The Thioredoxin System of the Filamentous Fungus Aspergillus nidulans

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase. Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase. Due to the metabolism of molecular oxygen as the final electron acceptor of the respiratory chain, all aerobic organisms are exposed to reactive oxygen intermediates (ROIs). 2The abbreviations used are:ROIreactive oxygen intermediateTrxthioredoxinTrxRthioredoxin reductaseAMMAspergillus minimal mediumAnTrxAA. nidulans thioredoxin AAnTrxRA. nidulans thioredoxin reductaseDTTdithiothreitolGSSGoxidized glutathionePrxperoxiredoxinALDHaldehyde dehydrogenaseDTNB or NBS25,5′-dithiobis(2-nitrobenzoic acid) Whereas low concentrations of ROI are supposed to function as secondary messengers, elevated ROI levels can lead to damage of biological macromolecules, like DNA, lipids, and proteins. However, there are several enzymatic and non-enzymatic defense mechanisms that are able to detoxify ROI efficiently. These mechanisms include superoxide dismutases, catalases, peroxidases, and the tripeptide glutathione. Glutathione is the most abundant intracellular thiol compound and serves as a powerful antioxidant and radical scavenger. Another important redox system is formed by the thioredoxin system. Thioredoxin systems are composed of two enzymes, i.e. thioredoxin (Trx) and thioredoxin reductase (TrxR) (1Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Thioredoxins are small, ubiquitously distributed proteins with a molecular mass of 12–13 kDa. Due to their redox-active cysteine pair in the active site (WCGPC), they are able to cycle between their oxidized disulfide (Trx-S2) and reduced dithiol [Trx-(SH)2] forms. In an NADPH-dependent protein disulfide reduction reaction TrxR catalyzes the reduction of oxidized thioredoxin using NADPH as electron donor, its own redox-active cysteine pair and FAD as cofactor. Reduced Trx directly reduces the disulfide in the target protein. This NADPH-dependent disulfide reduction mechanism is required for several intracellular processes. Since the discovery of the first Escherichia coli Trx, which was shown to be involved in DNA synthesis by acting as an electron donor for ribonucleotide reductase (3Laurent T.C. Moore E.C. Reichard P. J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar), a number of Trx target proteins were identified. Until now, the physiological functions assigned to Trx include protein disulfide reduction, sulfur assimilation, detoxification of reactive oxygen species, protein repair and redox regulation of enzymes and transcription factors. Also, regulatory effects of Trx on apoptosis as well as co-cytokine-, chemokine-, and growth-stimulating activities have been discussed (reviewed in Ref. 4Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar). reactive oxygen intermediate thioredoxin thioredoxin reductase Aspergillus minimal medium A. nidulans thioredoxin A A. nidulans thioredoxin reductase dithiothreitol oxidized glutathione peroxiredoxin aldehyde dehydrogenase 5,5′-dithiobis(2-nitrobenzoic acid) TrxRs are members of the larger family of pyridine nucleotide-disulfide oxidoreductases, which also includes enzymes like glutathione reductase, mercuric reductase, and lipoamide dehydrogenase (5Ghisla S. Massey V. Eur. J. Biochem. 1989; 181: 1-17Crossref PubMed Scopus (500) Google Scholar). Two classes of TrxRs have evolved, i.e. the low molecular weight TrxRs found in prokaryotes, archaea, plants, and fungi, and the high molecular weight TrxRs present in higher eukaryotes. Both classes have certain features in common. They are homodimeric flavoenzymes containing a redox active disulfide and binding sites for FAD and NADPH in each subunit (6Williams Jr., C.H. Arscott L.D. Müller S. Lennon B.W. Ludwig M.L. Wang P.-F. Veine D.M. Becker K. Schirmer R.H. Eur. J. Biochem. 2000; 267: 6110-6117Crossref PubMed Scopus (285) Google Scholar, 7Moore E.C. Reichard P. Thelander L. J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google Scholar). The basis of their reaction mechanism is the transfer of reducing equivalents from NADPH to an active disulfide by using FAD as cofactor (8Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (820) Google Scholar). However, low molecular mass TrxRs are homodimers of 35–36 kDa subunits, whereas the high molecular mass TrxRs from higher eukaryotes are composed of two subunits with a molecular mass of 55–58 kDa. In contrast to low molecular mass TrxRs, high molecular mass TrxRs possess an additional redox active site in the C-terminal extension, which is responsible for the interaction with the substrate Trx (6Williams Jr., C.H. Arscott L.D. Müller S. Lennon B.W. Ludwig M.L. Wang P.-F. Veine D.M. Becker K. Schirmer R.H. Eur. J. Biochem. 2000; 267: 6110-6117Crossref PubMed Scopus (285) Google Scholar, 9Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (773) Google Scholar). The rapidly growing literature on thioredoxin reductases, thioredoxins, and redox-regulated proteins indicates the deep impact of oxidoreductase systems on cellular processes. In microbial eukaryotes, ROIs are involved in development, cell differentiation (10Aguirre J. Rios-Momberg M. Hewitt D. Hansberg W. Trends Microbiol. 2005; 13: 111-118Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), and host-pathogen interaction (11Aguirre J. Hansberg W. Navarro R. Med. Mycol. 2006; 44: 101-107Crossref PubMed Scopus (102) Google Scholar). Also, a possible role of oxidoreductase systems in the penicillin biosynthesis has been discussed for Penicillium chrysogenum and Streptomyces clavuligerus (12Aharonowitz Y. Av-Gay Y. Schreiber R. Cohen G. J. Bacteriol. 1993; 175: 623-629Crossref PubMed Google Scholar, 13Cohen G. Argaman A. Schreiber R. Mislovati M. Aharonowitz Y. J. Bacteriol. 1994; 176: 973-984Crossref PubMed Google Scholar). In this report, we describe the isolation and characterization of a thioredoxin system from A. nidulans, which is an important model organism to study all kinds of biological questions, including development and the production of secondary metabolites (14Brakhage A.A. Al-Abdallah Q. Tüncher A. Spröte P. Phytochemistry. 2005; 66: 1200-1210Crossref PubMed Scopus (49) Google Scholar). As shown here, the thioredoxin system is essential for development of A. nidulans, and novel target proteins of thioredoxin were identified. Furthermore, the in vitro and in vivo data indicate that this thioredoxin system possesses a key role in the redox regulation of A. nidulans, because correlations with other redox systems, such as catalases, the glutathione system, and a thioredoxin-dependent peroxidase seem to exist. Strains and Molecular Genetic Techniques—Bacterial and fungal strains used in this study are listed in Table 1. A detailed oligonucleotide description can be found in the supplemental data (Table S1). Standard techniques in the manipulation of DNA were carried out as described by Sambrook et al. (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Genomic DNA from A. nidulans mycelia, grown for 24–48 h in Aspergillus minimal medium (AMM), was isolated by using the MasterPure™ Yeast DNA purification kit from Biozym Scientific (Oldendorf, Germany) according to a modified isolation protocol (16Wickes B. Epicentre Forum. 2004; 11: 7Google Scholar).TABLE 1Bacteria and fungi used in this studyStrainRelevant phenotype and/or genotypeSource/referenceE. coli DH5αF–, ϕ80d/lacZΔM15, Δ(lacZYA-argF)U169, recA1, endA1, hspR17(rk–mk+), supE44, λ–, thi-1, gyrA96, relA1Invitrogen TOP10F–, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, nupG, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(StrR), endA1 λ–Invitrogen BL21(DE3)F– ompT hsdSB (rB–mB) gal dcm (DE3)Novagen, Darmstadt, GermanyA. nidulans TN02A7 (ΔnkuA)pyrG89; pyroA4, nkuA::argB; riboB2(26Nayak T. Szewczyk E. Oakley C.E. Osmani A. Ukil L. Murray S.L. Hynes M.J. Osmani S.A. Oakley B.R. Genetics. 2006; 172: 1557-1566Crossref PubMed Scopus (489) Google Scholar) AnTrxAKOpyrG89; pyroA4, nkuA::argB; riboB2; trxA::pyr-4 N.crassaThis work AXB4A2pyrG89, pabaA1; argB2;fwA1; bga0; argB2::pAXB4A (acvAp-uidA, ipnAp-lacZ), ArgB+(28Weidner G. d'Enfert C. Koch A. Mol P.C. Brakhage A.A. Curr. Genet. 1998; 33: 378-385Crossref PubMed Scopus (181) Google Scholar) Open table in a new tab Media and Cultivation of Strains—AMM was prepared as previously described (17Brakhage A.A. Van den Brulle J. J. Bacteriol. 1995; 177: 2781-2788Crossref PubMed Google Scholar). If required, uridine (1 g/liter), p-aminobenzoic acid (3 mg/liter), riboflavin (2.5 mg/liter), pyridoxine-HCl (500 mg/liter), or reduced glutathione (307 mg/liter to 30.7 g/liter) was added to the medium. Isolation of Total RNA—A. nidulans strain AXB4A2 was grown at 37 °C in AMM supplemented with p-aminobenzoic acid and uridine. Mycelia were harvested, and cell extracts were obtained using liquid nitrogen as previously described (18Litzka O. Then Bergh K. Brakhage A.A. Mol. Gen. Genet. 1995; 249: 557-569Crossref PubMed Scopus (23) Google Scholar). Total RNA was isolated by using the RNeasy kit from Qiagen following the “RNeasy Mini Protocol for Isolation of Total RNA from Plant Cells and Tissues and Filamentous Fungi.” Aliquots (1–2 μg) of total RNA were used for the synthesis of AnTrxA-cDNA and AnTrxR-cDNA, as described in the following section. Synthesis of AnTrxA-cDNA and AnTrxR-cDNA—AnTrxA-cDNA was synthesized with the gene-specific primers AnTrxAf and AnTrxA-His6r by using the BioScript™ One-Step RT-PCR kit from Bioline (Luckenwalde, Germany) according to the manufacturer's protocol. AnTrxR-cDNA was synthesized by using the gene-specific primers AnTrxR-His6f and AnTrxRr. Generation of Recombinant Plasmids for AnTrxA and AnTrxR Overproduction—For the overproduction of the C-terminal His-tagged AnTrxA(wt) fusion protein AnTrxA-cDNA was cloned into the NdeI-NcoI site of the pET-39b(+) vector (Novagen) to generate the plasmid pET39-AnTrxA(wt)-H6. For the overproduction of the C-terminally His-tagged AnTrxA(C39S) fusion protein a cysteine residue (Cys-39) in AnTrxA(wt) was replaced by a two-step PCR amplification technique, as described by Ho et al. (19Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar) using pET39-AnTrxA(wt)-H6 as template and the primers AnTrxC39Sf and AnTrxC39Sr for mutagenesis. The resulting DNA fragment was cloned into the NdeI-NcoI site of the pET-39b(+) vector to generate the plasmid pET39-AnTrxA(C39S)-H6. For the overproduction of the N-terminally His-tagged AnTrxR fusion protein AnTrxR-cDNA was cloned into the NdeI-HindIII site of the pET-39b(+) vector to generate the plasmid pET39-H6-AnTrxR. The DNA sequence of the inserts was verified by sequence analysis. Purification of Recombinant Proteins—The recombinant soluble His6-tagged proteins AnTrxA(wt), AnTrxA(C39S), and AnTrxR were overproduced and purified by Ni2+ chelate and anion exchange chromatography, as described elsewhere (20Hortschansky P. Eisendle M. Al-Abdallah Q. Schmidt A.D. Bergmann S. Thön M. Kniemeyer O. Abt B. Seeber B. Werner E.R. Kato M. Brakhage A.A. Haas H. EMBO J. 2007; 26: 3086-3097Crossref PubMed Scopus (185) Google Scholar). For storage at -20 °C, the recombinant AnTrxA(wt), AnTrxA(C39S), and AnTrxR proteins were transferred into 50% (v/v) glycerol, 0.1 m potassium phosphate, pH 7.5, 2 mm EDTA, and 5 mm dithiothreitol (DTT), using a HiPrep desalting column (GE Healthcare, Freiberg, Germany). Protein concentrations were determined according to Bradford (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using the Coomassie Plus™ protein assay reagent (Pierce). Purity and Molecular Weight Determination—The purity and molecular weights of the recombinant proteins were determined by SDS-PAGE. In addition, the AnTrxA(wt) and AnTrxR proteins were subjected to gel filtration using a Superdex 200 HiLoad 16/60 column (GE Healthcare) equilibrated with a buffer containing 100 mm potassium phosphate, 150 mm NaCl, pH 7.0. FAD Content and Reconstitution of the AnTrxR Holo-enzyme with FAD—The concentration of enzyme-bound FAD was determined by measuring the absorbance at 454 nm with a molar extinction coefficient of 11.3 mm-1 × cm-1 for FAD (22Williams Jr., C.H. Zanetti G. Arscott L.D. McAllister K.J. J. Biol. Chem. 1967; 242: 5226-5231Abstract Full Text PDF PubMed Google Scholar). Due to the high production levels of AnTrxR in E. coli BL21(DE3) and the following purification procedures, the majority of the enzyme was present as an apo-enzyme. To reconstitute the AnTrxR holo-enzyme for further characterization, AnTrxR was incubated with a 60-fold molar excess of FAD for 20 min before adding on a NAP-5 column (GE Healthcare) to remove the excess of FAD. Thioredoxin Reductase Activity—TrxR activity of the purified AnTrxR was determined by using two different methods. In the NBS2 reduction assay AnTrxR activity was determined by the NADPH-dependent reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (23Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (285) Google Scholar). One enzyme unit is defined as the NADPH-dependent production of 2 μmol of 2-nitro-5-thiobenzoate (ϵ412 nm = 2 × 13.6 mm-1 × cm-1) per min. TrxR activity was also assayed based on the ability of AnTrxR to reduce AnTrxA(wt), which then reduces insulin disulfide bridges (24Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). AnTrxR activity was calculated from the decrease in absorbance at 340 nm using a molar extinction coefficient of 6.22 mm-1 × cm-1 for NADPH. One enzyme unit is defined as the amount of enzyme that leads to the consumption of 1 μmol of NADPH per minute. Trx Activity—Trx activity was determined by using the TrxR-dependent insulin precipitation assay (24Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). After starting the reaction by the addition of NADPH, the NADPH consumption was followed by recording the decrease in absorbance at 340 nm, until turbidity appeared. The increase of turbidity was measured at 650 nm. Trx-dependent GSSG Reduction Assay—The Trx-dependent GSSG-reduction assay was carried out as described elsewhere (25Kanzok S.M. Schirmer R.H. Türbachova I. Iozef R. Becker K. J. Biol. Chem. 2000; 275: 40180-40186Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). After addition of NADPH, the activity was calculated from the decrease in absorbance at 340 nm. Transformation of A. nidulans and Generation of trxA Deletion and Complemented Strains—As a parental strain for gene deletion, the uracil auxotrophic strain TN02A7 (ΔnkuA) was used (26Nayak T. Szewczyk E. Oakley C.E. Osmani A. Ukil L. Murray S.L. Hynes M.J. Osmani S.A. Oakley B.R. Genetics. 2006; 172: 1557-1566Crossref PubMed Scopus (489) Google Scholar). As a selectable marker, the pyr-4 gene, encoding orotidine-5′-monophosphate decarboxylase from Neurospora crassa, was applied. The trxA gene, including 1500-bp up-stream and downstream flanking regions, was amplified from genomic DNA of the wild-type strain AXB4A2 by the use of the oligonucleotides TrxA1500for and TrxA1500rev. The PCR product was cloned into the PCR2.1 vector (Invitrogen) to yield plasmid pAnTrxA-FLANK. Plasmid DNA of pAnTrxA-FLANK was cut with ClaI and BmgBI (blunt end cutter) to release a 1606-bp fragment, including the complete trxA gene, 650 bp of the upstream region, and 552 bp of the downstream region. For the introduction of the pyr-4 gene, plasmid DNA of pKTB (27Spröte P. Brakhage A.A. Arch. Microbiol. 2007; 188: 69-79Crossref PubMed Scopus (50) Google Scholar) was restricted with ClaI and PvuII (blunt end cutter). The resulting pyr-4-containing DNA fragment was then ligated with the ClaI- and BmgBI-restricted pAnTrxA-FLANK vector backbone to give plasmid pAnTrxAKO. Plasmid pAnTrxAKO was digested with NsiI and Acc65I to remove the PCR2.1 vector backbone. After gel purification (QIAquick gel extraction kit, Qiagen) the DNA fragment was directly used for transformation of A. nidulans TN02A7 (ΔnkuA) as previously described (28Weidner G. d'Enfert C. Koch A. Mol P.C. Brakhage A.A. Curr. Genet. 1998; 33: 378-385Crossref PubMed Scopus (181) Google Scholar). Transformants were pre-screened for their ability to sporulate on AMM agar plates containing 20 mm reduced glutathione and their inability to sporulate on AMM-agar plates without reduced glutathione. Genomic DNA of putative trxA deletion strains was subjected to Southern blot analysis. Complementation experiments were carried out by transformation of strain AnTrxAKO with a trxA-encoding PCR product, including 1.5-kb upstream and downstream flanking regions. Genomic DNA of transformants that behaved like the wild type was subjected to Southern blot analysis. For detection of DNA fragments, the digoxigenin system (Roche Applied Science) was used. Trx-affinity Chromatography—5 mg of AnTrxA(C39S) were coupled to a Hi-Trap NHS-activated 1-ml affinity column (GE Healthcare) according to the manufacturer's instructions. A. nidulans mycelia of the wild-type strain TN02A7 and the trxA deletion strain AnTrxAKO were ground in liquid nitrogen using mortar and pestle. The powder was resuspended in 100 mm potassium phosphate, pH 7.5, and 150 mm NaCl. After centrifugation (10,000 × g, 30 min) the soluble protein-containing supernatants were applied to the prepared thioredoxin-affinity column by injection at a flow rate of 1 ml/min. The column was washed with 100 mm potassium phosphate and 250 mm NaCl, pH 7.5, at 1 ml/min. Elution was carried out with 100 mm potassium phosphate containing 10 mm DTT, pH 7.5, and 150 mm NaCl. Aliquots of the supernatants, flow-through, wash, and elution fractions were analyzed by SDS-PAGE. Identification of AnTrxA Targets—Protein bands of the elution fraction were excised manually and digested with trypsin (Promega, Madison, WI). Peptides were extracted as described (29Kniemeyer O. Lessing F. Scheibner O. Hertweck C. Brakhage A.A. Curr. Genet. 2006; 49: 178-189Crossref PubMed Scopus (89) Google Scholar) and peptide mass fingerprint and fragmentation data were collected on a Bruker ultraflex TOF/TOF using Bruker Compass 1.2 software (FlexControl/FlexAnalysis 3.0). Obtained peak lists were sent to a Mascot in-house server (version 2.1.03) with the current NCBInr data base for protein identification. Search parameters were set as follows: mass tolerance of 200 ppm for peptide mass fingerprint and 0.5 Da for fragmentation, maximum of one missed cleavage by trypsin, taxonomy “fungi,” fixed carbamidomethyl modification, and optional methionine oxidation. The most significant hits were verified by comparison with the combined peptide mass fingerprint/fragmentation spectrum. With the chosen settings protein scores of >67 are significant (p < 0.05). Trx-dependent Peroxidase Activity—The elution fractions of AnTrx(C39S) affinity-purified protein solutions were applied to a NAP-10 column to remove the excess of DTT. Then aliquots of the DTT-free protein solution in 0.1 m potassium phosphate, 150 mm NaCl, pH 7.5, were incubated with or without the recombinant A. nidulans thioredoxin system and 200 μm NADPH. After addition of H2O2, the activity was calculated from the decrease in absorbance at 340 nm. Hydrogen Peroxide Sensitivity Assay—1.5 × 108 spores of the strains TN02A7 and AnTrxAKO were inoculated in 30 ml of liquid AMM agar (2% w/v) containing 0 mm, 1 mm, and 20 mm GSH. After the agar became solidified a hole of 1 cm in diameter in the center of the agar plate was created and filled with 150 μl of a 4.5% (v/v) H2O2 solution. The agar plates were incubated at 37 °C, and the zone of growth inhibition was measured after 48 h. Catalase Activity—A. nidulans mycelia and freshly harvested spores of the strains TN02A7 and AnTrxAKO were ground in liquid nitrogen using mortar and pestle. The obtained powder was resuspended in 100 mm potassium phosphate, pH 6.5. After centrifugation (10,000 × g, 30 min) the soluble protein extracts were diluted in 50 mm potassium phosphate, pH 6.5, to a final concentration of 5–50 μg of protein × ml-1. After adding H2O2 (20 mm) the decrease in absorbance at 240 nm was measured. Catalase activity was calculated from the decrease in absorbance at 240 nm using a molar extinction coefficient of 0.0436 mm-1 × cm-1 for H2O2 (30Beers R.F. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar). Catalase activity was also investigated by zymography (31Goldberg I. Hochman A. Biochim. Biophys. Acta. 1989; 991: 330-336Crossref PubMed Scopus (50) Google Scholar). Cloning and Sequence Analysis of the trxA and trxR Genes from A. nidulans—Two genes with the accession numbers XM_652682 and XM_656093 have been annotated to encode a classic cytoplasmatic thioredoxin (TrxA) and a hypothetical protein similar to thioredoxin reductase (TrxR), respectively. By using gene-specific primers for the reverse transcription and cDNA synthesis of the gene encoded by XM_652682, a DNA fragment was synthesized encoding a sequence identical to the deposited trxA cDNA. The deduced AnTrxA protein contains the thioredoxin-specific active site motif WCGPC and further highly conserved amino acids (see sequence alignment with other thioredoxins in supplemental Fig. S1A). AnTrxA exhibits all the characteristics of thioredoxins and represents the A. nidulans thioredoxin sequence (accession number AAB24444) described earlier (32Le Marechal P. Hoang B.M. Schmitter J.-M. Van Dorsselaer A. Decottignies P. Eur. J. Biochem. 1992; 210: 421-429Crossref PubMed Scopus (11) Google Scholar). Here, by reverse transcription of the gene designated with accession number XM_656093, we identified a shorter cDNA version for the A. nidulans thioredoxin reductase (accession number AM396558). This coding sequence is identical to an updated version of the trxR coding sequence deposited in the A. nidulans data base (AN3581.3). It contains a putative FAD-binding domain formed by the GXGXX(A/G) motif in the N-terminal region and the TXXXXVFAAGD motif at the C terminus of the protein (33Jeon S.J. Ishikawa K. Eur. J. Biochem. 2002; 269: 5423-5430Crossref PubMed Scopus (46) Google Scholar, 34Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 367: 491-497Crossref PubMed Scopus (47) Google Scholar). An NADPH-binding domain was also identified near the middle of the protein encoded by the motif GGGXXA (33Jeon S.J. Ishikawa K. Eur. J. Biochem. 2002; 269: 5423-5430Crossref PubMed Scopus (46) Google Scholar, 34Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 367: 491-497Crossref PubMed Scopus (47) Google Scholar). Furthermore, it contains the pyridine-nucleotide-disulfide oxidoreductases class-II active site motif, including a redox-active cysteine pair (CAVC). This motif was found by a pattern search using the “PROSITE data base of protein families and domains” (http://www.expasy.org/prosite/). The motif is characteristic of prokaryotic and eukaryotic thioredoxin reductases (8Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (820) Google Scholar, 9Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (773) Google Scholar, 35Kuriyan J. Krishna T.S. Wong L. Guenther B. Pahler A. Williams Jr., C.H. Model P. Nature. 1991; 352: 172-174Crossref PubMed Scopus (168) Google Scholar, 36Russel M. Model P. J. Biol. Chem. 1988; 263: 9015-9019Abstract Full Text PDF PubMed Google Scholar), bacterial alkyl hydroperoxide reductases (37Tartaglia L.A. Storz G. Brodsky M.H. Lai A. Ames B.N. J. Biol. Chem. 1990; 265: 10535-10540Abstract Full Text PDF PubMed Google Scholar), bacterial NADH:dehydrogenases (38Xu X.M. Koyama N. Cui M. Yamagishi A. Nosoh Y. Oshima T. J. Biol. Chem. (Tokyo). 1991; 109: 678-683Crossref Scopus (33) Google Scholar), and a probable oxidoreductase encoded by the Clostridium pasteurianum rubredoxin operon (39Mathieu I. Meyer J. Moulis J.M. Biochem. J. 1992; 285: 255-262Crossref PubMed Scopus (71) Google Scholar). An alignment of AnTrxR with other low molecular weight thioredoxin reductases can be found in the supplemental data (Fig. S1B). Both AnTrxA(wt) and AnTrxR were overproduced as His-tagged proteins in E. coli BL21(DE3) and purified to homogeneity. Additionally, an AnTrxA mutant version (AnTrxA(C39S)) was created, which had the second cysteine of the AnTrxA active site substituted by serine (Cys-39 → Ser-39). SDS-PAGE analysis of the purified proteins showed molecular masses of 12.7 and 37.6 kDa for AnTrxA and AnTrxR, respectively (Fig. 1A). After subtracting the molecular mass due to the His tag, the molecular masses of both proteins are in agreement with the values deduced from the respective cDNA sequences. The data obtained by gel filtration revealed apparent native molecular masses of 12.9 kDa for the AnTrxA(wt) and 88.0 kDa for the AnTrxR protein (Fig. 1B). These data indicate that, without the His tag, the native AnTrxA is a monomer of 11.6 kDa, whereas the native AnTrxR is a homodimer of 72.2 kDa. Consequently, the concentrations of AnTrxR given in the following refer to the homodimer. AnTrxR Is a Flavoenzyme—Both the sequence analysis and the yellow color of the purified AnTrxR led to the assumption that the enzyme is a flavoenzyme. Consistently, the UV-visible absorbance spectrum of the reconstituted AnTrxR holo-enzyme with absorbance maxima at 280, 380, and 460 nm and an absorbance ratio A280:A460 of 7.6 (Fig. 1C) is characteristic of a pure thioredoxin reductase with one FAD molecule per subunit (33Jeon S.J. Ishikawa K. Eur. J. Biochem. 2002; 269: 5423-5430Crossref PubMed Scopus (46) Google Scholar, 40Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (510) Google Scholar). The creation of the reduced form of AnTrxR by adding a12 m excess of NADPH resulted in a decreased absorbance at 460 nm (Fig. 1D). TrxR Substrate Specificity—For the determination of the kinetic parameters of the AnTrxR protein, we used the NBS2, insulin, and GSSG reduction assays, as described under “Experimental Procedures.” The Cys-39 → Ser-39 substitution in the active site led to an AnTrxA mutant protein (AnTrxA(C39S)), which was unable to cycle between its oxidized disulfide (Trx-S2) and reduced dithiol [Trx-(SH)2] form. Thus, this mutant protein did not serve as a substrate