Technology of China; Grant number: 2014BAI07B02; Grant sponsor: Ministry of Health of China; Grant number: 2013ZX10004-602; Grant sponsor: Beijing Nova Program; Grant number: Z141102001814020. Bing Meng and Dong Wu contributed equally to this work. *Correspondence to: Dong Wu, iHuman Institute, ShanghaiTech University, Shanghai 201210, China. E-mail: [email protected] and Zhi-Jie Liu, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. E-mail: [email protected] Received 19 March 2014; Revised 19 June 2014; Accepted 15 July 2014 Published online 26 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24653
C 2014 WILEY PERIODICALS, INC. V
Structure of Human Tryptophan 2,3-Dioxygenase
expressed in many tumor cells and is related to reduction of antitumor immune responses.4 Thus, TDO and IDO could be considered as novel drug targets for cancer and immune diseases. Although they catalyze the same reaction, TDO and IDO show low sequence identity and distinct substrate specificities. TDO is highly substrate-specific for L-Trp and the related derivatives 6-fluoro-Trp and 5-fluoroTrp,5 whereas IDO shows broad substrates specificity, recognizing L-Trp, D-Trp, 5-hydroxytryptophan, tryptamine, and melatonin.1 Moreover, the distribution of TDO differs from that of IDO in mammalian tissues. TDO is mainly expressed in the liver,1 whereas IDO exists in most tissues except the liver.2 Thus far, the crystal structures of two prokaryotic TDOs (Xanthomonas campestris TDO [XcTDO] and Ralstonia metallidurans TDO [RmTDO]), one eukaryotic TDO (Drosophila melanogaster TDO, DmTDO), and human IDO (hIDO) have been determined.6–9 All of these structures were solved in complex with the cofactor heme, which is indispensible for TDO enzymatic activity. XcTDO was also solved in apo form, as well as in complex with heme and L-Trp or its analogue. These structures have not only shed light on the molecular mechanisms of substrate binding and catalysis, but have also provided insight into the design of potential inhibitors of TDO and IDO. D21-methyltryptophan (D21-MT) is currently used clinically as a specific inhibitor of IDO, although its inhibitory activity is low.10 Just recently, dihydroquercetin was reported to be a selective inhibitor of TDO, with an in vitro inhibition constant of 16 lM.11 However, no potential TDO selective inhibitor has entered clinical trials thus far. Here, we report the crystal structure of human TDO (hTDO) in apo form to 2.90 A˚ resolution. The hTDO structure presents a tetrameric architecture similar to that of another eukaryotic TDO, DmTDO.8 However, hTDO shows different enzymatic characteristics from those of DmTDO, suggesting that there might be differences in substrate recognition and catalysis between them. Mutagenesis analysis has shown that some residues are critical for the enzymatic activity of hTDO. Our studies provide a framework for identifying more critical residues and designing new inhibitors of hTDO. MATERIALS AND METHODS Protein expression and purification
The gene encoding full-length hTDO (NP_005642) was amplified from a human cDNA library using the forward primer 50 -TACTTCCAATCCAATGCTATGAGTGGG 0 TGCC-3 and the reverse primer 50 -TTATCCACTTCCAA TGTTAATCTGATTCATCACTGC-30 . The amplified DNA was cloned into the vector pMCSG7 using a ligaseindependent cloning method,12 and the construct was transformed into Escherichia coli BL21 (DE3) cells. The
cells were cultured in LB medium at 37 C until the OD600 reached 0.8, and then isopropylthio-beta-D-galactoside (IPTG) was added at a final concentration of 0.2 mM. Hemin (at a final concentration of 5 mM) was introduced to ensure the recombinant protein to be a holoenzyme. The cultures were then incubated at 16 C for 20 h, and the cells were harvested by centrifugation at 4000 rpm. The protein was purified using an Ni-NTA column (Qiagen). The cells were suspended in buffer A (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5% glycerol) and lysed by ultrasonication. Cell debris was removed by centrifugation at 18,000g for 60 min. Protein was eluted with a linear gradient of 20–500 mM imidazole. The fractions were examined by SDS-PAGE, and the N-terminal 63 His-tag was removed by TEV protease on an extra Ni-NTA column. Purified protein was loaded onto a Superdex S200 column (GE Healthcare) equilibrated with buffer A. Fractions containing the protein were concentrated to 20 mg mL21 and stored at 280 C until further use. Full-length hTDO was unstable, and various truncations were constructed. A truncation containing amino acids 19– 388 (aa 19–388) was found to have good solubility and stability. Expression and purification of this truncation was performed in the same way as that for wild type, and the truncation was used for crystallization and further studies.
Site-directed mutagenesis
The Quick Change Site-Directed Mutagenesis Kit (Stratagene) was used to generate hTDO (aa 19–388) mutants. Eight mutants (Y42A, Y45A, F72A, H76A, F140A, R144A, S151A, and H328A) were constructed and verified by DNA sequencing. Expression and purification of the mutants were performed using the same method described above.
Kinetic analysis and spectroscopy
The enzymatic activity of hTDO was measured as described previously,5 with some minor modifications. The ferric hTDO (at a final concentration of 1 mM) was rapidly mixed with excess freshly prepared L-ascorbate (at a final concentration of 500 mM) in buffer A at 25 C and incubated for 120 s to maintain the enzyme in the ferrous state. The reaction was initiated by the addition of different concentrations of L-Trp, and the formation of NFK was monitored based on the absorbance increase at 321 nm (e321 5 3750 M21 cm21)5 using a U2010 spectrophotometer (Hitachi). The data were analyzed by Michaelis– Menten curve fitting with Origin software (OriginLab). The optical absorption spectra experiments were measured in buffer A at 25.0 C using a DU 800 UV–visible spectrophotometer (Beckman Coulter). The final concentrations of all samples were adjusted to 10 mM. In order to make hemin reduce to heme, excess freshly prepared PROTEINS
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L-ascorbate
was added before the spectrum scanning. The data were analyzed by Origin software (OriginLab).
Table I
Data Collection and Refinement Statisticsa hTDO (aa 19–388)
Isothermal titration calorimetry assay
Apo hTDO (aa 19–388) was prepared in the following way: Hemin was omitted from the culture medium during protein expression, so that only a basal level of hTDO could capture heme from the medium. After further purification, when no heme absorption was detected, the protein was defined as apo hTDO and used for the isothermal titration calorimetry (ITC) assay. ITC experiments were performed using a MicroCal iTC200 instrument (MicroCal). Hemin or L-Trp in a syringe was titrated into a sample cell containing apo hTDO at 25 C, and the resulting isotherm was fitted with Origin software (OriginLab). All the samples were prepared in buffer containing 100 mM Tris–HCl (pH 8.0) and 500 mM NaCl. Other components in the solution, except the titrant or titrate, were adjusted to the same levels as before titration. Crystallization and data collection
Full-length holo hTDO and various truncations were concentrated to 20 mg mL21 and screened for crystallization using the hanging drop vapor diffusion method at 16 C. Crystallization drops were made by mixing 1 lL of the protein solution with 1 lL of reservoir solution. Diffraction quality crystals were grown from a truncation consisting of aa 19–388. The crystals were grown for 3 days in the presence of 0.1 M sodium cacodylate (pH 6.0–6.5), 6–10% (w/v) MPEG 5000, and 10% (v/v) 2-propanol. Crystallization was then optimized using an additive screen kit (Hampton Research), and hexammine cobalt (III) chloride was found to improve the diffraction significantly. Crystals were harvested with 25% (v/v) glycerol as a cryoprotectant. X-ray diffraction data were collected at a wavelength of 0.9796 A˚ on beamline 22-ID at Advanced Photon Source, Argonne National Laboratory. Structure determination and refinement
The structure of hTDO (aa 19–388) was solved via the molecular replacement method with Phaser13 using the structure of XcTDO [Protein Data Bank (PDB) code, 2NW7] as the searching model.6 Model building and structure refinement were performed manually in Coot14 and automatically in PHENIX.15 Data collection and refinement statistics are presented in Table I. Atomic coordinates and structure factors have been deposited in the PDB with accession code 4PW8. RESULTS AND DISCUSSION Overall structure of hTDO
The crystal structure of hTDO (aa 19–388) was solved by molecular replacement and refined to 2.90 A˚ resolu-
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Data collection Beamline Wavelength () Space group Cell dimensions a, b, c () a, b, c ( ) Resolution () No. of observed reflections No. of unique reflections Completeness (%) Rmerge Redundancy Refinement Rwork/Rfree (%) RMSD Bond length () Bond angle ( ) Ramachandran plot (%) Favored Allowed Disallowed
APS, 22-ID 0.9796 P212121 134.33, 156.92, 160.33 90.00, 90.00, 90.00 50.00–2.90 (2.95–2.90) 492,077 75,459 99.9 (100.0) 0.044 (0.450) 27.84 (3.26) 6.5 (6.6) 20.39/22.22 0.012 1.380 97.26 2.36 0.38
RMSD, root-mean-square deviation. a Values in parentheses are for the highest resolution shell.
tion. The crystal belongs to space group P212121. One asymmetric unit consists of eight molecules of the protein based on the calculated solvent content of 49.1%. No systematic global differences are observed among the eight monomers, although they show variation at some loop regions. The electron densities of the major part are clear, whereas the densities of N-terminal residues 19–40 and C-terminal residues 383–388 are invisible. Residues 170–186, and 339–358 are also missing in the electron density map. In addition, the electron densities of residues 241–252 are invisible in three molecules. These residues are positioned at the loop regions or interacting surfaces of the active site. Furthermore, the cofactor heme is missing in the binding pocket, as its electron density is invisible. Thus, the structure is assumed as apo hTDO. The hTDO monomer comprises 15 a-helices and no b-strands [Fig. 1(A)], and, like DmTDO,8 can be divided into three major regions. The N-terminal region consists of residues 41–65 (residues 42–45 a1, 47–51 a2, and 58– 61 a3). The large domain contains residues 66–213 (residues 67–99 a4, 105–134 a5, 137–144 a6, 156–165 a7, 188–197 a8, and 201–211 a9) and 281–360 (residues 286–298 a13, and 305–335 a14). The small domain includes residues 214–280 (residues 222–242 a10, 251– 268 a11, and 270–279 a12) and residues 360–382 (residues 363–371 a15). The size exclusion chromatography profile suggests that holo hTDO (aa 19–388) exists as a tetramer in
Structure of Human Tryptophan 2,3-Dioxygenase
Figure 1 Overall structure of hTDO. (A) A ribbon representation of the hTDO monomer with secondary structural a helices (a1–15) labeled. The Nterminal region, large domain and small domain are colored in blue, cyan, and orange, respectively. The invisible residues are shown as dotted lines. (B) Overall structure of the hTDO tetramer viewed in two orientations. The four monomers A, B, C, and D are colored in green, red, yellow, and magenta, respectively. (C) Overall structure of the hTDO dimer viewed in two orientations. The two monomers are colored in green and red. Helices a1, a4, and a5 are labeled in monomers A and B. The swapped N-terminal segments are indicated by the oval circle.
solution, whereas apo hTDO (aa 19–388) exists as a dimer (Supporting Information Fig. S1). This result indicates that heme plays an important role in the oligomerization of hTDO. In our structure, the eight protein molecules in one asymmetric unit are packed as two tetramers, which was calculated by PDBePISA (www.ebi.ac. uk/msd-srv/prot_int/pistart.html). The tetramerization is similar to that observed in the structures of XcTDO, RmTDO and DmTDO.6–8 Generally, the four monomers (represented by ABCD) in the tetramer are related by
three mutually perpendicular twofold axes [Fig. 1(B)]. The total solvent-accessible surface area that is buried upon tetramerization is 16,360 A˚2, with the core of tetrameric surfaces formed by the a14 helices in each monomer. Adjacent monomers have more extensive interactions at the A–B interface than at the A–C interface, burying total solvent-accessible surface areas of 5370 and 2270 A˚2 upon dimerization, respectively. Therefore, the tetramer can be viewed as a dimer of dimers, in which the two C-shape dimers (AB and CD) are clamped PROTEINS
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perpendicularly to each other to form a tight tetramer. The dimerization of monomers A and B is shown in Figure 1(C). Helices a4 and a5 in the large domain of one monomer interact with a4 and a5 in the other monomer, respectively, which contributes to the majority of the dimer formation surfaces. In addition, the Nterminal segments of the two monomers are swapped, with each a1 helix extended into the active site of the other. Comparison of hTDO with known orthologues
Sequence alignment indicates that hTDO shares approximately 60% sequence identity with DmTDO, but only 30% identity with XcTDO and RmTDO [Fig. 2(A)]. A structural homology search using DaliLite (www.ebi.ac.uk/ Tools/structure/dalilite) shows that the overall structure of hTDO is highly similar to those of DmTDO8 [PDB code, 4HKA; Z-score, 33.1; root-mean-square deviation (RMSD), 1.4 A˚], XcTDO6 (PDB code, 2NW7; Z-score, 19.2; RMSD, 2.2 A˚), and RmTDO7 (PDB code, 2NOX; Zscore, 19.9; RMSD, 2.0 A˚). Superposition of the hTDO structure with those of DmTDO, XcTDO, and RmTDO shows that the core domain frameworks in these TDOs are highly conserved, whereas some loop regions surrounding the active site in hTDO are very flexible [Fig. 2(B)]. This finding indicates that these proteins may share the same molecular mechanism of substrate recognition and catalysis. Specifically, hTDO and DmTDO both contain an extra small domain, which interacts with the active site of an adjacent monomer and is the major difference between eukaryotic and prokaryotic TDOs. Because heme can be found in the structures of the other three TDOs but not in hTDO, we analyzed the regions around the heme binding site, which showed weak electron density and could not be fixed in our structure. Residues 170–186 are missing in the structure of hTDO; they form a short a-helix (6 residues) in XcTDO and RmTDO but a long insertion fragment (20 residues) in DmTDO. Residues 339–358 are also missing from hTDO; these residues form an a-helix and a flexible loop in the structures of the orthologues. Although hTDO and hIDO share only approximately 10% sequence identity, the large domains of hTDO and hIDO show similar folds. The overall structure and spatial orientation of the small domain differ between hTDO and hIDO [Fig. 2(C)]. In hIDO, the connecting loop and a-helix F in the small domain contribute to the formation of the active site. Both the active site and the small domain are located on the same end of the large domain.9 In hTDO, the active site is located on one end of the large domain, but the small domain is located on the other end and is far away from the active site. The complete formation of the active site requires the N-terminal region of an adjacent monomer and the small domain of another adjacent monomer. Therefore,
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hIDO can display activity as a monomer; however, hTDO must form an oligomer to exhibit activity. Enzymatic activity and mutagenesis of hTDO
The enzymatic activities of the wild type hTDO, the truncation aa 19–388 and eight point mutants were measured. The kinetic parameters are presented in Table II. The Km and kcat of wild type hTDO are 0.135 6 0.011 mM and 1.071 6 0.036 s21, respectively, which are comparable with the results reported previously.5 The truncation aa 19–388 shows 13.33% increase in Km and 8.87% decrease in kcat, suggesting that this truncation does not significantly affect the activity. All eight residues selected for mutagenesis are conserved [Fig. 2(A)] and are located around the active site. Compared with the template aa 19–388, all the mutants showed a major decrease in enzymatic activity. Mutants Y42A, Y45A, F140A, R144A, and S151A remain only 0.50%, 1.13%, 0.25%, 0.88%, and 9.08% relative activity of the wild type protein, respectively. In fact, no activity could be detected for mutants F72A, H76A, and H328A. The results indicate that these residues may play critical roles in catalytic reaction or substrate binding. To investigate the function of the eight residues, hTDO was superimposed with the DmTDO–heme complex8 and the XcTDO–heme–Trp complex (PDB code, 2NW8).6 The relative spatial position of these residues, heme, and L-Trp is shown in Figure 2(D), with heme and L-Trp from the DmTDO–heme complex and the XcTDO–heme–Trp complex, respectively. The result shows that the spatial conformations of these eight residues are almost the same in hTDO and DmTDO, except for S151 (S135 in DmTDO). Y42 and Y45 (Y27 and Y30 in DmTDO) are located in the outspread N-terminal segment and participate in the binding of the substrate L-Trp. R144 (R128 in DmTDO) also plays role in the recognition of L-Trp. F72, H76, and F140 (F57, H61, and F124 in DmTDO) are involved in binding both L-Trp and heme. In addition, H328 (H312 in DmTDO) binds with heme on the other side of the plane, which is consistent with the previous report.6 S151 takes part in binding with heme, whereas S135 in DmTDO does not. This difference may contribute to the difference in activity between hTDO and DmTDO. The kcat/Km ratios of hTDO and DmTDO are 7.93 mM21s21 and 1.05 mM21s21,8 respectively. Unfortunately, some residues involved in substrate recognition and heme binding are located in the invisible regions of the hTDO structure. Consequently, more detailed information regarding the missing residues must be obtained to clarify the significant activity difference between hTDO and DmTDO. Heme is indispensible for hTDO enzymatic activity [Supporting Information Fig. S2(A)], and the kinetic assays have confirmed the presence of heme in the holo hTDO solution. Unfortunately, the heme cofactor could
Structure of Human Tryptophan 2,3-Dioxygenase
Figure 2 Comparison of hTDO with other TDOs and hIDO. (A) Sequence alignment of hTDO with DmTDO, XcTDO, and RmTDO. The secondary structural elements of hTDO are depicted at the top of the alignment. Residues for mutagenesis are marked by green stars. (B) Upper: structural comparison of hTDO (magenta) with DmTDO (cyan), XcTDO (marine) and RmTDO (gray). Heme is shown as a ball-and-stick model in red. Lower: superimposition of the secondary structures around the active site between hTDO (magenta) and DmTDO (cyan). The helices are represented with the cylindrical mode. Two fragments that exist only in the structure of DmTDO are shown in green and yellow, respectively. Heme is from the DmTDO–heme complex and is shown as a ball-and-stick model in red. (C) Structural superimposition of hTDO with hIDO. The large domain and the N-terminal region of the adjacent monomer in hTDO are shown in magenta, and the small domain of another adjacent monomer is shown in bright pink. The large domain and the connecting loop of hIDO are shown in yellow, and the small domain is shown in orange. Heme is shown as a ball-and-stick model in red. (D) Comparison of the critical residues around active site between hTDO and DmTDO. Heme and L-Trp are shown as ball-and-stick models in red and yellow, respectively. The residues of hTDO are labeled and shown as a ball-and-stick model in magenta, and the residues of DmTDO are shown in cyan. Y42 and Y45 are from the N-terminal region of the adjacent monomer.
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Table II
Kinetic Parameters of hTDOa
Wild type aa 19–388 Y42A Y45A F140A R144A S151A F72A H76A H328A
Km (mM)
kcat (s21)
kcat/Km (mM21s21)
Relative activity (%)b
0.135 6 0.011 0.153 6 0.012 0.227 6 0.024 0.661 6 0.087 1.531 6 0.194 0.631 6 0.033 0.146 6 0.016 ND ND ND
1.071 6 0.036 0.976 6 0.033 0.010 6 0.001 0.062 6 0.004 0.032 6 0.002 0.045 6 0.001 0.105 6 0.004 ND ND ND
7.93 6.38 0.04 0.09 0.02 0.07 0.72
100.00 80.45 0.50 1.13 0.25 0.88 9.08
ND, not detected. a The kinetic parameters are the mean 6 SD of triplicate determinations. b The kcat/Km ratio of the wild type is defined as 100.00%.
not be located in the crystal structure, which means that in our condition the protein–heme interaction is probably too weak to anchor heme. Spectrum scanning results reveal red shift of heme absorbance when incubated with apo hTDO [Supporting Information Fig. S2(B)]. ITC results have shown that neither heme nor LTrp alone displays detectable interaction with apo hTDO [Supporting Information Fig. S3(A,B)], and hTDO can catalyze L-Trp only in the presence of heme [Supporting Information Fig. S3(C)]. These results indicate that hTDO and heme may not form a stable complex in solution. Many methods have been utilized in an attempt to obtain the hTDO–heme complex and the hTDO–heme– L-Trp complex, including the addition of extra hemin and the introduction of L-Trp in the purification procedure, cocrystallization of protein with L-Trp or its analogues, crystallization using hemin as an additive, substitution of hemin with its analogue zinc protoporphyrin, crystallization in an anaerobic environment, and a combination of these strategies. However, the results showed that heme was not identified in the crystal structure. More biophysical analyses are currently being performed to decipher the dynamics among hTDO, heme, and L-Trp.
ACKNOWLEDGMENTS The authors thank the staff at beamline 22-ID of the Advanced Photon Source for assisting with X-ray diffraction data collection. REFERENCES 1. Sono M, Roach MP, Coulter ED, Dawson JH. Heme-containing oxygenases. Chem Rev 1996;96:2841–2888. 2. Takikawa O. Biochemical and medical aspects of the indoleamine 2, 3-dioxygenase-initiated L-tryptophan metabolism. Biochem Biophys Res Commun 2005;338:12–19. 3. Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 2004;4:762–774.
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4. Pilotte L, Larrieu P, Stroobant V, Colau D, Dolusic´ E, Frederick R, De Plaen E, Uyttenhove C, Wouters J, Masereel B, Van den Eynde BJ. Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase. Proc Natl Acad Sci USA 2012;109:2497–2502. 5. Basran J, Rafice SA, Chauhan N, Efimov I, Cheesman MR, Ghamsari L, Raven EL. A kinetic, spectroscopic, and redox study of human tryptophan 2,3-dioxygenase. Biochemistry 2008;47:4752– 4760. 6. Forouhar F, Anderson JR, Mowat CG, Vorobiev SM, Hussain A, Abashidze M, Bruckmann C, Thackray SJ, Seetharaman J, Tucker T, Xiao R, Ma LC, Zhao L, Acton TB, Montelione GT, Chapman SK, Tong L. Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase. Proc Natl Acad Sci USA 2007;104: 473–478. 7. Zhang Y, Kang SA, Mukherjee T, Bale S, Crane BR, Begley TP, Ealick SE. Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis. Biochemistry 2007;46:145–155. 8. Huang W, Gong Z, Li J, Ding J. Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism. J Struct Biol 2013;181:291– 299. 9. Sugimoto H, Oda S-i, Otsuki T, Hino T, Yoshida T, Shiro Y. Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc Natl Acad Sci USA 2006;103:2611–2616. 10. L€ ob S, K€ onigsrainer A, Rammensee H-G, Opelz G, Terness P. Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees? Nat Rev Cancer 2009;9:445–452. 11. Pantouris G, Mowat CG. Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase. Biochem Biophys Res Commun 2014;443:28– 31. 12. Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr Purif 2002;25:8–15. 13. McCoy AJ. Solving structures of protein complexes by molecular replacement with phaser. Acta Crystallogr D Biol Crystallogr 2006; 63:32–41. 14. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004;60:2126–2132. 15. Adams PD, Afonine PV, Bunk oczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010;66:213–221.
STRUCTURE NOTE
Structural and functional analyses of human tryptophan 2,3-dioxygenase Bing Meng,1,2 Dong Wu,3* Jianhua Gu,4 Songying Ouyang,1 Wei Ding,1 and Zhi-Jie Liu1,5* 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 iHuman Institute, ShanghaiTech University, Shanghai 201210, China 4 Department of General Surgery, Tianjin First Center Hospital, Tianjin 300192, China 5 Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China
ABSTRACT Tryptophan 2,3-dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2-C3 bond of L-tryptophan. This is a rate-limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 A˚ resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in L-tryptophan oxidation. Proteins 2014; 82:3210–3216. C 2014 Wiley Periodicals, Inc. V
Key words: L-tryptophan metabolism; dioxygenase; crystal structure; enzymatic activity; TDO.
INTRODUCTION Tryptophan 2,3-dioxygenase (TDO, EC 1.13.11.11) and indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.52) are the only two heme-containing enzymes that catalyze the first and rate-limiting step of L-tryptophan (L-Trp) catabolism in the kynurenine pathway. This step converts LTrp to N-formyl kynurenine (NFK) by cleaving the C2– C3 bond in the indole moiety of L-Trp and incorporating one oxygen molecule.1 In addition to its role in protein synthesis, 95% of L-Trp in the human body is processed by the kynurenine pathway, leading to the production of nicotinamide adenine dinucleotide. A small amount of LTrp (1%) is used to synthesize the neurotransmitter serotonin.2 As key enzymes in this pathway, both TDO and IDO play important roles in biological processes and are associated with some diseases. IDO is involved in the
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regulation of immune system, including inhibition of T cell proliferation, suppression of adaptive immunity, and immune escape of tumors.3 TDO was found to be Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 31330019 and 31200559; Grant sponsor: Ministry of Science and Technology of China; Grant numbers: 2014CB910400 and 2013CB911103; Grant sponsor: National Key Technology Research and Development Program of the Ministry of Science and Technology of China; Grant number: 2014BAI07B02; Grant sponsor: Ministry of Health of China; Grant number: 2013ZX10004-602; Grant sponsor: Beijing Nova Program; Grant number: Z141102001814020. Bing Meng and Dong Wu contributed equally to this work. *Correspondence to: Dong Wu, iHuman Institute, ShanghaiTech University, Shanghai 201210, China. E-mail: [email protected] and Zhi-Jie Liu, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. E-mail: [email protected] Received 19 March 2014; Revised 19 June 2014; Accepted 15 July 2014 Published online 26 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24653
C 2014 WILEY PERIODICALS, INC. V
Structure of Human Tryptophan 2,3-Dioxygenase
expressed in many tumor cells and is related to reduction of antitumor immune responses.4 Thus, TDO and IDO could be considered as novel drug targets for cancer and immune diseases. Although they catalyze the same reaction, TDO and IDO show low sequence identity and distinct substrate specificities. TDO is highly substrate-specific for L-Trp and the related derivatives 6-fluoro-Trp and 5-fluoroTrp,5 whereas IDO shows broad substrates specificity, recognizing L-Trp, D-Trp, 5-hydroxytryptophan, tryptamine, and melatonin.1 Moreover, the distribution of TDO differs from that of IDO in mammalian tissues. TDO is mainly expressed in the liver,1 whereas IDO exists in most tissues except the liver.2 Thus far, the crystal structures of two prokaryotic TDOs (Xanthomonas campestris TDO [XcTDO] and Ralstonia metallidurans TDO [RmTDO]), one eukaryotic TDO (Drosophila melanogaster TDO, DmTDO), and human IDO (hIDO) have been determined.6–9 All of these structures were solved in complex with the cofactor heme, which is indispensible for TDO enzymatic activity. XcTDO was also solved in apo form, as well as in complex with heme and L-Trp or its analogue. These structures have not only shed light on the molecular mechanisms of substrate binding and catalysis, but have also provided insight into the design of potential inhibitors of TDO and IDO. D21-methyltryptophan (D21-MT) is currently used clinically as a specific inhibitor of IDO, although its inhibitory activity is low.10 Just recently, dihydroquercetin was reported to be a selective inhibitor of TDO, with an in vitro inhibition constant of 16 lM.11 However, no potential TDO selective inhibitor has entered clinical trials thus far. Here, we report the crystal structure of human TDO (hTDO) in apo form to 2.90 A˚ resolution. The hTDO structure presents a tetrameric architecture similar to that of another eukaryotic TDO, DmTDO.8 However, hTDO shows different enzymatic characteristics from those of DmTDO, suggesting that there might be differences in substrate recognition and catalysis between them. Mutagenesis analysis has shown that some residues are critical for the enzymatic activity of hTDO. Our studies provide a framework for identifying more critical residues and designing new inhibitors of hTDO. MATERIALS AND METHODS Protein expression and purification
The gene encoding full-length hTDO (NP_005642) was amplified from a human cDNA library using the forward primer 50 -TACTTCCAATCCAATGCTATGAGTGGG 0 TGCC-3 and the reverse primer 50 -TTATCCACTTCCAA TGTTAATCTGATTCATCACTGC-30 . The amplified DNA was cloned into the vector pMCSG7 using a ligaseindependent cloning method,12 and the construct was transformed into Escherichia coli BL21 (DE3) cells. The
cells were cultured in LB medium at 37 C until the OD600 reached 0.8, and then isopropylthio-beta-D-galactoside (IPTG) was added at a final concentration of 0.2 mM. Hemin (at a final concentration of 5 mM) was introduced to ensure the recombinant protein to be a holoenzyme. The cultures were then incubated at 16 C for 20 h, and the cells were harvested by centrifugation at 4000 rpm. The protein was purified using an Ni-NTA column (Qiagen). The cells were suspended in buffer A (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5% glycerol) and lysed by ultrasonication. Cell debris was removed by centrifugation at 18,000g for 60 min. Protein was eluted with a linear gradient of 20–500 mM imidazole. The fractions were examined by SDS-PAGE, and the N-terminal 63 His-tag was removed by TEV protease on an extra Ni-NTA column. Purified protein was loaded onto a Superdex S200 column (GE Healthcare) equilibrated with buffer A. Fractions containing the protein were concentrated to 20 mg mL21 and stored at 280 C until further use. Full-length hTDO was unstable, and various truncations were constructed. A truncation containing amino acids 19– 388 (aa 19–388) was found to have good solubility and stability. Expression and purification of this truncation was performed in the same way as that for wild type, and the truncation was used for crystallization and further studies.
Site-directed mutagenesis
The Quick Change Site-Directed Mutagenesis Kit (Stratagene) was used to generate hTDO (aa 19–388) mutants. Eight mutants (Y42A, Y45A, F72A, H76A, F140A, R144A, S151A, and H328A) were constructed and verified by DNA sequencing. Expression and purification of the mutants were performed using the same method described above.
Kinetic analysis and spectroscopy
The enzymatic activity of hTDO was measured as described previously,5 with some minor modifications. The ferric hTDO (at a final concentration of 1 mM) was rapidly mixed with excess freshly prepared L-ascorbate (at a final concentration of 500 mM) in buffer A at 25 C and incubated for 120 s to maintain the enzyme in the ferrous state. The reaction was initiated by the addition of different concentrations of L-Trp, and the formation of NFK was monitored based on the absorbance increase at 321 nm (e321 5 3750 M21 cm21)5 using a U2010 spectrophotometer (Hitachi). The data were analyzed by Michaelis– Menten curve fitting with Origin software (OriginLab). The optical absorption spectra experiments were measured in buffer A at 25.0 C using a DU 800 UV–visible spectrophotometer (Beckman Coulter). The final concentrations of all samples were adjusted to 10 mM. In order to make hemin reduce to heme, excess freshly prepared PROTEINS
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L-ascorbate
was added before the spectrum scanning. The data were analyzed by Origin software (OriginLab).
Table I
Data Collection and Refinement Statisticsa hTDO (aa 19–388)
Isothermal titration calorimetry assay
Apo hTDO (aa 19–388) was prepared in the following way: Hemin was omitted from the culture medium during protein expression, so that only a basal level of hTDO could capture heme from the medium. After further purification, when no heme absorption was detected, the protein was defined as apo hTDO and used for the isothermal titration calorimetry (ITC) assay. ITC experiments were performed using a MicroCal iTC200 instrument (MicroCal). Hemin or L-Trp in a syringe was titrated into a sample cell containing apo hTDO at 25 C, and the resulting isotherm was fitted with Origin software (OriginLab). All the samples were prepared in buffer containing 100 mM Tris–HCl (pH 8.0) and 500 mM NaCl. Other components in the solution, except the titrant or titrate, were adjusted to the same levels as before titration. Crystallization and data collection
Full-length holo hTDO and various truncations were concentrated to 20 mg mL21 and screened for crystallization using the hanging drop vapor diffusion method at 16 C. Crystallization drops were made by mixing 1 lL of the protein solution with 1 lL of reservoir solution. Diffraction quality crystals were grown from a truncation consisting of aa 19–388. The crystals were grown for 3 days in the presence of 0.1 M sodium cacodylate (pH 6.0–6.5), 6–10% (w/v) MPEG 5000, and 10% (v/v) 2-propanol. Crystallization was then optimized using an additive screen kit (Hampton Research), and hexammine cobalt (III) chloride was found to improve the diffraction significantly. Crystals were harvested with 25% (v/v) glycerol as a cryoprotectant. X-ray diffraction data were collected at a wavelength of 0.9796 A˚ on beamline 22-ID at Advanced Photon Source, Argonne National Laboratory. Structure determination and refinement
The structure of hTDO (aa 19–388) was solved via the molecular replacement method with Phaser13 using the structure of XcTDO [Protein Data Bank (PDB) code, 2NW7] as the searching model.6 Model building and structure refinement were performed manually in Coot14 and automatically in PHENIX.15 Data collection and refinement statistics are presented in Table I. Atomic coordinates and structure factors have been deposited in the PDB with accession code 4PW8. RESULTS AND DISCUSSION Overall structure of hTDO
The crystal structure of hTDO (aa 19–388) was solved by molecular replacement and refined to 2.90 A˚ resolu-
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Data collection Beamline Wavelength () Space group Cell dimensions a, b, c () a, b, c ( ) Resolution () No. of observed reflections No. of unique reflections Completeness (%) Rmerge Redundancy Refinement Rwork/Rfree (%) RMSD Bond length () Bond angle ( ) Ramachandran plot (%) Favored Allowed Disallowed
APS, 22-ID 0.9796 P212121 134.33, 156.92, 160.33 90.00, 90.00, 90.00 50.00–2.90 (2.95–2.90) 492,077 75,459 99.9 (100.0) 0.044 (0.450) 27.84 (3.26) 6.5 (6.6) 20.39/22.22 0.012 1.380 97.26 2.36 0.38
RMSD, root-mean-square deviation. a Values in parentheses are for the highest resolution shell.
tion. The crystal belongs to space group P212121. One asymmetric unit consists of eight molecules of the protein based on the calculated solvent content of 49.1%. No systematic global differences are observed among the eight monomers, although they show variation at some loop regions. The electron densities of the major part are clear, whereas the densities of N-terminal residues 19–40 and C-terminal residues 383–388 are invisible. Residues 170–186, and 339–358 are also missing in the electron density map. In addition, the electron densities of residues 241–252 are invisible in three molecules. These residues are positioned at the loop regions or interacting surfaces of the active site. Furthermore, the cofactor heme is missing in the binding pocket, as its electron density is invisible. Thus, the structure is assumed as apo hTDO. The hTDO monomer comprises 15 a-helices and no b-strands [Fig. 1(A)], and, like DmTDO,8 can be divided into three major regions. The N-terminal region consists of residues 41–65 (residues 42–45 a1, 47–51 a2, and 58– 61 a3). The large domain contains residues 66–213 (residues 67–99 a4, 105–134 a5, 137–144 a6, 156–165 a7, 188–197 a8, and 201–211 a9) and 281–360 (residues 286–298 a13, and 305–335 a14). The small domain includes residues 214–280 (residues 222–242 a10, 251– 268 a11, and 270–279 a12) and residues 360–382 (residues 363–371 a15). The size exclusion chromatography profile suggests that holo hTDO (aa 19–388) exists as a tetramer in
Structure of Human Tryptophan 2,3-Dioxygenase
Figure 1 Overall structure of hTDO. (A) A ribbon representation of the hTDO monomer with secondary structural a helices (a1–15) labeled. The Nterminal region, large domain and small domain are colored in blue, cyan, and orange, respectively. The invisible residues are shown as dotted lines. (B) Overall structure of the hTDO tetramer viewed in two orientations. The four monomers A, B, C, and D are colored in green, red, yellow, and magenta, respectively. (C) Overall structure of the hTDO dimer viewed in two orientations. The two monomers are colored in green and red. Helices a1, a4, and a5 are labeled in monomers A and B. The swapped N-terminal segments are indicated by the oval circle.
solution, whereas apo hTDO (aa 19–388) exists as a dimer (Supporting Information Fig. S1). This result indicates that heme plays an important role in the oligomerization of hTDO. In our structure, the eight protein molecules in one asymmetric unit are packed as two tetramers, which was calculated by PDBePISA (www.ebi.ac. uk/msd-srv/prot_int/pistart.html). The tetramerization is similar to that observed in the structures of XcTDO, RmTDO and DmTDO.6–8 Generally, the four monomers (represented by ABCD) in the tetramer are related by
three mutually perpendicular twofold axes [Fig. 1(B)]. The total solvent-accessible surface area that is buried upon tetramerization is 16,360 A˚2, with the core of tetrameric surfaces formed by the a14 helices in each monomer. Adjacent monomers have more extensive interactions at the A–B interface than at the A–C interface, burying total solvent-accessible surface areas of 5370 and 2270 A˚2 upon dimerization, respectively. Therefore, the tetramer can be viewed as a dimer of dimers, in which the two C-shape dimers (AB and CD) are clamped PROTEINS
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perpendicularly to each other to form a tight tetramer. The dimerization of monomers A and B is shown in Figure 1(C). Helices a4 and a5 in the large domain of one monomer interact with a4 and a5 in the other monomer, respectively, which contributes to the majority of the dimer formation surfaces. In addition, the Nterminal segments of the two monomers are swapped, with each a1 helix extended into the active site of the other. Comparison of hTDO with known orthologues
Sequence alignment indicates that hTDO shares approximately 60% sequence identity with DmTDO, but only 30% identity with XcTDO and RmTDO [Fig. 2(A)]. A structural homology search using DaliLite (www.ebi.ac.uk/ Tools/structure/dalilite) shows that the overall structure of hTDO is highly similar to those of DmTDO8 [PDB code, 4HKA; Z-score, 33.1; root-mean-square deviation (RMSD), 1.4 A˚], XcTDO6 (PDB code, 2NW7; Z-score, 19.2; RMSD, 2.2 A˚), and RmTDO7 (PDB code, 2NOX; Zscore, 19.9; RMSD, 2.0 A˚). Superposition of the hTDO structure with those of DmTDO, XcTDO, and RmTDO shows that the core domain frameworks in these TDOs are highly conserved, whereas some loop regions surrounding the active site in hTDO are very flexible [Fig. 2(B)]. This finding indicates that these proteins may share the same molecular mechanism of substrate recognition and catalysis. Specifically, hTDO and DmTDO both contain an extra small domain, which interacts with the active site of an adjacent monomer and is the major difference between eukaryotic and prokaryotic TDOs. Because heme can be found in the structures of the other three TDOs but not in hTDO, we analyzed the regions around the heme binding site, which showed weak electron density and could not be fixed in our structure. Residues 170–186 are missing in the structure of hTDO; they form a short a-helix (6 residues) in XcTDO and RmTDO but a long insertion fragment (20 residues) in DmTDO. Residues 339–358 are also missing from hTDO; these residues form an a-helix and a flexible loop in the structures of the orthologues. Although hTDO and hIDO share only approximately 10% sequence identity, the large domains of hTDO and hIDO show similar folds. The overall structure and spatial orientation of the small domain differ between hTDO and hIDO [Fig. 2(C)]. In hIDO, the connecting loop and a-helix F in the small domain contribute to the formation of the active site. Both the active site and the small domain are located on the same end of the large domain.9 In hTDO, the active site is located on one end of the large domain, but the small domain is located on the other end and is far away from the active site. The complete formation of the active site requires the N-terminal region of an adjacent monomer and the small domain of another adjacent monomer. Therefore,
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hIDO can display activity as a monomer; however, hTDO must form an oligomer to exhibit activity. Enzymatic activity and mutagenesis of hTDO
The enzymatic activities of the wild type hTDO, the truncation aa 19–388 and eight point mutants were measured. The kinetic parameters are presented in Table II. The Km and kcat of wild type hTDO are 0.135 6 0.011 mM and 1.071 6 0.036 s21, respectively, which are comparable with the results reported previously.5 The truncation aa 19–388 shows 13.33% increase in Km and 8.87% decrease in kcat, suggesting that this truncation does not significantly affect the activity. All eight residues selected for mutagenesis are conserved [Fig. 2(A)] and are located around the active site. Compared with the template aa 19–388, all the mutants showed a major decrease in enzymatic activity. Mutants Y42A, Y45A, F140A, R144A, and S151A remain only 0.50%, 1.13%, 0.25%, 0.88%, and 9.08% relative activity of the wild type protein, respectively. In fact, no activity could be detected for mutants F72A, H76A, and H328A. The results indicate that these residues may play critical roles in catalytic reaction or substrate binding. To investigate the function of the eight residues, hTDO was superimposed with the DmTDO–heme complex8 and the XcTDO–heme–Trp complex (PDB code, 2NW8).6 The relative spatial position of these residues, heme, and L-Trp is shown in Figure 2(D), with heme and L-Trp from the DmTDO–heme complex and the XcTDO–heme–Trp complex, respectively. The result shows that the spatial conformations of these eight residues are almost the same in hTDO and DmTDO, except for S151 (S135 in DmTDO). Y42 and Y45 (Y27 and Y30 in DmTDO) are located in the outspread N-terminal segment and participate in the binding of the substrate L-Trp. R144 (R128 in DmTDO) also plays role in the recognition of L-Trp. F72, H76, and F140 (F57, H61, and F124 in DmTDO) are involved in binding both L-Trp and heme. In addition, H328 (H312 in DmTDO) binds with heme on the other side of the plane, which is consistent with the previous report.6 S151 takes part in binding with heme, whereas S135 in DmTDO does not. This difference may contribute to the difference in activity between hTDO and DmTDO. The kcat/Km ratios of hTDO and DmTDO are 7.93 mM21s21 and 1.05 mM21s21,8 respectively. Unfortunately, some residues involved in substrate recognition and heme binding are located in the invisible regions of the hTDO structure. Consequently, more detailed information regarding the missing residues must be obtained to clarify the significant activity difference between hTDO and DmTDO. Heme is indispensible for hTDO enzymatic activity [Supporting Information Fig. S2(A)], and the kinetic assays have confirmed the presence of heme in the holo hTDO solution. Unfortunately, the heme cofactor could
Structure of Human Tryptophan 2,3-Dioxygenase
Figure 2 Comparison of hTDO with other TDOs and hIDO. (A) Sequence alignment of hTDO with DmTDO, XcTDO, and RmTDO. The secondary structural elements of hTDO are depicted at the top of the alignment. Residues for mutagenesis are marked by green stars. (B) Upper: structural comparison of hTDO (magenta) with DmTDO (cyan), XcTDO (marine) and RmTDO (gray). Heme is shown as a ball-and-stick model in red. Lower: superimposition of the secondary structures around the active site between hTDO (magenta) and DmTDO (cyan). The helices are represented with the cylindrical mode. Two fragments that exist only in the structure of DmTDO are shown in green and yellow, respectively. Heme is from the DmTDO–heme complex and is shown as a ball-and-stick model in red. (C) Structural superimposition of hTDO with hIDO. The large domain and the N-terminal region of the adjacent monomer in hTDO are shown in magenta, and the small domain of another adjacent monomer is shown in bright pink. The large domain and the connecting loop of hIDO are shown in yellow, and the small domain is shown in orange. Heme is shown as a ball-and-stick model in red. (D) Comparison of the critical residues around active site between hTDO and DmTDO. Heme and L-Trp are shown as ball-and-stick models in red and yellow, respectively. The residues of hTDO are labeled and shown as a ball-and-stick model in magenta, and the residues of DmTDO are shown in cyan. Y42 and Y45 are from the N-terminal region of the adjacent monomer.
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Table II
Kinetic Parameters of hTDOa
Wild type aa 19–388 Y42A Y45A F140A R144A S151A F72A H76A H328A
Km (mM)
kcat (s21)
kcat/Km (mM21s21)
Relative activity (%)b
0.135 6 0.011 0.153 6 0.012 0.227 6 0.024 0.661 6 0.087 1.531 6 0.194 0.631 6 0.033 0.146 6 0.016 ND ND ND
1.071 6 0.036 0.976 6 0.033 0.010 6 0.001 0.062 6 0.004 0.032 6 0.002 0.045 6 0.001 0.105 6 0.004 ND ND ND
7.93 6.38 0.04 0.09 0.02 0.07 0.72
100.00 80.45 0.50 1.13 0.25 0.88 9.08
ND, not detected. a The kinetic parameters are the mean 6 SD of triplicate determinations. b The kcat/Km ratio of the wild type is defined as 100.00%.
not be located in the crystal structure, which means that in our condition the protein–heme interaction is probably too weak to anchor heme. Spectrum scanning results reveal red shift of heme absorbance when incubated with apo hTDO [Supporting Information Fig. S2(B)]. ITC results have shown that neither heme nor LTrp alone displays detectable interaction with apo hTDO [Supporting Information Fig. S3(A,B)], and hTDO can catalyze L-Trp only in the presence of heme [Supporting Information Fig. S3(C)]. These results indicate that hTDO and heme may not form a stable complex in solution. Many methods have been utilized in an attempt to obtain the hTDO–heme complex and the hTDO–heme– L-Trp complex, including the addition of extra hemin and the introduction of L-Trp in the purification procedure, cocrystallization of protein with L-Trp or its analogues, crystallization using hemin as an additive, substitution of hemin with its analogue zinc protoporphyrin, crystallization in an anaerobic environment, and a combination of these strategies. However, the results showed that heme was not identified in the crystal structure. More biophysical analyses are currently being performed to decipher the dynamics among hTDO, heme, and L-Trp.
ACKNOWLEDGMENTS The authors thank the staff at beamline 22-ID of the Advanced Photon Source for assisting with X-ray diffraction data collection. REFERENCES 1. Sono M, Roach MP, Coulter ED, Dawson JH. Heme-containing oxygenases. Chem Rev 1996;96:2841–2888. 2. Takikawa O. Biochemical and medical aspects of the indoleamine 2, 3-dioxygenase-initiated L-tryptophan metabolism. Biochem Biophys Res Commun 2005;338:12–19. 3. Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 2004;4:762–774.
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