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Microbiology Letters RESEARCH LETTER A thioredoxin reductase-like protein from the thermophile, Thermus scotoductus SA-01, displaying iron reductase activity Phillip Armand Bester, Derek Litthauer, Lizelle A. Piater & Esta van Heerden Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa Downloaded from https://academic.oup.com/femsle/article/302/2/182/647366 by guest on 29 September 2021 Correspondence: Esta van Heerden, Abstract Department of Microbial, Biochemical and Food Biotechnology, University of the Free The transition metal iron is an important element for the sustenance of life – it can State, Bloemfontein 9300, South Africa. function either as an electron acceptor or as a donor and serves as a cofactor in Tel.: 12751 401 2472; fax: 12751 444 3219; many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase e-mail: [email protected] in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here we report the sequence of the ferric Received 9 June 2009; accepted 3 November reductase (which is typically annotated as a thioredoxin reductase-like protein) 2009. and a comparative kinetic study with the thioredoxin reductase from SA-01. Final version published online December 2009. Structurally, the most noteworthy difference, immediately apparent from the protein sequence, is the absence of the disulphide redox centre in the ferric DOI:10.1111/j.1574-6968.2009.01852.x reductase. This is the first report relating the attributes of such a redox protein to Editor: Christiane Dahl its ability to reduce a ferric substrate. Keywords ferric reductase; thioredoxin reductase; thermophilic; Thermus scotoductus SA-01. reductants include thiols, a-ketoacids, reduced flavins and Introduction NAD(P)H (Winterbourn, 1979; Rowley & Halliwell, 1982; The transition metal, iron, is an important element for most Fontecave et al., 1987; Imlay & Linn, 1987), whereas proteins organisms and is required for various physiological func- responsible for ferric reduction include flavin reductase, tions such as transport of molecular oxygen, involvement in lipoyl dehydrogenase, NADPH-glutathione reductase, NADH- electron transport and a cofactor for enzymes, and functions cytochrome c reductase and NADPH-cytochrome P450 reduc- either as an electron donor or as an acceptor in microbial tase (Cederbaum, 1989; Sevanian et al., 1990; Petrat et al., energy conservation. The dissimilatory reduction of ferric 2003). iron is considered the oldest form of respiration, thus In this paper, we describe the sequence determination and providing an electron sink while the earth’s atmosphere was characterization of a novel thermophilic ferric-reducing still anoxic (Vargas et al., 1998). Ironically, with the arrival of enzyme isolated from the metal-reducing bacterium (Kieft oxygen, iron posed a new threat to aerobically respiring et al., 1999; Balkwill et al., 2004), Thermus scotoductus organisms. Various redox-active biomolecules have been SA-01, which shares both notable primary and tertiary implicated in the cytotoxic effect of iron in aerobic respiring structural characteristics with that of prokaryotic thioredox- organisms by reducing the cellular ferric iron, which can in reductases, but differs fundamentally regarding the then participate in the Fenton reaction. The successive typical redox-active site for these enzymes. univalent reduction of molecular oxygen, during aerobic The striking similarities in these two enzymes led us À respiration, generates superoxide (O2 Á ), hydrogen perox- to compare their ability to reduce the ferric substrate ide (H2O2) and hydroxyl (HO Á ) radicals, with the latter Fe(III)–nitrilotriacetate (NTA). Prokaryotic thioredoxin being most cytotoxic. The Fenton reaction involves the reductase belongs to the pyridine nucleotide-disulphide MICROBIOLOGY LETTERS MICROBIOLOGY reduction of hydrogen peroxide by Fe(II), yielding hydroxyl oxidoreductase family of flavoenzymes, sharing this family radicals and Fe(III). Research on this subject has led to the with lipoamide dehydrogenase, glutathione reductase, discovery of various biomolecules that could be responsible mercury reductase and NADH peroxidase. Thioredoxin for ferric reduction. Examples of low-molecular-weight reductase contains a disulphide redox-active site as well as Journal compilation c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 302 (2010) 182–188 Published by Blackwell Publishing Ltd. No claim to original South African government works TrxR-like protein, displaying ferric reduction 183 noncovalently bound FAD. The mechanism of thioredoxin chromatography column (16 Â 1.3 cm) was equilibrated with reductase is similar to that of glutathione reductase with 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) regard to the flow of electrons, where the reducing power is buffer, pH 7, and the unbound protein was eluted with transferred from NADPH to FAD and the reduced FAD 90 mL of the same buffer (flow rate, 2 mL minÀ1). A NaCl then, in turn, reduces the disulphide redox-active centre, gradient ranging from 0 to 0.4 M was applied to elute the which ultimately serves as the reductant for the substrate ferric reductase activity. The active fractions were pooled and thioredoxin. When NADPH binds to glutathione reductase, used for further analysis. the pyridinium ring is adjacent to the isoalloxazine ring of FAD, thereby allowing for the transfer of electrons (Wil- Size estimation liams, 1995). However, this is not the case with thioredoxin A concentrated protein sample was loaded onto a Sephacryl reductase, where two conformational changes occur for Downloaded from https://academic.oup.com/femsle/article/302/2/182/647366 by guest on 29 September 2021 S-100 HR (Sigma-Aldrich, St. Louis, MO) column either the reduction of FAD by NADPH or the reduction of (62 Â 2.6 cm) equilibrated with 20 mM MOPS, pH 7, con- the disulphide redox centre by FADH (Lennon et al., 2000). 2 taining 50 mM NaCl. The column was eluted with the same Although the ferric reductase shares some remarkable buffer at a flow rate of 0.5 mL minÀ1. Cytochrome c features with that of prokaryotic thioredoxin reductases, the (12.4 kDa), chymotrypsin (25 kDa) and bovine serum albu- lack of a disulphide redox centre emphasizes that this redox min (66 kDa) were used as molecular mass standards, while enzyme has a yet unknown function in vivo. This is the first Blue Dextran was used to determine the exclusion volume. report ascribing activity to such an enzyme. Sodium dodecyl sulphate-polyacrylamide gel electro- phoresis (SDS-PAGE) was performed as described pre- Materials and methods viously (Laemmli, 1970) using a 10% resolving gel and a 4% stacking gel. Organism, growth conditions and plasmids Thermus scotoductus SA-01 (ATCC 700910; American Type N-terminal amino acid sequencing Culture Collection) was cultured in TYG media [5 g tryp- The N-terminal amino acid sequence was determined using tone (Biolab, Wadeville, South Africa), 3 g yeast extract an Applied Biosystems 4774A gas-phase amino acid sequen- (Saarchem, Wadeville, South Africa) and 1 g glucose in 1 L cer (Foster City, CA) at the protein chemistry facility of the double-distilled water], pH 6.5, at 65 1C under aerobic Centro de Investigaciones Bioligicas (CSIC, Madrid, Spain). conditions with aeration of 200 r.p.m. For the genomic library construction of T. scotoductus SA-01, pTrueBlue Genomic DNA library construction (GenomicsOne, Laval, Canada) vector (Slilaty & Lebel, 1998) and One Shot TOP10 Escherichia coli chemically Genomic DNA was isolated from T. scotoductus SA-01 using competent cells were used. Recombinant expression was a modified proteinase K/Phenol method (Towner, 1991). accomplished by the use of pET28b(1) (Novagen, Darm- Restriction digestion was accomplished with the endonu- stadt, Germany) and E. coli BL21(DE3) (Lucigen, Middle- clease Sau3AI (New England Biolabs, Beverly, MA). The ton, MI) chemically competent cells. digested DNA was size fractionated between 3 and 6 kbp from a 0.8% agarose gel and purified using the GFX PCR DNA and gel band purification kit (GE Healthcare, Buck- Ferric reductase assay for activity screening inghamshire, UK). A continuous ferrozine-based assay was used to monitor the Ligation was conducted with a 1 : 2 vector to insert formation of ferrous iron as described by Moller¨ & Van ratio (6 Weiss units, 12 h at 16 1C) into the plasmid Heerden (2006) at 65 1C. pTrueBlue. This was transformed into One Shot TOP10 chemically competent E. coli according to the manufac- Purification of the soluble ferric reductase turer’s instructions. The purification was adapted as described previously (Moller¨ Screening of the genomic library & Van Heerden, 2006) with the addition of a final Blue Sepharose CL-6B (Sigma-Aldrich, Steinheim, Germany) dye An oligonucleotide probe (ATG GAG CAC ACS GAC GTG affinity chromatography step using the Acta Prime purifica- ATY ATY ATY GGS, where S = G/C, Y =C/T) was designed tion system (GE Healthcare AB, Uppsala, Sweden). Har- from the N-terminal sequence with the aid of a codon usage vested cells of T. scotoductus SA-01 were fractionated into table. Codon usage was obtained from four complete ORFs cytoplasmic, periplasmic and membrane fractions as de- from T. scotoductus that were available at the time following scribed previously (Park et al., 2000). The Blue Sepharose analysis with EMBOSS (EUROPEAN MOLECULAR BIOLOGY OPEN SOFT- CL-6B (Sigma-Aldrich, Steinheim,
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