Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress Danila Limauro 1 , Emilia Pedone 2 , Luciano Pirone 2 and Simonetta Bartolucci 1 1 Dipartimento Biologia Strutturale e Funzionale, University of Naples ‘Federico II’, Complesso Universitario Monte S. Angelo, Naples, Italy 2 Istituto di Biostrutture e Bioimmagini, C.N.R., Naples, Italy Reactive oxygen species (ROS) are either generated by incomplete oxygen reduction during respiration, or by exposure to environmental factors such as light, radiation or increased oxygen pressure. ROS, notably, superoxide (O 2 • ) ) and hydroxyl radical (OH • ), hydro- gen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ) cause damage to all major classes of biological macromole- cules, leading to protein oxidation, lipid peroxidation, DNA base modifications and strand breaks [1]. In order to protect against toxic ROS, aerobic organisms are equipped with a full array of defence mechanisms, among which are antioxidative enzymes and antioxid- ant molecules (e.g. superoxide dismutases, catalases, peroxidases, thioredoxins, and glutathione) which are developed by most cells [2–4]. Most aerobes have multiple enzymes with overlapping ROS detoxification pathways but different expression and regulation times. For example, many bacteria induce catalase or other protective enzyme expressions during the trans- ition from the exponential to the stationary growth phase, presumably as an adaptation to protect the genome and other cellular components against oxida- tion during a prolonged nongrowth phase. In recent years, much attention has been given to peroxiredox- ins (Prxs) [5–7], a new family of thiol-specific anti- oxidant proteins. These include alkyl hydroperoxide Keywords Archaea; Sulfolobus solfataricus; oxidative stress; ROS; peroxiredoxin Correspondence S. Bartolucci, Dipartimento di Biologia Strutturale e Funzionale, Complesso Universitario di Monte S. Angelo, Universita ` di Napoli ‘Federico II’, Via Cinthia, 80126 Naples, Italy Fax: +39 081679053 Tel: +39 081679052 E-mail: bartoluc@unina.it (Received 9 November 2005, revised 14 December 2005, accepted 16 December 2005) doi:10.1111/j.1742-4658.2006.05104.x Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome data- base of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Its role in oxidative stress was investigated by transcriptional analysis of RNA isolated from cultures that had been stressed with various oxidant agents. Its specific involvement was confirmed by a considerable increase in the bcp2 transcript following induction with H 2 O 2 . The 5¢ end of the transcript was mapped by primer extension analysis and the promoter region was characterized. bcp2 was cloned and expressed in Escherichia coli, the recombinant enzyme was purified and the predicted molecular mass was confirmed. Using dithiothreitol as an electron donor, this enzyme acts as a catalyst in H 2 O 2 reduction and protects plasmid DNA from nicking by the metal-catalysed oxidation system. Western blot analysis revealed that the Bpc2 expression was induced as a cellular adaptation in response to the addition of exogenous stressors. The results obtained indicate that Bcp2 plays an important role in the peroxide-scavaging system in S. solfataricus. Mutagenesis studies have shown that the only cysteine, Cys 49 , present in the Bcp2 sequence, is involved in the catalysis. Lastly, the presence of this Cys in the sequence confirms that Bcp2 is the first archaeal 1-Cysteine per- oxiredoxin (1-Cys Prx) so far identified. Abbreviations Bcp, Bacterioferritin comigratory protein; LB, Luria–Bertani; MCO, metal catalysed oxidation; Prx, peroxiredoxin; ROS, reactive oxygen species. FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 721 reductase, thiol-specific peroxidase, and bacterioferr- itin comigratory protein (Bcp), which perform their protective role in cells through antioxidant activity (ROOH +2e – fiROH + H 2 O), i.e. by reducing and detoxifying H 2 O 2 , peroxynitrite and numerous organic hydroperoxides. They are ubiquitous enzymes found in every domain of life such as eukarya, bacteria and archaea. Prxs use a redox-active cysteine to reduce peroxides. Based on the number of cysteinyl residues involved in catalysis they can be divided into two groups, 1-Cys and 2-Cys Prxs [8]. Structural and mechanistic data actually support the division of 2-Cys Prxs into two subclasses, ‘typical’ and ‘atypical’ 2-Cys Prxs. All three Prxs classes are found to have in common an initial catalytic step, at which peroxid- atic cysteine (Cys-S p H), generally near residue 50, attacks the peroxide substrate and is oxidized to cys- teine sulfenic acid (Cys-SOH). The second step of the peroxidase reaction, the resolution of cysteine sulfenic acid, varies according to the class considered. The Cys-SOH of 1-Cys Prx, is presumably reduced by a thiol-containing electron donor, although the physio- logical partners have not ever been identified [9]. The main characteristic of 2-Cys Prxs, the largest Prx class, is their second redox-active cysteine, the resol- ving cysteine (Cys-S R ). In typical 2-Cys, S p at the N terminus and S R at the C terminus of the protein belong to different subunits and condense to form an intersubunit disulfide bond; in atypical 2-Cys Prxs, S p and S R belong to the same subunit and establish an intrasubunit disulfide bond. The successive reduction of 2-Cys Prxs involves a flavoprotein disulfide reduc- tase and at least one additional protein or domain with a CXXC motif, which is oxidized from the di- thiol to the disulfide state during Prx reduction (e.g. thioredoxin reductase and thioredoxin) (Fig. 1). Two archaeal Prxs from Aeropyrum pernix APE2278 [10] and Pyrococcus horikoshii PH1217 [11,12] have recently been characterized and subsumed within the 2-Cys family. APE2278 was found to have a hexadeca- meric structure and it showed the ability to reduce H 2 O 2 using the NADPH ⁄ thioredoxin reductase ⁄ thio- redoxin system as electron donor partner [13]. Also PH1217 has peroxidase activity but the electron donor partner might be different from that found in A. pernix because in the genome data base of P. horikoshii nei- ther a homologue of A. pernix thioredoxin-like nor other types of thioredoxins were found. In this study we examined the involvement of the peroxiredoxin Bcp2 in oxidative stress in the hyper- thermophilic aerobic archaeon Sulfolobus solfataricus. Furthermore, we report the cloning, the expression and the characterization of the recombinant protein rBcp2 in order to shed light on its role in the detoxifi- cation process and on its catalytic mechanism. Results Identification of the bcp2 gene encoding putative peroxiredoxin The analysis of the complete sequenced genome of S. solfataricus P2 [14] (http://www-archbac.u-psud.fr/ projects/sulfolobus/) revealed four ORFs homologues of Prxs and annotated as Bcp1 (SSO2071), Bcp2 (SSO2121), Bcp3 (SSO225) and Bcp4 (SSO2613). The S. solfataricus Bcp which bears the greatest similarity to other Prxs in the GenBank Database is Bcp2, which encodes a putative protein of 215 amino acids with a predicted molecular mass of 24744.79 Da and a theor- etical pI of 6.85. The deduced amino acid sequence shows 61% identity with the archaeal Prx (APE2278) from the aerobic hyperthermophilic archaeon A. pernix [10], 61% with the putative bacterial Prx (Q9WZR4) derived from the hyperthermophilic bacterium Thermo- toga maritima, 57% with the Prx (PH1217) from the XH 2 X + H 2 O 1-Cys Prx S p OH 1-Cys Prx S p H H 2 O H 2 O 2 RSH Flavoprotein disulfide reductase RSSR 2-Cys Prx S p 2-Cys Prx S p H H 2 O H 2 O 2 S R H 2 O 2-Cys Prx S p OH S R H B S R H A Fig. 1. Peroxiredoxin mechanisms. (A) 1-Cys Prx. (B) 2-Cys Prx. S p peroxidatic cysteine; X unidentified electron donor; S R resolving cysteine; RSH protein or domain with CXXC motif (e.g. thioredoxin). In typical 2-Cys, S p at the N terminus and S R at the C terminus belong to different subunits and condense to form an intersubunit disulfide bond; in atypical 2-Cys, S p at the N terminus and S R at C terminus, originate from the same subunit. Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al. 722 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS anaerobic hyperthermophilic archaeon P. horikoshii [11,12], all of which belong to the 2-Cys Prx family; in addition, Bcp2 reveals 40% of identity with 1-Cys Human PRDX6 (Fig. 2). Following primary structure analysis, Bcp2 was clas- sified as a 1-Cys Prx with only one conserved cysteine residue (Cys 49 ) in a consensus surrounding sequence DFTPVCTTE which is also found both in prokaryotic and eukaryotic Prxs. It is the first of all Prxs analysed so far in archaea that has only one cysteine residue in the sequence. Transcriptional analysis of bcp2 under oxidative stress and characterization of mRNA 5¢ end In order to understand the involvement of bcp2 in oxi- dative stress, the levels of bcp2 mRNA were assessed after treatment of S. solfataricus cells with paraquat, which was used to generate O 2 • ) , with H 2 O 2 and tert- butyl hydroperoxide as direct oxidants [15]. To estab- lish the concentrations of agents whose effect can be to slow down or otherwise affect growth, in the expo- nential phase the cells were treated with varying amounts of stressors (data not shown). Therefore, the S. solfataricus P2 strain was grown until the early exponential phase (0.3 OD 600 nm ) and then induced with 0.05 mm H 2 O 2 , 0.1 mm paraquat or 0.05 mm tert- butyl hydroperoxide for different periods of time (Fig. 3). As shown, the addition of stressors to the cul- tures inhibits growth without killing the cells. The hybridizing band in the northern analysis showed the expected size of about 680 bp indicating that the gene is transcribed as a monocistronic mRNA. When S. solfataricus cells were incubated with H 2 O 2 , paraquat and tert-butyl hydroperoxide, the bcp2 Fig. 2. Multiple sequence alignment (CLUSTAL W 1.82) of Bcp2 from S. solfataricus and Prxs from A. pernix (APE2278), T. maritima (Q9WZR4), P. horikoshii (PH1217), and human (PRDX6). 0 0,1 0,2 0,3 0,4 0,5 0,6 0246810 Time (h) OD m n 0 0 6 Fig. 3. S. solfataricus P2 cultures treated with different oxidative stress agents. Sulfolobus solfataricus P2 cultures were grown until 0.3 OD 600 nm then the cultures were treated with 0.1 mM paraquat (n), 0.05 m M H 2 O 2 (m), 0.05 mM tert-butyl hydroperoxide(*), or con- trol (r). The arrow indicates the OD 600 nm value at which the anti- oxidant agents were added. D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 723 mRNA levels increased considerably (Fig. 4A, B and C ), i.e. a 10-fold increase in transcriptional levels was observed 15 min after the addition of H 2 O 2 , and a fourfold increase within 30 min after paraquat treat- ment; when the tert-butyl hydroperoxide was used, the induction observed was less marked. To evaluate bcp2 expression in response to growth phases, the RNA obtained from cultures harvested at 0.3, 0.6 and 1.0 OD 600 nm corresponding to early, mid and station- ary growth phases was analysed. The data obtained suggest that bcp2 transcriptional levels were independ- ent of the growth phase (Fig. 5). Primer extension analysis was performed in order to characterize the promoter region (Fig. 6). Figure 6B shows the nucleotide sequence 5¢ of the upstream regu- latory region of the bcp2 gene. A consensus sequence, GGUG, with Shine–Dalgarno motifs of the Sulfolobus species was observed upstream of the ATG start codon [16]. The 5¢ end of the bcp2 transcript begins with an adenine and maps 10 nucleotides upstream of the ATG translation start codon. The presence of cis-act- ing regulatory sequences typical of archaeal promoters had been observed. These sequences are part of the basal transcriptional apparatus, such as the TATA box, centered at )27 from the transcriptional start site, and the BRE motif, targets for the general transcrip- tion factors TBP and TFB, respectively [17]. Purification and characterization of recombinant Bcp2 (rBcp2) In order to overproduce rBcp2, the gene was amplified by PCR from S. solfataricus genomic DNA, as des- cribed in Experimental procedures, and cloned into pET-30c(+), rBcp2 was highly overexpressed in Escherichia coli in soluble form, as a fusion with a C-terminal eight-residue histidine tag (LEHHHHHH) with a yield of 12.8% of homogeneous protein. To purify the recombinant protein, the soluble frac- tion (140 mg) of the cell extract was heated at 80 °C for 15 min; this heat treatment removed about 40% of E. coli proteins. rBcp2 was purified to homogeneity in a two-stage process using affinity chromatography on HisTrap HP and gel filtration on HiLoad Superdex 75 obtaining 30 mg and 18 mg, respectively. The SDS ⁄ PAGE of the final preparation revealed a single band with a molecular mass of 25 ± 1 kDa (Fig. 7). The molecular mass of rBcp2, 25 678 Da, was deter- mined using mass spectrometric analysis as reported in Experimental procedures; the 131 shortfall compared to the predicted 25 809 Da molecular mass suggested the removal of the N-terminal methionine. To assess the quaternary structure of the enzyme, analytical gel filtration on PC 75 and Biosep-SEC-4000 of purified rBcp2 were performed. The protein was eluted at a volume accounting for a monomeric struc- ture, but it was observed that increasing protein 16S rRNA bcp2 mRNA 0 15 30 45 60 CBA min H 2 O 2 0 15 30 45 60 paraquat 0 15 30 45 60 tert-butyl hydroperoxide Fig. 4. Northern hybridization analysis of S. solfataricus P2 bcp2 transcripts: effect of oxidative stress agents. Cultures of S. solfataricus P2 were grown until the mid-exponential phase and treated with (A) 0.05 m M H 2 O 2 (B) 0.1 mM paraquat (C) 0.05 mM tert-butyl hydroperoxide. RNAs were obtained from cultures harvested at time shown. On the bottom 16S rRNA were reported as normalization. A B 16S rRNA bcp2 mRNA 1 2 3 1 2 3 Fig. 5. Northern hybridization analysis of S. solfataricus P2 bcp2 transcripts at different growth phases. (A) RNA was extracted from cultures harvested at 0.3 OD 600 nm (1), at 0.6 OD 600 nm (2), at 1.0 OD 600 nm (3). (B) 16S rRNA levels were used as a control. Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al. 724 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS concentration (0.4–2 lgÆlL )1 ) or prolonged storage time (24 h at 4 °C), produced changes in the quater- nary structure with the appearance of the dimeric and multimeric forms (data not shown). Homogeneous rBcp2 was tested for its capacity to serve as an antioxidant enzyme. One of the most widely used test for detecting Prx activity is the ability to pro- tect plasmids against the metal catalysed oxidation (MCO) system (DTT ⁄ Fe 3+ ⁄ O 2 ); in the presence of an electron donor, such as dithiothreitol (DTT), Fe 3+ catalyses the reduction of O 2 to H 2 O 2 , which is further converted to OH • by the Fenton reaction [18]. The MCO system causes damage to DNA by producing OH • , which in turn can nick the intact supercoiled plas- mid DNA [19] as shown in Fig. 8 (lane 4). The damage was averted when rBcp2 was included in the reaction mixture, showing that the enzyme is an active Prx and can remove H 2 O 2 generated by the MCO system in vitro (Fig. 8A). BSA was used as negative control. The antioxidant activity of rBcp2 was then tested for its ability to remove exogenously added H 2 O 2 in a more quantitative in vitro spectrophotometric assay. rBcp2 was capable of catalysing the removal of H 2 O 2 in a concentration-dependent manner using DTT as the electron donor (Fig. 8B). In order to characterize the thermophilicity of rBcp2, peroxidase activity was investigated by measuring the H 2 O 2 removal at increasing temperature. rBcp2 showed maximum activity between 80 and 90 °C, which is in the optimum temperature range for the growth of A B Fig. 6. (A) Primer extension analysis and sequence of the S. solfataricus bcp2 gene. Total RNA was isolated from a culture of S. solfataricus P2. Primer extension was carried out as described in Experimental procedures, and the products were separated by electrophoresis under denaturing conditions alongside sequencing reactions with the same primer. (B) Nucleotide sequence of bcp2. The transcriptional start point is shown by the bent arrow above the underlined boldface A nucleotide. The Shine–Dalgarno sequence is underlined by dotted line. A puta- tive TATA box and BRE sequence are underlined. The ATG start codon is in bold and the TAA stop codon is marked by an asterisk. D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 725 S. solfataricus. We also analysed the thermoresistance of the enzyme by incubating rBcp2 for varying periods of time at 80, 90, and 95 °C and then assaying the resid- ual peroxidase activity. Following incubation for 6 h at 80 °C, the activity retained was 63%; the enzyme dis- played a half-life of 3 h at 90 °C, while after 30 min at 95 °C the activity measured was 30% (Fig. 9). Bcp2 expression in S. solfataricus We investigated how the expression of Bcp2 was induced by H 2 O 2 , paraquat and tert-butyl hydroper- oxide. A polyclonal rBcp2-specific rabbit antiserum was used to conduct a quantitative analysis of the Bcp2 expression in S. solfataricus. The anti-Bcp2 anti- serum was used in a western blot analysis on cytoplas- mic extracts from cells harvested in the exponential growth phase before and after addition of the stressor agents at the identical concentrations utilized in the northern analysis (Fig. 10). Twenty-five kDa signals corresponding to Bcp2 were detected in noninduced and induced cells. The increased amount of Bcp2 in the cytoplasmic fraction of S. solfataricus correlated with increased bcp2 mRNA level after treatment with the stressors. In particular, the maximum Bcp2 expres- sion ) a sevenfold increase compared to that monit- ored for controls ) was observed after H 2 O 2 addition as in the northern analysis. Fig. 7. SDS ⁄ PAGE of different steps in the purification of rBcp2. Lane 1, E. coli BL21-CodonPlus (DE3)-RIL ⁄ pETBcp2 cellular extract not induced by IPTG; lane 2, E. coli BL21-CodonPlus (DE3)- RIL ⁄ pETBcp2 induced by 1 m M isopropyl-thio-b-d-galactopyrano- side; lane 3, heat-treated sample; lane 4, molecular weight markers; lane 5, sample after affinity chromatography; lane 6, sam- ple after size-exclusion chromatography. A B Fig. 8. (A) rBcp2 assayed as antioxidant enzyme: DNA cleavage protection assay performed by rBcp2. Supercoiled pUC19 plasmid was exposed to the MCO system (DTT ⁄ Fe 3+ ⁄ O 2 ) alone and with different rBcp2 concentrations. Nicked form (NF) and supercoiled form (SF) of pUC19 are indicated on the left by arrows. (B) rBcp2 was assayed for its ability to remove H 2 O 2 in an in vitro assay system in the presence (r) and absence (d) of DTT. Peroxidase activity was measured at 80 °C using the ferrithiocyanate complex as described in experimental procedures. The nonenzymatic removal of H 2 O 2 by heat was performed in parallel. Fig. 9. Thermoresistance was measured as residual peroxidase activity with 50 lgÆmL )1 of rBcp2 after incubation for different times at 80 °C(n), 90 °C(r), 95 °C(m). Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al. 726 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS Role of the conserved cysteine residue in rBcp2 To investigate the catalytic role of the Cys residue we constructed a mutant enzyme in which the cysteine at position 49 was replaced by serine (C49S). The mutant Bcp2 protein was expressed in E. coli BL21-CodonPlus (DE3)-RIL cells and purified from the soluble fraction of bacterial cells as described in Experimental proce- dures. The yield of C49S was the same of that obtained for the wild-type protein. The activity of C49S was tested by peroxidase (Fig. 11A) and DNA cleavage protection (Fig. 11B) assays and compared to that of the wild-type protein. In both cases the mutant showed no peroxidase or plasmid DNA protection activity. These findings indi- cate that Cys 49 is required for the proceeding of the enzymatic reaction and that Cys 49 -SOH can be conver- ted back to Cys-SH using DTT, and that Cys 49 -SH is responsible for scavenging the OH • induced by the MCO system. Discussion The natural environment in which S. solfataricus lives is strongly oxidative, in addition ROS can be generated by naturally occurring phenomena such as ultraviolet irradiation of water, autoxidations and aeration turbulence. Consequently, to survive in this harsh habitat S. solfataricus should have developed antioxidant enzymes and molecules that protect it from ROS. At the present time the investigation of response to oxidative stress in S. solfataricus is at an initial stage and has been focused mainly on the superoxide dismutase (Fe-SOD) (EC 1.15.1.1) [20,21]. This enzyme represents the primary defense against O 2 • ) as suggested by its ubiquitous location in the membrane and in the cytoplasm [22], by its constitu- tive level [21] and by the long half-life (2 h) of the mRNA [23]. The dismutation of O 2 • ) by SOD deve- lops H 2 O 2 that can go freely through the membrane CBA Fig. 10. Bcp2 expression in S. solfataricus cells in response to H 2 O 2 (A), paraquat (B) and tert-butyl hydroperoxide (C). Twenty micrograms of cytoplasmic proteins extracted from nonexposed and exposed culture for 30, 60 and 120 min were analysed by western blot with anti- rBcp2 IgG. AB Fig. 11. Effect on peroxidase activity of replacement of Cys 49 of rBcp2 with serine. (A) At different enzyme concentrations the peroxidase activity was assayed as previously reported. H 2 O 2 removal by rBcp2 (r) and C49S (m) was measured over a range of concentrations (0– 100 lgÆmL )1 ). (B) Effect on protection against DNA cleavage of the replacement of Cys 49 of rBcp2 with serine. Lane 1, pUC19; lane 2, pUC19 and 10 m M DTT; lane 3, pUC19 and 3 lM FeCl 3 ; lane 4, pUC19, 10 mM DTT, and 3 lM FeCl 3 ; lane 5, pUC19, 10 mM DTT, 3 lM FeCl 3 , and 50 lgÆmL )1 rBcp2; lane 6, pUC19, 10 mM DTT, 3 lM FeCl 3 , and 50 lgÆmL )1 C49S. D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 727 in the cell, where it must be scavenged to prevent damage of biological molecules. In S. solfataricus the peroxide detoxification system has not yet been studied. Genome analysis has shown the absence of putative catalases and the presence of four putative Bcps proteins: Bcp1, Bcp2, Bcp3, Bcp4 whose roles should be clarified in detail and could play a key role in the detoxification processes. In this study we examined the role of Bcp2 in order to increase the knowledge of the enzymatic activity involved in the oxidative stress in S. solfataricus. To detect differences in the response to various agents we induced oxidative stress with paraquat, an O 2 • ) generating compound, H 2 O 2 and tert-butyl hydroperoxide, an alkyl hydroperoxide. Our results show that compounds acting both indirectly and directly as oxidants can induce transcription of bcp2. Data reveal that transcription of bcp2 in S. solfataricus is upregulated by the various stressors, and the differ- ent kinetics in response to these agents imply that sev- eral regulatory mechanisms or at least variations on the same mechanism could be involved in controlling the expression of bcp2. Western analysis performed after treatment with H 2 O 2 showed a slower and more slight increase in protein level than mRNA level. This could imply that post-transcriptional processes, such as lower rate of translational or protein instability caused by oxidative stress conditions, are important in deter- mining the level of Bcp2 protein. Similar results are observed for genes and related proteins involved in oxidative stress in other microrganisms [24]. Moreover, the basal level of bcp2 transcript in the early and mid-exponential, and the stationary phase of growth suggests that bcp2 is not involved in the control of endogenous peroxides that are produced during aero- bic respiration. In contrast with Prxs discovered in the aerobic hyperthermophilic archaeon A. pernix and the anaer- obic hyperthermothilic archaeon P. horikoshii, the ana- lysis of the primary structure of Bcp2 shows only one cysteine (Cys 49 ). This residue is positioned inside the DFTPVCTTE sequence in the N-terminal region of the protein that is conserved both in 1-Cys and 2-Cys classes of Prxs. Site-directed mutagenesis showed that Cys 49 is required for peroxidase activity. Both func- tional data and analysis of homologous sequences sup- ported the classification of Bcp2 in the family of peroxiredoxin in the 1-Cys Prx class and Bcp2 could be considered the first ancient Prx developed in the early stages of evolution. The results indicate that Bcp2 displays peroxidase activity with a temperature optimum between 80 and 90 °C which is the temperature range for growth of S. solfataricus. The enzyme appears to be less thermo- stable (60% of activity after 15 min at 90 °C) than the Prx of P. horikoshii that retains full activity on heating at 90 °C for 20 min; this difference reflects the differ- ence in the optimum growth temperature between the two organisms. The enzyme can function at 37 °Cas verified by the protection of DNA in MCO system and by removal of H 2 O 2 (data not shown) but it has the maximum activity in the range 80–90 °C. The peroxi- dase activity of Bcp2 is DTT dependent, suggesting a mechanism in which Cys 49 residue is firstly oxidized by H 2 O 2 and successively reduced by DTT that could be the electron donor partner in vitro. The physiological partner has not yet been found. Recently, a thioredox- in reductase has been characterized in S. solfataricus; the presence in the genome of two thioredoxins and two other thioredoxin reductases suggest their involve- ment as physiological partners as electron donor. These speculations require experimental evidence and studies are underway in our laboratory. Finally size-exclusion chromatography has shown that the protein can shift from a monomer to a multi- meric form depending on the protein concentration and the temperature. On the basis of the data repor- ted in the literature on the structure of Prxs [25] ionic interactions play an important role in oligomerization; further analyses are in procress to define completely the quaternary structure of the enzyme. Experimental procedures Strains, media and growth conditions Sulfolobus solfataricus P2 strain liquid cultures were grown aerobically at 80 °C in mineral medium supplemented with 0.1% Bacto TM yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 0.1% tryptone (Oxoid, Basingstoke, Hampshire, UK) and 0.2% sucrose (TYS medium) in an orbital shaker. Oxidative stresses were created by adding H 2 O 2 , paraquat, or tert-butyl hydro- peroxide at a final concentration of 0.05 mm, 0.1 mm and 0.05 mm, respectively, to S. solfataricus cultures in early exponential growth phase (0.3 OD 600 nm ). Escherichia coli Top F¢10 was used as a general host for DNA manipulation, E. coli XL1-Blue (Invitrogen SRL, Milan, Italy) was used to transform the mutagenesis products. Escherichia coli BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA, USA) was used for expression of the recombinant Bcp2. These strains were cultivated in Luria–Bertani (LB) medium at 37 ° C. When necessary 100 lgÆmL )1 ampicillin, or 50 lgÆmL )1 kanamycin and 33 lgÆmL )1 chloramphenicol were added to the medium to maintain plasmids. Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al. 728 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS RNA extraction Sulfolobus solfataricus cultures, grown to early exponential phase, were stressed with H 2 O 2, paraquat or tert -butyl hydroperoxide. Aliquots were collected at different times by centrifugation at 5000 g for 10 min at 4 °C. Total RNA was extracted by the guanidinium isothiocyanate method as described in Sambrook et al. [26]. The integrity and concen- tration of total RNA were verified by electrophoretic analy- sis by separating total RNA on 1% agarose gel containing formaldehyde. Northern hybridization Northern blot analysis was used to quantify the amount of bcp-2 mRNA in different stress conditions and to determine the size of the specific transcript. Genomic DNA from S. sol- fataricus P2 was used as a template for PCR amplification of bcp2 using HF Taq DNA polymerase (Roche Applied Science, Monza, Italy) and the following primers: forward primer (the inserted Nde I restriction site is underlined) 5¢-CTAGGTGAA CATATGAGTGAGGAAAGAATTCC-3¢ and the reverse primer 5¢-GGAGCTGGATTAATG CTC GAGTCTCCTATTAG-3¢ (the inserted XhoI restriction site is underlined). The PCR products obtained were purified from agarose gel, and the NdeI–XhoI fragment was labelled with a 32 P(dATP) and with Random primed DNA labelling kit (Roche). Primer extension Primer extension analysis was carried out with avian myelo- blastosis virus reverse transcriptase (Roche) as described in Limauro et al. [27] using the synthetic oligonucleotide 5¢-CATCAGGTAGTTTTATCCTGCC-3¢ complementary to nucleotides 1801–1822 of DB source AE006819. The sequencing reaction of the corresponding DNA fragment cloned, which had been primed with the same synthetic oligonucleotide, was used as a marker to locate the prod- ucts on 6% urea ⁄ polyacrylamide gel. Construction and expression of recombinant protein Genomic DNA of S. solfataricus was prepared as described by Arnold et al. [28]. bcp2 was amplified by PCR using chromosomal DNA as template and the same two primers as used to generate the northern blot probe. Amplification by PCR was carried out at 94 °C for 1 min, 45 °C for 1 min, and 72 °C for 1 min, for 35 cycles using HF Taq DNA polymerase (Roche). The PCR product was purified with QIAquick PCR purification kit (Quiagen Spa, Milan, Italy) and cloned in pGEMTeasy vector (Promega Italia srl, Milan, Italy). The nucleotide sequence of the inserted gene was determined to ensure that no mutations were pre- sent in the gene. Then, the NdeI–XhoI fragment was cloned into pET-30c(+) (Novagen, Darmstadt, Germany) giving the recombinant plasmid pETBcp2 that was used to trans- form the E. coli BL21-CodonPlus (DE3)-RIL. Purification of the recombinant Bcp2 protein (rBcp2) BL21-CodonPlus (DE3)-RIL ⁄ pETBcp2 cells were grown at 37 °C in 1000 mL LB medium supplemented with kanamycin and chloramphenicol to an OD 600 of 1 was reached. Induc- tion was carried out by the addition of 1 mm isopropyl- thio-b-d-galactopyranoside to the medium and growth was continued for 12 h. Cells were harvested by centrifugation, suspended in 20 mm Tris ⁄ HCl pH 8.0 and distrupted by ultrasonication (Sonicator Ultrasonic liquid processor; Heat System Ultrasonics Inc., Plainview, NY, USA). The suspen- sion was ultracentrifugated at 160 000 g for 30 min. The crude extract obtained was heated at 80 °C for 15 min, and the denaturated proteins were removed by centrifugation (15 000 g for 30 min). The extract was concentrated (Am- icon, Millipore Corp., Bedford, MA) and loaded on a Hi- sTrap HP (Amersham Biosciences Europe GmbH, Milan, Italy) equilibrated with 50 m m Tris ⁄ HCl pH 8.0 ⁄ 0.3 m NaCl (buffer A). After the column was washed with buffer A containing 20 mm imidazole, proteins were eluted with the same buffer A supplemented with 250 mm imidazole. The active fractions were pooled and dialyzed against 20 mm Tris ⁄ HCl pH 8.0 ⁄ 1mm DTT. The concentrated sample was applied to HiLoad Superdex 75 column (1.6 cm · 60 cm, Amersham) connected to an FPLC system (Amersham) and eluted with 50 mm Tris ⁄ HCl pH 8.0 ⁄ 0.2 m KCl at a flow rate of 1 mLÆmin )1 . The active fractions were pooled, concentra- ted and extensively dialysed against 20 mm Tris ⁄ HCl pH 8.0. Determination of quaternary structure The molecular mass of the protein was determined by gel-filtration chromatography on a Superdex 75 PC (0.3 cm · 3.2 cm) and a Biosep-SEC-S4000 (30 cm · 0.78 cm, Phenomenex Inc., St Torrance, CA, USA) connected to AKTA system (Amersham). Protein was eluted with buffer 50 mm Tris ⁄ HCl pH 8.0 ⁄ 0.2 m KCl at a flow rate of 0.04 mLÆmin )1 and with 20 mm NaPO 4 pH 7.2 at a flow rate of 0.5 mLÆ min )1 . b-Amylase (200 kDa), alcohol dehy- drogenase (150 kDa), BSA (65.4 kDa), ovalbumin (48.9 kDa), chymotrypsinogen (22.8 kDa) and the RNA- se A (15.6 kDa) were used as molecular weight standards. Analytical methods for protein characterization Protein concentration was determined using BSA as the standard [29]. Protein homogeneity was estimated by D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 729 SDS ⁄ PAGE [30] using a 12.5% (w ⁄ v) acrylamide resolving gel and a 5% acrylamide stacking gel. Samples were heated at 100 °C for 5 min in 2% SDS ⁄ 2% 2-mercaptoethanol and run in comparison with molecular weight standards. Gels were stained with the Coomassie blue. The molecular mass of the protein was also estimated using electrospray MS recorded on a Bio-Q triple quadrupole instrument (Thermofinnigan, San Jose, CA, USA). Samples were dissolved in 1% (v ⁄ v) acetic acid ⁄ 50% (v ⁄ v) acetonitrile and injected into the ion source at a flow rate of 10 lLÆmin )1 using a Phoenix syringe pump. Spectra were collected and elaborated using MASSLYNX software provided by the manufacturer. Calibration of the mass spectrometer was performed with horse heart myoglobin (16.9 kDa). Assay of peroxidase activity rBcp2 was tested for its ability to remove H 2 O 2 in an in vitro assay system as fol- lows. The reaction was started adding H 2 O 2 at a final concentration of 0.2 mm to the reaction mixture contain- ing 50 mm Hepes pH 7.0 ⁄ 10 mm DTT in the presence of different concentrations of Bcp2 in a final volume of 0.1 mL. The reaction was incubated at 80 °C for 1 min and stopped by the addition of 0.9 mL trichloroacetic acid (10% w ⁄ v) [19]. Peroxidase activity was determined from the amount of peroxide remaining, which was detec- ted by measurement of the purple-coloured ferrithiocya- nate complex developed after the addition of 0.2 mL 10 mm Fe(NH 4 ) 2 (SO 4 ) 2 and 0.1 mL 2.5 n KSCN using H 2 O 2 as standard. The amount of ferrithiocyanate com- plex was determined by absorbance at 490 nm. The per- centage of H 2 O 2 removed was calculated on the basis of the change in A 490 obtained with Bcp2 relative to that obtained without Bcp2. Thermophilicity and thermoresistance Thermophilicity was evaluated in the temperature range 50–90 °C by measuring peroxidase activity by the ferrithio- cyanate method. rBcp2 thermoresistance was estimated by measuring the residual peroxidase activity at 80 °C for 1 min after heat treatment at 80 °C, 90 °C, 95 °C for differ- ent times. DNA cleavage assay by the MCO system The ability of Bcp2 to protect DNA from oxidative nick- ing by OH • was determined as described previously [19]. A reaction mixture of 50 lL included 3 lm FeCl 3 ,10mm DTT for the thiol MCO system, 100 mm Hepes pH 7.0, different concentrations of rBcp2 or BSA as a negative control. The reaction was initiated incubating the mixture for 40 min at 37 °C before adding 2 lg plasmid pUC19 and developed for an additional 4 h at the same tempera- ture. DNA bands were evaluated on a 0.8% (w ⁄ v) agarose gel after staining with ethidium bromide (5 lgÆmL )1 ). Site-directed mutagenesis The mutant rBcp2 C49S was obtained by following the pro- tocol outlined in the QuickChange II site directed mutagen- esis kit (Stratagene) using primers complementary to the coding and noncoding template sequence (pET30Bcp2) con- taining a double mismatch. To generate the C49S mutant, the forward primer 5 ¢-GATTTCACACCGGTG AGCA CTACGGAGTTCTAC-3¢ and a complementary reverse pri- mer were used (the underlined letter indicates the base pair mismatch). The reaction (50 lL) contained 50 ng template DNA (pET30Bcp2), 125 ng each primer, 200 lm dNTP and 2.5 U Pfu Ultra HF DNA polymerase. Twelve cycles of 95 °C for 30 s, 55 °C for 1 min and 68 °C for 6 min were carried out in a Mini Cycler followed by one cycle at 4 °C for 2 min. To digest methylated template, each reaction mixture was treated with 10 U DnpIat37°C for 1 h. Muta- genesis products were transformed into XL-1 Blue cells. Sin- gle colonies were selected on LB plates containing kanamicin, and isolated plasmid DNA was sequenced throughout the coding region at Primm (DNA sequencing service Naples, Italy). The plasmid pETBcp2Mut containing the mutation inserted was used to transform BL21-Codon- Plus (DE3)-RIL competent cells. C49S was expressed and purified with the same procedure reported above for rBcp2. Western blot analysis Sulfolobus solfataricus was stimulated during early exponen- tial growth with 0.05 mm H 2 O 2 , 0.1 mm paraquat or 0.05 mm tert-butyl hydroperoxide for 30, 60, and 120 min. Cells were harvested by centrifugation, suspended in 20 mm Tris ⁄ HCl pH 8.0 and the crude extracts were prepared by so- nication, disrupting the cells with 3 · 1-min pulses at 4 °Cat 20 Hz and ultracentrifugated at 160 000 g for 30 min at 4 °C. Following SDS ⁄ PAGE of homogenates, proteins were electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk powder, 0.1% Tween in TBS (20 mm Tris ⁄ HCl pH 7.5, 0.9% NaCl) and then incubated with rBcp2-specific rabbit antibodies (Igtech, Paestum, Salerno, Italy) for 2 h, followed by peroxidase-conjugated secondary antibodies for 1 h. Antibodies to rBcp2 were detected by enhanced chemiluminescence using ECL Plus western blotting Detec- tion system (Amersham Biosciences). Acknowledgements This work was supported by grants from MIUR (PRIN 2002). References 1 Halliwell B & Gutteridge JMC (1989) Free Radicals in Biology and Medicine. Oxford: Clarendon Press. Antioxidant activity of Bcp2 in S. solfataricus D. 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Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress Danila. thioredoxin-like nor other types of thioredoxins were found. In this study we examined the involvement of the peroxiredoxin Bcp2 in oxidative stress in the