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• W2023999919 abstract "Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin. The apparent K m value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity. A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity. In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in growing E. coli cells as shown by Western blot analysis. Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin. The apparent K m value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity. A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity. In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in growing E. coli cells as shown by Western blot analysis. Thioredoxin (Trx) 1The abbreviations used are: Trx, thioredoxin; RNR, ribonucleotide reductase; Trx-S2, oxidized thioredoxin; Trx-(SH)2, reduced thioredoxin; Grx, glutaredoxin; DTT, dithiothreitol; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); PCR, polymerase chain reaction; PEG, polyethylene glycol; BSA, bovine serum albumin. 1The abbreviations used are: Trx, thioredoxin; RNR, ribonucleotide reductase; Trx-S2, oxidized thioredoxin; Trx-(SH)2, reduced thioredoxin; Grx, glutaredoxin; DTT, dithiothreitol; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); PCR, polymerase chain reaction; PEG, polyethylene glycol; BSA, bovine serum albumin. is a small protein (M r 12,000) with a conserved active site sequence Trp-Cys-Gly-Pro-Cys that catalyzes many redox reactions through the reversible oxidation of its active site dithiol to a disulfide. Oxidized thioredoxin, Trx-S2, can be reduced by NADPH and the flavoenzyme thioredoxin reductase, the so-called thioredoxin system (Reaction 1) (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Reduced thioredoxin, Trx-(SH)2, contains two thiol groups and can efficiently catalyze the reduction of many exposed disulfides, thus being a general protein-disulfide reductase, Reaction 2. Trx­S2+NADPH+H+↔TRTrx­(SH)2+NADP+Reaction 1 Trx­(SH)2+Protein­S2↔Trx­S2+Protein­(SH)2Reaction 2 Thioredoxin is present in all living organisms and has been isolated and characterized from a wide variety of prokaryotic and eukaryotic cells (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). In Escherichia coli, thioredoxin was first identified as an electron donor for ribonucleotide reductase (RNR), the enzyme that reduces ribonucleotides to deoxyribonucleotides for DNA synthesis and repair (2Laurent T.C. Moore E.C. Reichard P. J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar). E. coli thioredoxin can also function as a hydrogen donor for 3′-phosphoadenosine 5′-phosphosulfate reductase in the sulfate assimilation pathway as well as methionine sulfoxide reductase (3Tsang M.L. Schiff J.A. J. Bacteriol. 1976; 125: 923-933Crossref PubMed Google Scholar, 4Ejiri S.I. Weissbach H. Brot N. J. Bacteriol. 1979; 139: 161-164Crossref PubMed Google Scholar). Apart from these functions,E. coli thioredoxin is necessary for the life cycle of some bacteriophages such as T7, M13, and f1 (5Chamberlin M. J. Virol. 1974; 14: 509-516Crossref PubMed Google Scholar, 6Russel M. Model P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 29-33Crossref PubMed Scopus (102) Google Scholar, 7Lim C.J. Haller B. Fuchs J.A. J. Bacteriol. 1985; 161: 799-802Crossref PubMed Google Scholar). In eukaryotic cells, thioredoxin can also function as a hydrogen donor for RNR, 3′-phosphoadenosine 5′-phosphosulfate, and methionine sulfoxide reductases, similar to the prokaryotic thioredoxin (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). In addition, thioredoxin can (a) facilitate refolding of disulfide-containing proteins (8Lundstrom J. Holmgren A. J. Biol. Chem. 1990; 265: 9114-9120Abstract Full Text PDF PubMed Google Scholar); (b) activate the interleukin-2 receptor (9Tagaya Y. Maeda Y. Mitsui A. Kondo N. Matsui H. Yodoi J. EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (516) Google Scholar); (c) modulate the DNA binding activity of some transcription factors, e.g. NF-κB (10Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (724) Google Scholar); and (d) stimulate proliferation of lymphoid cells and a variety of human solid tumors (11Wakasugi N. Tagaya Y. Wakasugi H. Mitsui A. Maeda M. Yodoi J. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8282-8286Crossref PubMed Scopus (256) Google Scholar, 12Gasdaska J.R. Berggren M. Powis G. Cell Growth Differ. 1995; 6: 1643-1650PubMed Google Scholar). Furthermore, thioredoxin is an efficient antioxidant able to reduce hydrogen peroxide (13Spector A. Yan G.Z. Huang R.R. McDermott M.J. Gascoyne P.R. Pigiet V. J. Biol. Chem. 1988; 263: 4984-4990Abstract Full Text PDF PubMed Google Scholar), scavenge free radicals (14Schallreuter K.U. Wood J.M. Biochem. Biophys. Res. Commun. 1986; 136: 630-637Crossref PubMed Scopus (149) Google Scholar), and protect cells against oxidative stress (15Nakamura H. Matsuda M. Furuke K. Kitaoka Y. Iwata S. Toda K. Inamoto T. Yamaoka Y. Ozawa K. Yodoi J. Immunology Lett. 1994; 42: 75-80Crossref PubMed Scopus (200) Google Scholar). In photosynthetic organisms, three types of thioredoxins have been identified, two forms in chloroplasts (f and m) involved in regulatory systems in oxygen photosynthesis and one form in cytosol and endoplasmic reticulum (h) (16Besse I. Buchanan B.B. Botanical Bulletin of Academia Sinica (Taipei). 1997; 38: 1-11Google Scholar). E. coli Trx is a well studied enzyme, and its three-dimensional structure has been determined by NMR for both oxidized and reduced forms, as well as by x-ray crystallography (17Katti S.K. LeMaster D.M. Eklund H. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (534) Google Scholar,18Jeng M.F. Campbell A.P. Begley T. Holmgren A. Case D.A. Wright P.E. Dyson H.J. Structure. 1994; 2: 853-868Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). E. coli thioredoxin contains 109 amino acids and has a central core of five strands of twisted β-pleated sheet flanked by four α-helices and the active site located in a protrusion of the protein (19Gleason F.K. Holmgren A. FEMS Microbiol. Rev. 1988; 4: 271-297Crossref PubMed Google Scholar). Thioredoxin-negative mutants (trxA −) ofE. coli are viable (5Chamberlin M. J. Virol. 1974; 14: 509-516Crossref PubMed Google Scholar), and analysis of these mutants led to the identification of a novel cofactor, glutaredoxin-1 (Grx1), as an efficient substitute of thioredoxin for RNR and 3′-phosphoadenosine 5′-phosphosulfate reductase enzymatic activity (20Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2275-2279Crossref PubMed Scopus (361) Google Scholar, 21Tsang M.L. J. Bacteriol. 1981; 146: 1059-1066Crossref PubMed Google Scholar). However, Grx1 could neither substitute for thioredoxin in methionine sulfoxide reduction nor in bacteriophage growth or assembly (5Chamberlin M. J. Virol. 1974; 14: 509-516Crossref PubMed Google Scholar, 6Russel M. Model P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 29-33Crossref PubMed Scopus (102) Google Scholar, 7Lim C.J. Haller B. Fuchs J.A. J. Bacteriol. 1985; 161: 799-802Crossref PubMed Google Scholar, 22Russel M. Model P. J. Biol. Chem. 1986; 261: 14997-15005Abstract Full Text PDF PubMed Google Scholar), which thus remained typical phenotypes of E. coli thioredoxin mutants. The isolation of an E. coli double mutant in thioredoxin/glutaredoxin-1 allowed the identification of two novel glutaredoxins, Grx2 and Grx3, but only Grx3 is able to serve as a hydrogen donor for RNR (23Miranda-Vizuete A. Martinez-Galisteo E. Åslund F. López-Barea J. Pueyo C. Holmgren A. J. Biol. Chem. 1994; 269: 16631-16637Abstract Full Text PDF PubMed Google Scholar, 24Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar). Thus, it seemed that only one thioredoxin did exist in E. coli. However, the existence of another thioredoxin has been suggested to be necessary for the maintenance of the reducing environment in E. coli cytoplasm (25Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (373) Google Scholar). In addition, a triple Trx, Grx1, and Grx3 mutant was viable (26Prinz W.A. Åslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: 15661-15667Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar), indicating the presence of an alternative protein capable of reducing RNR in vivo. We report here the cloning of a DNA sequence coding for a novelE. coli thioredoxin (Trx2) based upon biological activity data and protein homology. We also present evidence that the protein is normally expressed in E. coli cells and that the N-terminal sequence of the protein contains a novel domain with four cysteine residues that partly regulates its enzymatic activity as protein-disulfide reductase. E. coli K-12 was a stock from our laboratory. K38 (wild type) and A179 (trxA::kan) (27Russel M. Model P. J. Bacteriol. 1984; 159: 1034-1039Crossref PubMed Google Scholar) strains were a kind gift from Prof. Arne Holmgren (Karolinska Institutet, Stockholm). Cells were grown in LB medium supplemented (when necessary) with 50 μg/ml ampicillin or kanamycin. A thioredoxin-like sequence (Trx2) containing an open reading frame coding for a protein of 139 amino acids (GenBankTMaccession number 1788936), 2The sequence is available on the World Wide Web at the server for the E. coli data base collection (http://susi.bio.uni-giessen.de/ecdc.html). was used to design the specific mutagenic primers EcTrx2-NdeI, 5′-TCCCGAGGTTACATATGAATACCGTTTG-3′ (forward), and EcTrx2-BamHI, 5′-CAAGATGGGATCCGGTAAGATTAAAGAGATTC-3′ (reverse), that introduce an NdeI site and aBamHI site at the N terminus and C terminus of the coding sequence, respectively. These primers were used to amplify E. coli K-12 genomic DNA by polymerase chain reaction (PCR) with the ExpandTM Long Template PCR System (Boehringer Mannheim) (30 cycles at 94 °C for 20 s, 58 °C for 30 s, and 68 °C for 2 min, linked to a 68 °C for 30 min cycle). The PCR product was cloned into the pGEM-T Easy Vector System I (Promega) and sequenced. The insert of Trx2 cloned into the pGEM-T vector was digested with NdeI and BamHI and cloned into theNdeI/BamHI sites of the pET-15b expression vector (AMS Biotechnology). The recombinant plasmid was designated pET-Trx2.E. coli BL21 (DE3) was transformed with the pET-Trx2 construct, and a single positive colony was inoculated in 1 liter of LB medium plus ampicillin and grown at 37 °C untilA 600 = 0.5. Then fusion protein was induced by the addition of 0.5 mmisopropyl-1-thio-β-d-galactopyranoside, and growth was continued for another 3.5 h. The cells were harvested by centrifugation at 10,000 × g for 10 min, and the pellet was resuspended in 50 ml of 20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride. Lysozyme was added to a final concentration of 0.5 mg/ml with stirring for 30 min on ice. Subsequently, MgCl2 (10 mm), MnCl2 (1 mm), DNase I (10 μg/ml), and RNase (10 μg/ml) were added, and the incubation was continued for another 45 min on ice. The cells were sonicated, and the supernatant was cleared by centrifugation at 15,000 ×g for 30 min and loaded onto a Talon Resin Column (CLONTECH). The His-Trx2 protein was eluted with 50 mm imidazole, dialyzed against 20 mm HEPES, pH 8.0, and concentrated with Centricon concentrators (Amicon Inc.), and the size and purity of His-Trx2 was determined by SDS-polyacrylamide gel electrophoresis. When indicated, the His tag was removed by thrombin incubation. Protein concentrations were determined from the absorbance at 280 nm using a molar extinction coefficient of 14,180m−1 cm−1 for E. coliTrx1 and 17,790 m−1 cm−1 forE. coli Trx2. The truncated form of Trx2 (ΔTrx2) lacking the first 32 amino acids at the N-terminal part of the protein was amplified using the primer EcΔTrx2-NdeI 5′-GCGGTCACGACCATATGGACGGAGAGGTG-3′ as forward primer and EcTrx2-BamHI as a reverse primer. The cloning, overexpression, and purification of this truncated form was identical to that described above for the full-length one. Protein concentration was determined from the absorbance at 280 nm using a molar extinction coefficient of 13,310 m−1cm−1 for ΔTrx2. For antibody production, we used a modification of the method described by Song et al. (28Song C.S. Yu J.H. Bai D.H. Hester P.Y. Kim K.H. J. Immunol. 1985; 135: 3354-3359PubMed Google Scholar). Female chickens were injected (once every 15 days during 6 weeks) subcutaneously at multiple sites with 100 μg (in 0.25 ml) of His-Trx2 in complete adjuvant. After the second injection, eggs were collected daily, and when a suitable number of eggs were obtained antibodies were purified as follows. Egg yolks were separated from whites by dropping the egg contents on a funnel placed in a graduated cylinder. An equal volume of buffer S (10 mm phosphate, pH 7.5, 0.1 m NaCl, containing 0.01% sodium azide) was added to the yolks and stirred. Next, 10.5% PEG 8000 (Sigma) in buffer S was added to a final concentration of 3.5%. The mixture was stirred for 30 min at room temperature and centrifuged at 12,000 × g for 20 min. The supernatant was filtered through two layers of 3MM Whatman chromatography paper, and 42% PEG 8000 in buffer S was added to a final concentration of 12%. The mixture was stirred thoroughly and centrifuged at 12,000 × g for 20 min. The supernatant was discharged, and the pellet was redissolved in 12% PEG in buffer S to the original yolk volume. After centrifugation, the pellet was dissolved in 30 ml of buffer S and dialyzed overnight against buffer S without NaCl. Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column where 1 mg of Trx2, in which the His tag had been removed by thrombin, had been coupled following the procedure recommended by the manufacturer (Pharmacia Biotech Inc.). The specificity of the antibodies was tested by Western blotting using recombinant Trx2 and total cell extracts. For immunoblotting, samples were subjected to 15% SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-C Super, Amersham Corp.). The membranes were blocked with phosphate-buffered saline containing 8% dry fat-free milk powder, 2% bovine serum albumin (BSA), 150 mm NaCl, and 0.1% Tween 20 and further incubated with affinity-purified anti-Trx2 antibodies. Immunodetection was performed with horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma) diluted 1:5000, following the ECL protocol (Amersham). Cells were harvested atA 600 = 0.5 and washed three times in 50 mm Tris, pH 8.0. The pellet was resuspended in the same buffer plus 2 mm MgCl2 and 2 mg/ml lysozyme at an A 600 = 40, incubated for 30 min on ice, and centrifuged at 14,000 rpm at 4 °C for 1 min. The supernatant after centrifugation was collected as the periplasmic lysate fraction. The pellet was resuspended in the same volume of 50 mm Tris, pH 8.0, 2 mm MgCl2 and kept as the cytosolic fraction. Cells were harvested and washed as in the previous treatment. The pellet was resuspended in 50 mmTris, pH 8.0, 2 mm MgCl2 at the sameA 600 and quickly frozen in a dry ice/ethanol bath. The frozen suspension was slowly thawed in an ice bucket, and this cycle was repeated three times. The suspension was centrifuged, and periplasmic and cytosolic fractions were collected as described above. For crude bacteria extracts, exponentially growing cells were harvested by centrifugation, and the pellet was resuspended in 50 mm Tris-HCl, pH 8.0, 1 mm EDTA. Cells were sonicated, and the supernatant was cleared by centrifugation for 15,000 × g at 4 °C for 30 min. Heated extracts were prepared by heating crude extracts for 5 min, placing them on ice and spinning as described above. The activity of E. coli Trx2 was determined by the DTNB and insulin assays. The DTNB assay was performed essentially as described elsewhere (29Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (815) Google Scholar). Briefly, the reaction mix contained 200 mm phosphate buffer, pH 7.0, 2 mm EDTA, 0.1 mg/ml BSA, 1 mm DTNB, and 0.5 mm NADPH. The reactions containing 0.5–8 μm Trx1, Trx2, or ΔTrx2 were started by the addition of 10 nm E. colithioredoxin reductase (IMCO, Sweden). The reaction was followed at 412 nm against a blank containing thioredoxin reductase in a SpectraMaxTM 250 Microplate Spectrophotometer (Molecular Devices Corp.) for 7 min at 25 °C in a final volume of 100 μl. Insulin was used to determine the protein-disulfide reductase activity of thioredoxin as described previously (29Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (815) Google Scholar). The rate of DTNB reduction was calculated from the increase in A412 using a molar extinction coefficient of 27,200 m−1 cm−1, since reduction of DTNB by 1 mol of Trx-(SH)2 yields 2 mol of 3-carboxy-4-nitrobenzenethiol each with a molar extinction coefficient of 13,600 m−1 cm−1(29Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (815) Google Scholar). Values of ΔA412 were multiplied by a factor of 4.3 to give the ΔA412 of a cuvette with a path length of 1 cm. Reduction by DTT was carried out by preincubation of aliquots of Trx2 at 37 °C for 20 min with 2 μl of 50 mm HEPES, pH 7.6, 100 μg/ml BSA, and 2 mm DTT. The ability of E. coli Trx1 and Trx2 to serve as hydrogen donor for NrdAB and EF reductases was determined using the standard ribonucleotide reductase assay (30Thelander L. Eriksson S. Sjöberg B.M. Methods Enzymol. 1978; 51: 227-237Crossref PubMed Scopus (71) Google Scholar). Stoichiometric amounts (1 μm final concentration) of R1A/R2B and R1E (1.8 μg)/R2F (0.6 μg) were incubated for 20 min at 37 °C with Trx1 (IMCO) or Trx2 in a final volume of 50 μl containing 0.5 mm[3H]CDP (19,100 cpm/nmol), 10 mmMgCl2, 50 mm Tris-HCl, pH 8.0, 1 mmNADPH, 0.1 μm E. coli thioredoxin reductase, and 0.3 mm dATP (for NrdEF) or 1.5 mm ATP (for NrdAB). The reaction was stopped with 0.5 ml of HClO4, and the [3H]dCDP formed was determined by liquid scintillation after ion exchange chromatography on Dowex 50 columns. One enzyme unit is the activity that produces 1 nmol of dCDP during 1 min under these conditions. The identification of a novel thioredoxin sequence in the E. coli genome appeared serendipitously when using the recently described mammalian Trx2 (31Spyrou G. Enmark E. Miranda-Vizuete A. Gustafsson J.-Å. J. Biol. Chem. 1997; 272: 2936-2941Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) cDNA as probe in a Northern blot analysis with E. coligenomic DNA as a negative control. The strong signal obtained (data not shown) prompted us to search for the tentative E. colihomologue of the mammalian Trx2. We identified a DNA sequence in theE. coli genome (not completed at the time of this search) that displayed high homology with mammalian Trx2, but when translated to protein in the three possible reading frames only short peptide sequences were obtained. These short peptide sequences could be due to a possible sequencing mistake (an additional deoxycytidine) that, when removed, resulted in an open reading frame of 417 base pairs encoding a protein of 139 amino acids with the typical thioredoxin active site WCGPC. To further confirm the existence of this sequence coding for a new thioredoxin in the E. coli genome, we used two primers, EcTrx2-NdeI and EcTrx2-BamHI (see “Materials and Methods”) to amplify a fragment of 420 base pairs from genomicE. coli K-12 DNA. This PCR fragment was further cloned into pGEM-T Easy vector, and sequencing confirmed the absence of the additional deoxycytidine. The sequence of the Trx2 is positioned at min 58.5 in the E. coli chromosome map downstream of the uracyl DNA glycosylase gene. The nucleotide sequence of the E. coli Trx2 gene and flanking regions is given in Fig. 1 together with the deduced amino acid sequence. The open reading frame extends from the methionine codon, ATG, at coordinate +1 to the termination codon, TAA, at coordinate 417. This region codes for a protein of 139 amino acids with a estimated molecular mass of 15.5 kDa, containing the classical active site of thioredoxins WCGPC. The methionine +1 codon is preceded by a potential ribosome binding site at position −9 including six out of nine bases of the consensus ribosome binding sequence (32Chen H. Bjerknes M. Kumar R. Jay E. Nucleic Acids Res. 1994; 22: 4953-4957Crossref PubMed Scopus (262) Google Scholar) and an upstream in frame TAA stop codon. There is a putative −10 region centered at position −73 that conforms quite well to the consensus Pribnow box ofE. coli. If we assume a standard 16–18-base pair spacing region, a putative −35 region, TTGTCT, can be defined (Fig. 1). The DNA sequence following the coding part of the Trx2 gene contains a putative transcriptional Rho-independent terminator detected as an inverted repeat of 10 nucleotides followed by a stretch of T bases (33Rosenberg M. Court D. Annu. Rev. Genet. 1979; 13: 319-353Crossref PubMed Scopus (1701) Google Scholar). This is followed by another open reading frame of 723 nucleotides coding for a putative 27-kDa protein with no clear homology with any other protein in the data base. The main difference in Trx2 protein sequence with respect to the well known Trx1 is the presence of an extra stretch of 32 amino acids at the N terminus. The C-terminal half of the protein contains the active site found in all thioredoxins (Fig. 2). Recently, Lim et al. (34Lim C.J. Sa J.H. Fuchs J.A. Biochim. Biophys. Acta. 1996; 1307: 13-16Crossref PubMed Scopus (5) Google Scholar) have described a third thioredoxin inCorynebacterium nephridii, which displays a similar structure to E. coli Trx2 with an extra N-terminal domain of 32 amino acids and a C-terminal domain homologous to the rest of the prokaryotic thioredoxins (Fig. 2). E. coli Trx2 displays a 29% identity with E. coli Trx1 and 37% with C. nephridii Trx3 and is less similar to the mammalian Trx1 and Trx2. A phylogenetic analysis of several thioredoxins places E. coli Trx2 and C. nephridii Trx3 in the same branch of the tree between E. coli Trx1 and mammalian Trx2 (data not shown). To establish that the putative Trx2 does in fact encode a protein with thioredoxin activity, we cloned the Trx2 gene into the pET-15b expression vector under the control of a T7 promoter. The resulting plasmid pET-Trx2 was transformed in E. coli BL21 (DE3), and the expression of His-Trx2 was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside for 3.5 h. The recombinant protein was expressed to levels of approximately 20% of the total soluble protein and was purified almost to homogeneity by affinity chromatography with a Talon column (Fig.3, inset). Recombinant Trx2 (with or without the His tag) was used to examine the reduction of insulin, a classical assay in which thioredoxin catalyzes disulfide reduction of insulin in a coupled reaction with NADPH in the presence of E. coli thioredoxin reductase. As shown in Fig. 3, oxidized Trx2 was active as a disulfide reductase; however, it exhibited approximately 5-fold lower activity than Trx1 at concentrations between 0.2 and 0.5 μm and 1.5–2-fold lower activity between 0.5 and 2 μm. At these higher thioredoxin concentrations, the efficiency of thioredoxin reductase is the rate-limiting step, since the reactivity of Trx-(SH2) and insulin is known to be very fast (35Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar, 36Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). To understand the role of the extra cysteines present at the N terminus of Trx2, we preincubated Trx2 with DTT. As shown in Fig.4, the ability of Trx2 to reduce insulin was increased to levels similar to that of Trx1 (compare with Fig. 3). The fact that Trx2 activity was enhanced after reduction of the protein suggested that cysteine residues in Trx2 other than those in the active site could be involved in regulating its enzymatic activity. We used the truncated form of Trx2 (ΔTrx2) lacking the N-terminal portion, including the four cysteine residues, to further test this possibility. Fig. 4 shows that ΔTrx2 has thioredoxin activity independent of DTT reduction, similar to Trx1. Furthermore, the activity is similar to the reduced Trx2 and higher than the one displayed by the full-length oxidized protein, indicating that the N-terminal portion of the protein partly regulates the activity of Trx2. DTNB is an artificial disulfide substrate and a fast oxidant of Trx-(SH)2, which keeps the concentration of Trx-S2 constant. DTNB is often used in the assay of thioredoxin reductase, where one molecule of DTNB is split into two 5′-thionitrobenzoic acid molecules by reduced Trx1 (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). The DTNB assay and low concentrations (10 nm) of thioredoxin reductase to obtain saturation Michaelis-Menten kinetics were used to calculate theK m of Trx2 for E. coli thioredoxin reductase at pH 7.0 and 25 °C. The values obtained were used to calculate the k cat as well as thek cat/K m or apparent second order rate constant for the reaction between thioredoxin reductase and Trx-S2. The K m value for Trx2 and thek cat/K m were similar to Trx1. The ΔTrx2 has a lower K m value, and thek cat is approximately halved. TableI shows the comparison of Trx1 and Trx2 kinetic parameters.Table IKinetics parameters of Trx1 and Trx2 for thioredoxin reductaseK mK catk cat/K mμms −1m −1 s −1Trx11.9 ± 0.211.3 ± 0.66.3 × 106Trx22.4 ± 0.412.8 ± 1.05.4 × 106Δ Trx21.5 ± 0.16.5 ± 0.54.7 × 106The assay was carried out as described under “Materials and Methods.” Three separate measurements were made for each protein, and the mean value derived from Lineweaver-Burk plots of 1/[S]versus 1/V is shown. Open table in a new tab The assay was carried out as described under “Materials and Methods.” Three separate measurements were made for each protein, and the mean value derived from Lineweaver-Burk plots of 1/[S]versus 1/V is shown. E. coli contains genetic information for three different ribonucleotide reductases. The NrdAB is active during aerobiosis. NrdDG is active during anaerobiosis and uses formate as an electron donor. NrdEF is a cryptic enzyme that uses Grx1 but not Trx1 as an electron donor (37Fontecave M. Nordlund P. Eklund H. Reichard P. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 147-183PubMed Google Scholar, 38Ollagnier S. Mulliez E. Gaillard J. Eliasson R. Fontecave M. Reichard P. J. Biol. Chem. 1996; 271: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 39Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar). The viability of a triple mutant Trx1, Grx1 and Grx3, the three known electron donors for the NrdAB protein when growing in aerobiosis, prompted us to assay Trx2 as a tentative reductant for this enzyme. As shown in Fig.5, Trx2 was found to be a functional electron donor for the NrdAB enzyme. However, compare" @default.
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