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Subunit structure of recombinant rat liver l -tryptophan 2,3-dioxygenase
International Congress Series 1233 (2002) 161 – 169
Subunit structure of recombinant rat liver l-tryptophan 2,3-dioxygenase Surya Prasad Manandhar *, Hideo Shimada, Shingo Nagano, Tsuyoshi Egawa, Yuzuru Ishimura Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal Department of Biochemistry, School of Medicine, Keio University, Shinanomachi 35, Shinjyuku-ku, Tokyo 160-8582, Japan
Abstract Rat liver L-tryptophan 2,3-dioxygenase (TDO) with a histidine tag at the N-terminus was expressed in Escherichia coli JM109 harboring plasmid pUC18 carrying the full-length cDNA of TDO. The recombinant enzyme was purified to near homogeneity by employing conventional purification methods including nickel-chelate immobilized resin column chromatography. The purified enzyme had a turnover number per heme 303 min 1 with similar spectral properties to those of native rat liver enzyme. SDS-PAGE of purified TDO preparation showed two distinct bands with molecular masses of 49 and 46 kDa. N-terminal sequence analysis of the components revealed that the 46-kDa species is shorter than the 49-kDa one by 19 amino acid residues including six histidine residues at the end. Thus, a limited proteolysis appeared to occur between Tyr13 and Thr14 of the original polypeptide chain. The construct of recombinant TDO with deletion of the N-terminal 13 residues gave a single band on SDS-PAGE with a molecular mass of about 46 kDa. The N-terminal truncation had no effect on the catalytic activity nor on the spectral properties. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rat liver; L-tryptophan 2,3-dioxygenase; Recombinant protein; Expression in Escherichia coli; Limited proteolysis
* Corresponding author. Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. Tel.: +977-1-331869/483498; fax: +977-1-331964. E-mail address: [email protected] (S.P. Manandhar).
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1. Introduction L-Tryptophan 2,3-dioxygenase (EC 1.13.1.12, TDO) is a hemoprotein that catalyzes the conversion of tryptophan to N-formylkynurenine by the introduction of two atoms of oxygen between the second and third carbon atoms of indole ring of L-tryptophan. TDO had been isolated and characterized from different sources, including bacteria [1,2], rat and other mammalian livers [3,4], Streptomyces parvulus [5], yeast [6] and human [7]. Among them, the enzymes from rat liver and Pseudomanas were reported to be purified to near homogeneity. In spite of these studies, it remained elusive about subunit composition, heme content and reaction mechanism of this enzyme. Based on the results of PAGE at pH 12, which showed double bands, Schutz and Feigelson [3] have concluded that the rat liver TDO is a tetrameric enzyme with a2h2-structure containing two hemes per tetramer. Similarly, mouse liver [4] and recombinant rat liver TDO [8] showed double bands on SDS-PAGE. However, only single gene for TDO has been so far identified in rat, as well as in human [7,9]. In order to understand the subunit structure of rat liver TDO, we established an expression system for rat liver TDO in E. coli here and characterized the recombinant enzyme by creating a truncated enzyme.
2. Experimental procedures 2.1. Construction of expression vector Plasmid pTO14B carrying a full-length cDNA of rat liver tryptophan 2,3-dioxygenase was a generous gift of Prof. T. Nakamura of Osaka University, Japan. For the construction of an expression vector of TDO, the cDNA was first amplified with introduction of a six-histidine tag and restriction sites (Nde1 and Sal1 at 5V- and 3V-ends, respectively) by using polymerase chain reaction (PCR). The PCR product was digested with Nde1 and Sal1 and then ligated into pUC 18 along with the DNA fragment of 5Vuntranslated region from the cytochrome P450cam gene with restriction sites EcoRI and Nde1 at 5V and 3V, respectively, using ligation solution I version 2.0 (TaKaRa, Japan). The ligated plasmid was transformed into E. coli JM109. Truncated TDO was obtained by eliminating the sequences encoding the first 13 amino acid residues from the fulllength cDNA by PCR using the following primers: 5VGCCATATGCATCACCATCATCATCACACT TTGAAA AACTTATCT ATG G3V as forward and M13 universal primer as reverse primer. The forward primer contained Nde1 site (underlined) and sequence encoding six histidine residues as a tag (doubly underlined). The sequence of the PCR amplified cDNA of TDO was confirmed by the conventional DNA sequencing. 2.2. Expression of TDO TDO was expressed in E. coli JM109 by inoculating into terrific broth, supplemented with 100 Ag/ml ampicillin and 1 mM isopropyl-h-D-thiogalactopyranoside and incubated
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at 30 jC in a rotary shaker with a speed of 150 rpm for 13– 14 h. TDO in the E. coli cells was monitored by taking a difference spectrum of ferrous –carbon monoxide minus ferrous form of TDO in the culture. Cells were harvested by centrifugation at 12,000g for 20 min at 4 jC and stored at 20 jC until use. 2.3. Purification of TDO Fifty grams of E. coli cell paste frozen at 20 jC was thawed at 4 jC for overnight and cell paste was lightly suspended in 500 ml of 0.05 M potassium phosphate buffer, pH 7.8, containing 10 mM L-tryptophan (buffer A) in an electrical blender and treated with 50 mg lysozyme (Sigma) and 1 mg of pancreatic DNAse I (Merck). Ten tabs of protease inhibitor cocktail (Roche Biochemical) were added to the cell suspension. The mixture was stirred for about 1.5 h at room temperature to lyse the bacterial cells and then next 30 min at 4 jC. The following procedures were performed at 4 jC unless stated otherwise. The cell lysate was then centrifuged at 12,000 rpm for 30 min. The supernatant thus obtained was passed through a DEAE –cellulose column (415 cm) equilibrated with buffer A. TDO was eluted with a linear gradient formed between equal volume of buffer A and buffer A containing 0.6 M NaCl and 20 ml fractions were collected. Fractions containing enzyme activity more than 0.2 U/ml were pooled together and subjected to 20% and 35% ammonium sulfate fractionation. Fraction precipitated with 35% ammonium sulfate was recovered by centrifugation at 12,000 rpm for 20 min. The pellet was dissolved in minimal volume of buffer A and dialyzed overnight against buffer A to remove ammonium sulfate. The dialysate was then centrifuged at 5000 rpm for 10 min and further purified by Nichelate immobilized column chromatography (Ni-NTA superflow agarose, QIAGEN) by stepwise elution with 20, 50, 100 and 300 mM imidazole in 20 mM potassium phosphate buffer, pH 8.0, containing 10 mM L-tryptophan and 300 mM NaCl. The highly concentrated brown red colored TDO was then dialyzed against buffer A to remove imidazole. The dialysate was concentrated using Centricon 30. The highly concentrated solution of TDO was then passed through a Sephadex G-25 column equilibrated with 0.05 M potassium phosphate buffer, pH 7.8 to remove L-tryptophan. This solution devoid of Ltryptophan was used as enzyme preparation for all assays, spectral measurements and SDS-PAGE. 2.4. Western blot analysis Expression of recombinant TDO was checked by Western blot analysis. Cell lysate proteins or purified proteins (2.5 Ag) were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Milipore) with the transfer buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol at 8 V for 45 min on a semidry electroblot apparatus (Bio-Rad). The membrane was blocked in 20 mM Tris –HCl, pH 7.5, containing 0.15 M NaCl (TBS) supplemented with 3% bovine serum albumin. The membrane was washed several times with TBS supplemented with 0.05% Tween 20 and 0.1% Triton X-100 (TBSD) and incubated with monoclonal anti-His tag antibody (Qiagen) for 1 h at room temperature. The membrane was washed again with TBSD and then incubated with secondary antibody conjugated with horseradish perox-
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idase for overnight. Protein was visualized by incubating with a solution containing diaminobenzidine as chromogenic substrate. 2.5. Enzyme assays L-Tryptophan 2,3-dioxygenase activity was measured in an assay mixture containing 100 mM potassium phosphate, pH 7.0, and 10 mM L-tryptophan under aerobic conditions at 25jC. The reaction was started by adding the enzyme reduced with a minimum amount of dithionite under anaerobic conditions, and the formation of N-formylkynurenine was monitored spectrophotometrically at 321 nm. The enzyme activity was expressed as turnover number per heme and minute. Alternatively, the enzyme activities of cell extract were measured in the above reaction mixture supplemented with ascorbic acid and expressed in units per milligram of protein, where 1 U is defined as micromole of Nformylkynurenine formed per minute. Km value for L-tryptophan was determined from a curve of the activities (initial velocities) vs. concentrations of L-tryptophan added into the assay mixture by best fitting to the curve using IgorPro software (WaveMetrics, Lake Oswego, OR).
2.6. Optical spectrophotometry Absorption spectra of ferric, ferrous and ferrous– CO complex of purified wild-type and truncated forms of TDOs were measured on a Hitachi spectrophotometer U-3000 or Perkin Elmer spectrophotometer Lambda 18 in a wavelength region between 380 and 700 nm in the presence and absence of L-tryptophan. 2.7. Other determinations Protein content was determined by Bradford method using Bradford reagent (BioRad) after removal of L-tryptophan. BSA (Sigma) was used as a standard. Heme content was determined by pyridine hemochromogen method [10]. Protein content of purified TDO preparation was determined by amino acid analysis after hydrolysis in 5.7 N HCl in vacuo Table 1 Properties of recombinant and nativea rat liver tryptophan 2,3-dioxygenases Properties
Recombinant
Native1
Molecular mass (kDa) Subunit molecular mass (kDa) Number of subunits Heme Heme content Turnover number Km for l-tryptophan (mM) Optimum pH
200 46 and 49b 4 (a2h2) Protoheme IX 2 303 0.05 7.6 – 8.0
168 45 – 47 4 (a2h2) Protoheme IX 2 200 – 323 0.1 – 0.2 7.2
a Data of the native enzyme were taken from Schutz and Feigelson [3] except for the turnover number of the enzyme, which were from Makino et al. [11] and Uchida et al. [12]. b cDNA sequence of rat liver TDO by Maezono et al. [9] predicts 48 kDa as a subunit molecular mass.
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at 110 jC. The analysis was performed on Beckman amino acid analyzer at Toray Research Center, Japan. N-terminal amino acid sequence of the proteolytically cleaved fragment of TDO was analyzed on a protein sequencer at Takara Biomedical, Japan. For this, the fractionated protein bands were transferred onto PVDF membrane from polyacrylamide gel and visualized by staining with Coomassie brilliant blue. The protein bands with molecular mass of 46 and 49 kDa were excised from the membrane and analyzed.
Fig. 1. Absorption spectra of recombinant rat liver L-tryptophan 2,3-dioxygenase. The spectra of TDO were measured at 25 jC in K phosphate buffer, pH 7.0. Ferrous form of the enzyme was produced by the reduction with dithionite and ferrous-CO form was obtained by gentle blowing of CO gas into the surface of solution containing the ferrous form of enzyme. (A) In the absence of L-tryptophan. (B) In the presence of 10 mM L-tryptophan.
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2.8. Chemicals and reagents L-Tryptophan was purchased from Sigma, USA. Plasmid pUC 18, pyrobest buffers and Taq polymerase, restriction enzymes, ligation solution I version II were purchased from TaKaRa Biomedicals, Japan.
3. Results 3.1. Purification and characterization of recombinant L-tryptophan 2,3-dioxygenase In addition to the recombinant TDO with a N-terminal His tag, we also constructed untagged and C-terminal His-tagged TDOs. Among the three, TDO with the N-terminal His tag was most highly expressed under the present expression conditions, and therefore employed throughout this study. TDO showed purity more than 90% as estimated by SDSPAGE and turnover number about 303 min 1, which is slightly higher than reported previously for native rat liver TDO [11]. Other properties of the recombinant enzyme were compared with native rat liver enzyme [3,11,12] and summarized in Table 1. Optical measurements of TDO showed spectral properties characteristic to those of rat liver TDO as shown in Fig. 1A. Ferric form of this enzyme showed Soret band at 406 nm and other bands at 498 and 632 nm, whereas ferrous form showed Soret band at 430 nm and another band at 557 nm. Ferrous – CO complex exhibited its Soret band at 420 nm and a- and hbands at 569 and 538 nm, respectively. In the presence of 10 mM L-tryptophan (Fig. 1B),
Fig. 2. SDS-PAGE of wild-type and truncated L-tryptophan 2,3-dioxygenases. Lane 1: purified wild type TDO. Lane 2: purified truncated TDO.
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the Soret band of ferric form shifted to 407 nm, whereas that of ferrous form to 421 nm. Ferrous – CO complex did not show any significant change in Soret and other bands. 3.2. Subunit structure of recombinant L-tryptophan 2,3-dioxygenase SDS-PAGE showed the presence of two distinct bands at 49 and 46 kDa (Fig. 2, Lane 1). The latter band was, however, not detected by anti-His-tag antibody (data not shown), suggesting a loss of N-terminal sequence carrying the His tag. N-terminal amino acid analysis of this band revealed that it is shorter by 19 amino acid residues from the Nterminus due to cleavage of peptide bond between Tyr19 and Thr20 in the polypeptide chain (Tyr13 and Thr14 in the original peptide). Construction of truncated TDO with elimination of this fragment revealed a single band on SDS-PAGE (Fig. 2, Lane 2), suggesting that doubling of TDO band was a result of limited proteolysis. Interestingly, truncated TDO did not show any alteration in enzyme activities and spectral properties compared to those of full-length wild type. The specific activity of truncated TDO in the crude cell extracts was 0.113 U/mg protein, which is very close to that of the wild-type enzyme (0.137 U/mg protein). Physiological significance of such a truncation remains unknown at present.
4. Discussion We have expressed, purified and characterized rat liver TDO in terms of its activity and spectral properties. The wild-type recombinant TDO showed many properties similar to native rat liver enzyme (Table 1). Its turnover number per heme was found to be 303 min 1, which was higher than the reported value (200 – 250 min 1) by Schutz and Feigeson [3] for rat liver enzyme, but was considerably lower than that of Pseudomonas enzyme (1100 min 1) [11]. It was close to 323 min 1, the highest value so far reported for rat liver TDO by Uchida et al. [13]. Although, both rat liver and Pseudomonas enzymes show maximal activity around pH 7.0 – 7.2, recombinant TDO was active at a pH range between 7.6 and 8.0 at both saturating (10 mM), as well as subsaturating concentration of L-tryptophan. Km value for L-tryptophan was 0.05 mM, which is slightly lower than reported; 0.1– 0.2 mM for rat liver [13] and 0.21 for mouse liver [4]. The recombinant TDO had spectral properties indistinguishable from those of native enzyme. Thus, in many aspects recombinant TDO showed properties very similar to those of native enzyme, suggesting that recombinant enzyme does not undergo undesirable modifications in host cells and the addition of an Nterminal His tag does not affect enzyme properties in a significant manner. One of the significant findings about TDO structure in this paper is that we have found the reason for the doubling of TDO band in polyacrylamide gel electrophoresis. As previously observed for other TDO preparations [3,4,8], recombinant TDO showed double bands in SDS-PAGE with molecular masses of 49 and 46 kDa. For native rat liver enzyme, such double bands have been ascribed to the presence of different subunits in the enzyme, i.e. to an a2h2-structure [3]. Similar double bands were reported for the case of recombinant rat liver TDO expressed in E. coli DH5a [8]. It is worthy of note that the results obtained by Watanabe et al. [4] on mouse liver TDO showed a single band on 7.5%
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native PAGE, but two bands on 10% SDS-PAGE. The authors are, however, in agreement with the concept that TDO is composed of two pairs of identical subunits (a2h2). We also observed two distinct bands from purified recombinant TDO on 10% SDS-PAGE (Fig. 2, Lane 1). No such double bands were however noticed on Western blot analysis using antiHis tag antibody with which only a single band with a molecular mass around 49 kDa was observed. The result suggests that the observed second band at 46 kDa was devoid of the N-terminal sequence containing His tag. Base on these findings we concluded that the second band was a product of limited proteolysis of the 49-kDa band. N-terminal sequence analysis of this second band, recovered from PVDF membrane after having transferred from polyacrylamide gel showed that this band had lost 19 amino acid residues from Nterminus as a result of cleavage occurring between amino acid residues Tyr19 and Thr20 (Tyr13 and Thr14 in the original sequence). A truncated form of TDO constructed by eliminating the fragment showed a single band confirming that the appearance of double bands was a result of the proteolytic cleavage at the site mentioned. We further analyzed rat liver extract along with purified native and recombinant rat liver TDO by Western blot using anti-TDO antibody and found that the location of the bands was very similar to that of purified ones (unpublished observation). In addition to this, only one gene for TDO has so far been known for both rat as well as human [7,9]. These data taken together indicate that there is only one gene for TDO and the doubling of SDS-PAGE band is associated with limited proteolysis. It is however of interest to note that if this is nonspecific proteolysis occurring with TDO subunits, then question may arise why not all of TDO molecules (or all of its subunits) undergo this kind of cleavage. The physiological significance of this proteolysis is also unknown at present; truncation of this fragment did not cause any detectable change in spectral properties and enzyme activity. Based on these data we assumed that TDO subunits have different orientation in the tetrameric enzyme structure and accordingly have variable susceptibility to protease digestion. In any case, further studies (including crystal structure) will be required to elucidate the biological significance of such a kind of proteolytic modification.
5. Conclusion Based on the above data we conclude that we have established expression system for Ltryptophan 2,3-dioxygenase in E. coli JM109 and our recombinant enzyme resembles native rat liver enzyme in many aspects. It can be used for further investigation regarding native properties and also for mechanism of catalysis, etc. It is a tetrameric enzyme consisting of four identical subunits, encoded by single gene. The doubling of the enzyme on SDS-PAGE is associated with proteolytic cleavage of the original native subunit as deduced by Maezono et al. [9] at peptide bond between Tyr13 and Thr14.
Acknowledgements The authors are thankful to Professor T. Nakamura of Osaka University, Japan, for providing us the full-length cDNA of TDO. The authors are highly indebted to Drs. K.
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Mukai, Y. Katayama and K. Shimokata for their technical advice and discussions throughout this research, as well as during the preparation of this manuscript. We also thank the Japan Society for Promotion of Science (JSPS) for the support that enabled Dr. Surya P. Manandhar to stay in Japan during the period of May 01, 1999– April 30, 2000. This investigation was supported in part by grants from Ministry of Education, Science, Culture, Sports and Technology of Japan, and Keio University.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
Y. Ishimura, Methods Enzymol. 17A (1970) 429 – 434. M. Matsumura, K. Osada, S. Aiba, Biochim. Biophys. Acta 786 (1984) 9 – 17. G. Schutz, P. Feigelson, J. Biol. Chem. 242 (1972) 5327 – 5332. Y. Watanabe, M. Fujiwara, R. Yoshida, O. Hayaishi, Biochem. J. 189 (1980) 393 – 405. M.J.M. Hitchcock, E. Katz, Arch. Biochem. Biophys. 261 (1980) 148 – 160. Y. Iwamoto, I.-S. Lee, M. Tsubaki, R. Kido, Can. J. Microbiol. 41 (1995) 19 – 26. D.E. Comings, D. Muhleman, G. Diez, M. Sherman, G.L. Forest, Genomics 29 (1995) 390 – 396. S. Res, H. Liu, E. Licad, M.A. Correia, Arch. Biochem. Biophys. 333 (1996) 96 – 102. K. Maezono, K. Tashiro, T. Nakamura, Biochem. Biophys. Res. Commun. 170 (1990) 176 – 181. K.G. Paul, H. Theorell, A. Akesson, Acta Chem. Scand. 7 (1953) 1284 – 1287. R. Makino, K. Sakaguchi, T. Iizuka, Y. Ishimura, J. Biol. Chem. 255 (1980) 11883 – 11891. K. Uchida, T. Shimizu, R. Makino, K. Sakaguchi, T. Iizuka, Y. Ishimura, T. Nozawa, M. Hatano, J. Biol. Chem. 258 (1983) 2519 – 2525. [13] K. Uchida, M. Usami, H. Bandow, I. Harada, Biochim. Biophys. Acta 1121 (1992) 153 – 159.
Subunit structure of recombinant rat liver l-tryptophan 2,3-dioxygenase Surya Prasad Manandhar *, Hideo Shimada, Shingo Nagano, Tsuyoshi Egawa, Yuzuru Ishimura Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal Department of Biochemistry, School of Medicine, Keio University, Shinanomachi 35, Shinjyuku-ku, Tokyo 160-8582, Japan
Abstract Rat liver L-tryptophan 2,3-dioxygenase (TDO) with a histidine tag at the N-terminus was expressed in Escherichia coli JM109 harboring plasmid pUC18 carrying the full-length cDNA of TDO. The recombinant enzyme was purified to near homogeneity by employing conventional purification methods including nickel-chelate immobilized resin column chromatography. The purified enzyme had a turnover number per heme 303 min 1 with similar spectral properties to those of native rat liver enzyme. SDS-PAGE of purified TDO preparation showed two distinct bands with molecular masses of 49 and 46 kDa. N-terminal sequence analysis of the components revealed that the 46-kDa species is shorter than the 49-kDa one by 19 amino acid residues including six histidine residues at the end. Thus, a limited proteolysis appeared to occur between Tyr13 and Thr14 of the original polypeptide chain. The construct of recombinant TDO with deletion of the N-terminal 13 residues gave a single band on SDS-PAGE with a molecular mass of about 46 kDa. The N-terminal truncation had no effect on the catalytic activity nor on the spectral properties. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rat liver; L-tryptophan 2,3-dioxygenase; Recombinant protein; Expression in Escherichia coli; Limited proteolysis
* Corresponding author. Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. Tel.: +977-1-331869/483498; fax: +977-1-331964. E-mail address: [email protected] (S.P. Manandhar).
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1. Introduction L-Tryptophan 2,3-dioxygenase (EC 1.13.1.12, TDO) is a hemoprotein that catalyzes the conversion of tryptophan to N-formylkynurenine by the introduction of two atoms of oxygen between the second and third carbon atoms of indole ring of L-tryptophan. TDO had been isolated and characterized from different sources, including bacteria [1,2], rat and other mammalian livers [3,4], Streptomyces parvulus [5], yeast [6] and human [7]. Among them, the enzymes from rat liver and Pseudomanas were reported to be purified to near homogeneity. In spite of these studies, it remained elusive about subunit composition, heme content and reaction mechanism of this enzyme. Based on the results of PAGE at pH 12, which showed double bands, Schutz and Feigelson [3] have concluded that the rat liver TDO is a tetrameric enzyme with a2h2-structure containing two hemes per tetramer. Similarly, mouse liver [4] and recombinant rat liver TDO [8] showed double bands on SDS-PAGE. However, only single gene for TDO has been so far identified in rat, as well as in human [7,9]. In order to understand the subunit structure of rat liver TDO, we established an expression system for rat liver TDO in E. coli here and characterized the recombinant enzyme by creating a truncated enzyme.
2. Experimental procedures 2.1. Construction of expression vector Plasmid pTO14B carrying a full-length cDNA of rat liver tryptophan 2,3-dioxygenase was a generous gift of Prof. T. Nakamura of Osaka University, Japan. For the construction of an expression vector of TDO, the cDNA was first amplified with introduction of a six-histidine tag and restriction sites (Nde1 and Sal1 at 5V- and 3V-ends, respectively) by using polymerase chain reaction (PCR). The PCR product was digested with Nde1 and Sal1 and then ligated into pUC 18 along with the DNA fragment of 5Vuntranslated region from the cytochrome P450cam gene with restriction sites EcoRI and Nde1 at 5V and 3V, respectively, using ligation solution I version 2.0 (TaKaRa, Japan). The ligated plasmid was transformed into E. coli JM109. Truncated TDO was obtained by eliminating the sequences encoding the first 13 amino acid residues from the fulllength cDNA by PCR using the following primers: 5VGCCATATGCATCACCATCATCATCACACT TTGAAA AACTTATCT ATG G3V as forward and M13 universal primer as reverse primer. The forward primer contained Nde1 site (underlined) and sequence encoding six histidine residues as a tag (doubly underlined). The sequence of the PCR amplified cDNA of TDO was confirmed by the conventional DNA sequencing. 2.2. Expression of TDO TDO was expressed in E. coli JM109 by inoculating into terrific broth, supplemented with 100 Ag/ml ampicillin and 1 mM isopropyl-h-D-thiogalactopyranoside and incubated
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at 30 jC in a rotary shaker with a speed of 150 rpm for 13– 14 h. TDO in the E. coli cells was monitored by taking a difference spectrum of ferrous –carbon monoxide minus ferrous form of TDO in the culture. Cells were harvested by centrifugation at 12,000g for 20 min at 4 jC and stored at 20 jC until use. 2.3. Purification of TDO Fifty grams of E. coli cell paste frozen at 20 jC was thawed at 4 jC for overnight and cell paste was lightly suspended in 500 ml of 0.05 M potassium phosphate buffer, pH 7.8, containing 10 mM L-tryptophan (buffer A) in an electrical blender and treated with 50 mg lysozyme (Sigma) and 1 mg of pancreatic DNAse I (Merck). Ten tabs of protease inhibitor cocktail (Roche Biochemical) were added to the cell suspension. The mixture was stirred for about 1.5 h at room temperature to lyse the bacterial cells and then next 30 min at 4 jC. The following procedures were performed at 4 jC unless stated otherwise. The cell lysate was then centrifuged at 12,000 rpm for 30 min. The supernatant thus obtained was passed through a DEAE –cellulose column (415 cm) equilibrated with buffer A. TDO was eluted with a linear gradient formed between equal volume of buffer A and buffer A containing 0.6 M NaCl and 20 ml fractions were collected. Fractions containing enzyme activity more than 0.2 U/ml were pooled together and subjected to 20% and 35% ammonium sulfate fractionation. Fraction precipitated with 35% ammonium sulfate was recovered by centrifugation at 12,000 rpm for 20 min. The pellet was dissolved in minimal volume of buffer A and dialyzed overnight against buffer A to remove ammonium sulfate. The dialysate was then centrifuged at 5000 rpm for 10 min and further purified by Nichelate immobilized column chromatography (Ni-NTA superflow agarose, QIAGEN) by stepwise elution with 20, 50, 100 and 300 mM imidazole in 20 mM potassium phosphate buffer, pH 8.0, containing 10 mM L-tryptophan and 300 mM NaCl. The highly concentrated brown red colored TDO was then dialyzed against buffer A to remove imidazole. The dialysate was concentrated using Centricon 30. The highly concentrated solution of TDO was then passed through a Sephadex G-25 column equilibrated with 0.05 M potassium phosphate buffer, pH 7.8 to remove L-tryptophan. This solution devoid of Ltryptophan was used as enzyme preparation for all assays, spectral measurements and SDS-PAGE. 2.4. Western blot analysis Expression of recombinant TDO was checked by Western blot analysis. Cell lysate proteins or purified proteins (2.5 Ag) were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Milipore) with the transfer buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol at 8 V for 45 min on a semidry electroblot apparatus (Bio-Rad). The membrane was blocked in 20 mM Tris –HCl, pH 7.5, containing 0.15 M NaCl (TBS) supplemented with 3% bovine serum albumin. The membrane was washed several times with TBS supplemented with 0.05% Tween 20 and 0.1% Triton X-100 (TBSD) and incubated with monoclonal anti-His tag antibody (Qiagen) for 1 h at room temperature. The membrane was washed again with TBSD and then incubated with secondary antibody conjugated with horseradish perox-
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idase for overnight. Protein was visualized by incubating with a solution containing diaminobenzidine as chromogenic substrate. 2.5. Enzyme assays L-Tryptophan 2,3-dioxygenase activity was measured in an assay mixture containing 100 mM potassium phosphate, pH 7.0, and 10 mM L-tryptophan under aerobic conditions at 25jC. The reaction was started by adding the enzyme reduced with a minimum amount of dithionite under anaerobic conditions, and the formation of N-formylkynurenine was monitored spectrophotometrically at 321 nm. The enzyme activity was expressed as turnover number per heme and minute. Alternatively, the enzyme activities of cell extract were measured in the above reaction mixture supplemented with ascorbic acid and expressed in units per milligram of protein, where 1 U is defined as micromole of Nformylkynurenine formed per minute. Km value for L-tryptophan was determined from a curve of the activities (initial velocities) vs. concentrations of L-tryptophan added into the assay mixture by best fitting to the curve using IgorPro software (WaveMetrics, Lake Oswego, OR).
2.6. Optical spectrophotometry Absorption spectra of ferric, ferrous and ferrous– CO complex of purified wild-type and truncated forms of TDOs were measured on a Hitachi spectrophotometer U-3000 or Perkin Elmer spectrophotometer Lambda 18 in a wavelength region between 380 and 700 nm in the presence and absence of L-tryptophan. 2.7. Other determinations Protein content was determined by Bradford method using Bradford reagent (BioRad) after removal of L-tryptophan. BSA (Sigma) was used as a standard. Heme content was determined by pyridine hemochromogen method [10]. Protein content of purified TDO preparation was determined by amino acid analysis after hydrolysis in 5.7 N HCl in vacuo Table 1 Properties of recombinant and nativea rat liver tryptophan 2,3-dioxygenases Properties
Recombinant
Native1
Molecular mass (kDa) Subunit molecular mass (kDa) Number of subunits Heme Heme content Turnover number Km for l-tryptophan (mM) Optimum pH
200 46 and 49b 4 (a2h2) Protoheme IX 2 303 0.05 7.6 – 8.0
168 45 – 47 4 (a2h2) Protoheme IX 2 200 – 323 0.1 – 0.2 7.2
a Data of the native enzyme were taken from Schutz and Feigelson [3] except for the turnover number of the enzyme, which were from Makino et al. [11] and Uchida et al. [12]. b cDNA sequence of rat liver TDO by Maezono et al. [9] predicts 48 kDa as a subunit molecular mass.
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at 110 jC. The analysis was performed on Beckman amino acid analyzer at Toray Research Center, Japan. N-terminal amino acid sequence of the proteolytically cleaved fragment of TDO was analyzed on a protein sequencer at Takara Biomedical, Japan. For this, the fractionated protein bands were transferred onto PVDF membrane from polyacrylamide gel and visualized by staining with Coomassie brilliant blue. The protein bands with molecular mass of 46 and 49 kDa were excised from the membrane and analyzed.
Fig. 1. Absorption spectra of recombinant rat liver L-tryptophan 2,3-dioxygenase. The spectra of TDO were measured at 25 jC in K phosphate buffer, pH 7.0. Ferrous form of the enzyme was produced by the reduction with dithionite and ferrous-CO form was obtained by gentle blowing of CO gas into the surface of solution containing the ferrous form of enzyme. (A) In the absence of L-tryptophan. (B) In the presence of 10 mM L-tryptophan.
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2.8. Chemicals and reagents L-Tryptophan was purchased from Sigma, USA. Plasmid pUC 18, pyrobest buffers and Taq polymerase, restriction enzymes, ligation solution I version II were purchased from TaKaRa Biomedicals, Japan.
3. Results 3.1. Purification and characterization of recombinant L-tryptophan 2,3-dioxygenase In addition to the recombinant TDO with a N-terminal His tag, we also constructed untagged and C-terminal His-tagged TDOs. Among the three, TDO with the N-terminal His tag was most highly expressed under the present expression conditions, and therefore employed throughout this study. TDO showed purity more than 90% as estimated by SDSPAGE and turnover number about 303 min 1, which is slightly higher than reported previously for native rat liver TDO [11]. Other properties of the recombinant enzyme were compared with native rat liver enzyme [3,11,12] and summarized in Table 1. Optical measurements of TDO showed spectral properties characteristic to those of rat liver TDO as shown in Fig. 1A. Ferric form of this enzyme showed Soret band at 406 nm and other bands at 498 and 632 nm, whereas ferrous form showed Soret band at 430 nm and another band at 557 nm. Ferrous – CO complex exhibited its Soret band at 420 nm and a- and hbands at 569 and 538 nm, respectively. In the presence of 10 mM L-tryptophan (Fig. 1B),
Fig. 2. SDS-PAGE of wild-type and truncated L-tryptophan 2,3-dioxygenases. Lane 1: purified wild type TDO. Lane 2: purified truncated TDO.
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the Soret band of ferric form shifted to 407 nm, whereas that of ferrous form to 421 nm. Ferrous – CO complex did not show any significant change in Soret and other bands. 3.2. Subunit structure of recombinant L-tryptophan 2,3-dioxygenase SDS-PAGE showed the presence of two distinct bands at 49 and 46 kDa (Fig. 2, Lane 1). The latter band was, however, not detected by anti-His-tag antibody (data not shown), suggesting a loss of N-terminal sequence carrying the His tag. N-terminal amino acid analysis of this band revealed that it is shorter by 19 amino acid residues from the Nterminus due to cleavage of peptide bond between Tyr19 and Thr20 in the polypeptide chain (Tyr13 and Thr14 in the original peptide). Construction of truncated TDO with elimination of this fragment revealed a single band on SDS-PAGE (Fig. 2, Lane 2), suggesting that doubling of TDO band was a result of limited proteolysis. Interestingly, truncated TDO did not show any alteration in enzyme activities and spectral properties compared to those of full-length wild type. The specific activity of truncated TDO in the crude cell extracts was 0.113 U/mg protein, which is very close to that of the wild-type enzyme (0.137 U/mg protein). Physiological significance of such a truncation remains unknown at present.
4. Discussion We have expressed, purified and characterized rat liver TDO in terms of its activity and spectral properties. The wild-type recombinant TDO showed many properties similar to native rat liver enzyme (Table 1). Its turnover number per heme was found to be 303 min 1, which was higher than the reported value (200 – 250 min 1) by Schutz and Feigeson [3] for rat liver enzyme, but was considerably lower than that of Pseudomonas enzyme (1100 min 1) [11]. It was close to 323 min 1, the highest value so far reported for rat liver TDO by Uchida et al. [13]. Although, both rat liver and Pseudomonas enzymes show maximal activity around pH 7.0 – 7.2, recombinant TDO was active at a pH range between 7.6 and 8.0 at both saturating (10 mM), as well as subsaturating concentration of L-tryptophan. Km value for L-tryptophan was 0.05 mM, which is slightly lower than reported; 0.1– 0.2 mM for rat liver [13] and 0.21 for mouse liver [4]. The recombinant TDO had spectral properties indistinguishable from those of native enzyme. Thus, in many aspects recombinant TDO showed properties very similar to those of native enzyme, suggesting that recombinant enzyme does not undergo undesirable modifications in host cells and the addition of an Nterminal His tag does not affect enzyme properties in a significant manner. One of the significant findings about TDO structure in this paper is that we have found the reason for the doubling of TDO band in polyacrylamide gel electrophoresis. As previously observed for other TDO preparations [3,4,8], recombinant TDO showed double bands in SDS-PAGE with molecular masses of 49 and 46 kDa. For native rat liver enzyme, such double bands have been ascribed to the presence of different subunits in the enzyme, i.e. to an a2h2-structure [3]. Similar double bands were reported for the case of recombinant rat liver TDO expressed in E. coli DH5a [8]. It is worthy of note that the results obtained by Watanabe et al. [4] on mouse liver TDO showed a single band on 7.5%
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native PAGE, but two bands on 10% SDS-PAGE. The authors are, however, in agreement with the concept that TDO is composed of two pairs of identical subunits (a2h2). We also observed two distinct bands from purified recombinant TDO on 10% SDS-PAGE (Fig. 2, Lane 1). No such double bands were however noticed on Western blot analysis using antiHis tag antibody with which only a single band with a molecular mass around 49 kDa was observed. The result suggests that the observed second band at 46 kDa was devoid of the N-terminal sequence containing His tag. Base on these findings we concluded that the second band was a product of limited proteolysis of the 49-kDa band. N-terminal sequence analysis of this second band, recovered from PVDF membrane after having transferred from polyacrylamide gel showed that this band had lost 19 amino acid residues from Nterminus as a result of cleavage occurring between amino acid residues Tyr19 and Thr20 (Tyr13 and Thr14 in the original sequence). A truncated form of TDO constructed by eliminating the fragment showed a single band confirming that the appearance of double bands was a result of the proteolytic cleavage at the site mentioned. We further analyzed rat liver extract along with purified native and recombinant rat liver TDO by Western blot using anti-TDO antibody and found that the location of the bands was very similar to that of purified ones (unpublished observation). In addition to this, only one gene for TDO has so far been known for both rat as well as human [7,9]. These data taken together indicate that there is only one gene for TDO and the doubling of SDS-PAGE band is associated with limited proteolysis. It is however of interest to note that if this is nonspecific proteolysis occurring with TDO subunits, then question may arise why not all of TDO molecules (or all of its subunits) undergo this kind of cleavage. The physiological significance of this proteolysis is also unknown at present; truncation of this fragment did not cause any detectable change in spectral properties and enzyme activity. Based on these data we assumed that TDO subunits have different orientation in the tetrameric enzyme structure and accordingly have variable susceptibility to protease digestion. In any case, further studies (including crystal structure) will be required to elucidate the biological significance of such a kind of proteolytic modification.
5. Conclusion Based on the above data we conclude that we have established expression system for Ltryptophan 2,3-dioxygenase in E. coli JM109 and our recombinant enzyme resembles native rat liver enzyme in many aspects. It can be used for further investigation regarding native properties and also for mechanism of catalysis, etc. It is a tetrameric enzyme consisting of four identical subunits, encoded by single gene. The doubling of the enzyme on SDS-PAGE is associated with proteolytic cleavage of the original native subunit as deduced by Maezono et al. [9] at peptide bond between Tyr13 and Thr14.
Acknowledgements The authors are thankful to Professor T. Nakamura of Osaka University, Japan, for providing us the full-length cDNA of TDO. The authors are highly indebted to Drs. K.
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Mukai, Y. Katayama and K. Shimokata for their technical advice and discussions throughout this research, as well as during the preparation of this manuscript. We also thank the Japan Society for Promotion of Science (JSPS) for the support that enabled Dr. Surya P. Manandhar to stay in Japan during the period of May 01, 1999– April 30, 2000. This investigation was supported in part by grants from Ministry of Education, Science, Culture, Sports and Technology of Japan, and Keio University.
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