A 35 kDa NAD(P)H oxidase previously isolated from the archaeonSulfolobus solfataricus is instead a thioredoxin reductase

M.R. Ruocco a, A. Ruggiero b, L. Masullo a, P. Arcari a,c, M. Masullo a,d,*a Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, via S. Pansini 5, I-80131 Napoli, Italia

b Dipartimento di Chimica Biologica, Sezione di Biostrutture, Università di Napoli Federico II, via Mezzocannone 6, 80134 Napoli, Italiac CEINGE Biotecnologie Avanzate S.C. a r.l., via Comunale Margherita 482, 80145 Napoli, Italia

d Dipartimento di Scienze Farmacobiologiche, Università degli Studi di Catanzaro “Magna Graecia”, 88021 Roccelletta di Borgia, Catanzaro, Italia

Received 28 July 2004; accepted 15 October 2004

Available online 10 November 2004

Abstract

A thioredoxin reductase (TrxR) has been identified in the hyperthermophilic archaeon Sulfolobus solfataricus (Ss). This enzyme is ahomodimeric flavoprotein that was previously identified as NADH oxidase in the same micro-organism (‘Biotechnol. Appl. Biochem. 23(1996) 47’). The primary structure of SsTrxR is made of 323 amino acid residues and contains two putative bab regions for the binding ofFAD, and a NADP(H) binding consensus sequence in the proximity of a CXXC motif. These findings indicate that SsTrxR is structurallyrelated to the class II of the pyridine nucleotide-disulphide oxidoreductases family. Moreover, the enzyme exhibits a NADP(H) dependentthioredoxin reductase activity requiring the presence of FAD. Surprisingly, the reductase activity of SsTrxR is reduced in the presence of aspecific inhibitor of mammalian TrxR. This finding demonstrates that the archaeal enzyme, although structurally related to eubacterial TrxR,is functionally closer to eukaryal enzymes. Experimental evidences indicate that a disulphide bridge is required for the reductase but also forthe NADH oxidase activity of the enzyme. These results are further supported by the significantly reduced activities exerted by the C147Amutant. The integrity of the CXXC motif is also involved in the stability of the enzyme.© 2004 Elsevier SAS. All rights reserved.

Keywords: Thioredoxin reductase; NAD(P)H oxidase; Archaea; Sulfolobus solfataricus; mutagenic analysis

1. Introduction

Thioredoxin reductase (TrxR) is a flavoenzyme thatcatalyses the NADPH-dependent electron transfer to theactive site disulphide of oxidised thioredoxin (Trx) to forma dithiol [1]. The reduced form of Trx in turn transfers itsreducing equivalents to several cellular targets. This redoxsystem is widely distributed in nature and participates to dif-ferent cellular functions [2]. In particular, the activity of TrxR

has been involved in ribonucleotide metabolism [3], controlof cellular redox state [4,5], regulation of transcriptionalactivity [6] and protein folding [7]. TrxR is organised as ahomodimer [8], and on the basis of the monomer size, thisenzyme can be classified into two structurally unrelatedgroups. Type I high molecular mass TrxR (55–58 kDa persubunit), isolated from higher eukaryotes [9–11] and type IIlow molecular mass TrxR (around 35 kDa per subunit) iso-lated from lower eukaryotes [12] and prokaryotes [13]. Bothenzymes belong to the family of pyridine nucleotide-disulphide oxidoreductases that includes also lipoamidedehydrogenase, glutathione reductase and mercuric reduc-tase [14], but class I TrxR exhibits a broader substrate speci-ficity, and an additional redox active motif at their C-terminalregion [2]. In archaea, only recently TrxR has been isolatedand characterised in Aeropyrum pernix K1 [15] and Pyro-coccus horikoshii [16] belonging to the crenarchaeota andeuryarchaeota branches of the archaeal domain, respectively[17]. Regarding the enzymes, that in Sulfolobales are in-

Abbreviations: TrxR, thioredoxin reductase (E.C. 1.8.1.9); Trx, thiore-doxin; Ss, Sulfolobus solfataricus; At, Arabidopsis thaliana; Ec, Escheri-chia coli; DTNB, 5,5′dithiobis (2,2′-dinitrobenzoate).

* Corresponding author. Dipartimento di Scienze Farmacobiologiche,Università degli Studi di Catanzaro “Magna Graecia”, 88021 Roccellettadi Borgia, Catanzaro, Italia. Tel.: +39-081-7463127; fax:+39-081-7463653.

E-mail address: masullo@unicz.it (M. Masullo).

Biochimie 86 (2004) 883–892

www.elsevier.com/locate/biochi

0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.biochi.2004.10.008

volved in the control of the intracellular redox balance, wehave previously reported the isolation and characterisation ofa NADH oxidase from the archaea Sulfolobus solfataricusand Sulfolobus acidocaldarius [18]. The S. solfataricus en-zyme was a homodimer composed of two 35 kDa subunitscontaining one molecule of FAD per subunit, whereas theS. acidocaldarius enzyme was a 27 kDa monomer which waspurified with no flavin nucleotide bound. More recently, wehave also reported the cloning and the characterisation of a38 kDa NADH oxidase from S. solfataricus which was dif-ferent from that previously identified [19], but with a highsequence homology to a putative eubacterial thioredoxinreductase [20]. However, this enzyme showed a ho-modimeric structure that did not contain the canonical con-sensus sequence CXXC typical of the enzymes belonging tothe TrxR family. These findings prompted us to investigatewhether the previously reported S. solfataricus 35 kDaNADH oxidase [18] could belong to the TrxR family. In thiswork, we show that this enzyme contains the CXXC motif inthe active site and possesses thioredoxin reductase activity.

2. Materials and methods

2.1. Materials

Restriction enzymes, modifying enzymes and labelledcompounds were from Amersham. Plasmid DNA, genomicDNA and labelled probes were prepared as described [21].b-NADH, b-NADPH, FAD, ATP, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), isopropyl-b-thiogalactopyra-noside (IPTG), auranofin, azelaic acid, dithiothreitol (DTT),b-mercaptoethanol (b-ME), Escherichia coli thioredoxin(EcTrx), bovine trypsin treated with tosyl-L-phenylalaninechloromethyl ketone, and soybean trypsin inhibitor wereobtained from Sigma. The following buffers were used.Buffer A: 25 mM Mes/KOH, pH 5.5; buffer B: 20 mMTris/HCl, pH 7.8, 5 mM MgCl2, 50 mM KCl; buffer C:20 mM Tris/HCl, pH 7.8; buffer D: 20 mM Tris/HCl, pH 7.8,50% glycerol.

2.2. Purification and trypsin digestion of SsTrxR

SsTrxR, formerly identified as 35 kDa NADH oxidase waspurified from S. solfataricus cells as reported [18]. The tryp-tic digestion of the enzyme was performed in buffer B at aconcentration of 0.2 mg/ml in the presence of 0.4 mg/mltrypsin. The mixture was incubated at 37 °C overnight andthe reaction was stopped by adding soybean trypsin inhibitor(25 µg/ml final concentration). The mixture was then analy-sed on a C18 column connected to an HPLC system equili-brated in 0.1% trifluoracetic acid containing 5% acetonitrile.Peptides were eluted by a linear 5–55% acetonitrile gradientin 0.1% trifluoracetic acid. The most abundant peak, eluted atabout 35% acetonitrile, was dried and its N-terminal aminoacid sequence was determined.

2.3. Cloning of the SsTrxR encoding gene

A molecular probe for the isolation of the gene encodingSsTrxR was amplified by PCR, in a Perkin Elmer DNAThermal Cycler, using the S. solfataricus genomic DNA astemplate, and two primers derived from the amino acid se-quence of the enzyme. In particular, the forward primerGA(AG)AA(AG)TT(CT)GA(CT)GT(AT)AT(AT)AT (TrxR1)was deduced from the N-terminal amino acid sequenceE14KFDVI of the purified protein [18], whereas the reverseprimer AA(AT)CC(AT)AT(TC)TC(AT)AT(GA)AA(AT)AC(TrxR2) was designed from the N-terminal sequenceV243FIEIG of the purified tryptic fragment of the enzymedescribed above. Degenerations on the third codon positionwere reduced on the basis of the preferential codon usage inS. solfataricus [22] and indicated in parentheses. A S. solfa-taricus DNA library was prepared into pUC19 cloning vec-tor, using EcoR I and HindIII as cloning sites. About 2 × 103

colonies were analysed using the 32P labelled PCR fragment,as a probe and a recombinant clone containing a 4 kbp insert,including the entire SsTrxR gene, was isolated and partiallysequenced. This plasmid was named pUCTrxR.

2.4. Construction of the expression vector

The complete coding sequence of the SsTrxR gene wassynthesised by PCR using as template pUCTrxR. The prim-ers used were 5′-GTATAGccaTGgGTCTTCTACCAA-3′(forward) and 5′-TATCGTGaaGCtTCTATAGCAAC-3′ (re-verse), where the underlined letters indicate the initiationcodon and lower case letters indicate the base substitutionsinserted to create the Nco I and Hind III cloning sites. Theamplified fragment was digested with Nco I and Hind III andinserted into the prokaryotic expression vector pET-28(Novagen), digested with the same enzymes. Restrictionanalysis and sequencing confirmed the correct sequence ofthe recombinant clone, which was designated as pSsTrxR.

2.5. Site-directed mutagenesis

Cysteine 147 in SsTrxR was mutated to alanine (C147A)by site-directed mutagenesis [23]. The primers used were:5′-CTGCTCAGTCGCTGATGCACCTC-3′ (forward), and5′-GAGGTGCATCAGCGACTGAGCAG-3′ (reverse); theunderlined triplet GCT coding for Ala147 replaced the wild-type triplet TGT coding for Cys147. Double-strandedpSsTrxR and primers were added to a reaction mixture con-taining deoxyribonucleotides, buffer, and PfuTurbo™ DNApolymerase according to the manufacturer’s protocol (Strat-agene). The correct substitutions and the absence of otherundesired mutations were confirmed by sequence analysis.

2.6. SsTrxR expression and purification

E. coli BL21 (DE3) competent cells were transformedwith the pSsTrxR expression vector and grown in Luria–

884 M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

Bertani medium at 37 °C overnight. The addition of 1 mMIPTG for induction led to the accumulation of a large amountof insoluble recombinant protein; therefore, the IPTG induc-tion was omitted. Cells were harvested by centrifugation at3000 × g for 15 min at 4 °C, and re-suspended in 20 ml ofbuffer B supplemented with 1 mM PMSF. Cells were dis-rupted by a constant cell disruption system (Constant Sys-tems Ltd., UK) and the suspension was centrifuged at100,000 × g for 2 h to remove cellular debris. The supernatantwas incubated at 70 °C for 30 min to denaturate most of theE. coli proteins that were then removed by centrifugation at22,000 × g for 30 min. The supernatant was dialysed againstbuffer A and then applied to a Mono S cation-exchangecolumn equilibrated at room temperature with buffer A andconnected to an FPLC apparatus (Amersham). RecombinantSsTrxR was eluted with a linear 0–200 mM KCl gradient inbuffer A. Fractions, showing a single protein band onSDS/PAGE, were pooled, concentrated by Aquacide II, di-alysed against buffer D and stored at –20 °C. Under theseconditions, the yield from 1 l culture was 5–10 mg of purifiedrecombinant SsTrxR (rSsTrxR) or its C147A mutant.

2.7. Enzymatic assays

All assays were carried out at 60 °C and were followedkinetically. NADH oxidase activity was determined spectro-photometrically by measuring the initial rate of NADH oxi-dation as reported [18].

Thioredoxin reductase activity was evaluated by bothDTNB- and thioredoxin reduction methods [24,25]. In theDTNB reduction, the formation of the product 2-nitro-5-thiobenzoate (TNB) was followed spectrophotometrically bythe increase in absorbance at 412 nm. A different molarextinction coefficient than that usually employed in theDTNB-method of 7680 M–1 cm–1, was determined in bufferA and used to calculate the enzymatic activity. The reactionmixture contained 5 mM DTNB, 0.25 mM NADPH,0.05 mM FAD, and 10 mM EDTA, in 1 ml final volume ofbuffer A. In the thioredoxin reduction assay, the reactionmixture contained 0.2 mM E. coli Trx (EcTrx), 0.05 mMFAD, 2 mM EDTA, and an appropriate amount of SsTrxR in1 ml final volume of buffer A; the reaction was started byadding 0.1 mM NADPH. The activity was calculated fromthe decrease in absorbance at 340 nm using a molar extinc-tion coefficient of 6220 M–1 cm–1. Blanks run in the absenceof the enzyme were carried out in parallel and were sub-tracted.

The inhibition of rSsTrxR activities was measured in thepresence of the specific TrxR inhibitors auranofin or azelaicacid [25] as well as in the presence of reducing agents (DTTor b-ME). Stock solutions of auranofin (9 mM) and azelaicacid (7 mM) were prepared in DMSO and water, respec-tively. The inhibitory effect was evaluated on both DTNB-and thioredoxin reduction methods. Each inhibitor was incu-bated with rSsTrxR for 1 min at 60 °C prior substrate addi-tion.

2.8. Ellman assay

The number of disulphide bonds was evaluated by theEllman assay [26] on both the oxidised or reduced rSsTrxR.Reduced rSsTrxR was obtained by incubating 25 µM proteinwith 6 M guanidine•HCl, and 50 mM DTT for 20 min at25 °C in 0.1 M sodium phosphate pH 7.2. Following thereduction, the sample was dialysed against 6 Mguanidine•HCl in 0.1 M acetic acid, in order to remove DTTexcess. To titrate free cysteines, 1.25–5 µM of either oxidisedor reduced rSsTrxR in 0.1 M sodium phosphate pH 7.2 con-taining 6 M guanidine•HCl, was incubated with 0.2 MDTNB at 25 °C. Quantification of DTNB-modified cysteineswas carried out monitoring the time-dependent increase inthe absorbance at 412 nm (TNB molar extinction coefficient= 13,600 M–1 cm–1).

2.9. Stability of rSsTrxR

The chemical stability of rSsTrxR or its C147A mutantwas evaluated by fluorescence spectra using 295 nm as exci-tation wavelength, after exposure of the enzymes to 6 Mguanidine•HCl in buffer C for 20 h at 25 °C. The proteinconcentration was 0.2 µM for both rSsTrxR and its C147Amutant.

The thermal denaturation of rSsTrxR or its C147A mutantwas followed by fluorescence melting curves in the tempera-ture range 25–100 °C, using a computer assisted CaryEclipse Spectrofluorimeter (Varian), equipped with an elec-tronic temperature control. The increase in temperature wasset at 0.2 °C/min; the fluorescence in the aromatic region wasmonitored at every degree centigrade increase, using 280 and345 nm excitation and emission wavelength, respectively.The enzyme concentration was 1.42 µM for both rSsTrxRand its C147A mutant. The values of the fluorescence inten-sity were corrected for the temperature quenching, norma-lised between 0% and 100% and plotted vs. temperature [27].In both fluorimetric methods, excitation and emission slitswere set to 10 nm.

2.10. Other methods

Purity of protein samples was assessed on 12%SDS/PAGE using appropriate Mr standards; protein concen-tration was determined using the protein assay system ofBio-Rad, using BSA as the standard. Nucleotide sequencingwas performed on both DNA strands, by using the T7 se-quencing kit (Promega) and synthetic oligonucleotides asprimers. A Southern blot on S. solfataricus genomic DNAwas performed as described [28]. The amino acid sequenceof the N-terminal region and that of a tryptic fragment of theprotein was determined by automated Edman degradation ona pulsed liquid sequencer (Applied Biosystems) connectedon line to an HPLC apparatus for amino acid identification.The molecular mass of proteins in non-denaturing conditionswas determined by a Superdex 75 gel-filtration column cali-

885M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

brated as reported [29]. Query for sequence similarities wasaddressed to the Swiss-Prot Database using the BLASTPprogram [30]. Sequence analyses and alignments were estab-lished using the CLUSTAL program [31]. A structurallybased alignment was obtained from the Swiss-Model server[32] using the structural coordinates of TrxR from E. coli(Protein Data Bank code 1tde) and Arabidopsis thaliana(PDB code 1vdc) as templates.

3. Results

3.1. Cloning and sequencing of the gene encoding SsTrxR

The 710 bp DNA fragment amplified by PCR from theS. solfataricus genomic DNA using the primers TrxR1 andTrxR2 (see Section 2), was cloned in the pGEM-T easyvector and sequenced. The primary structure translated from

the nucleotide sequence of this fragment contained theN-terminal sequence of both intact and tryptic fragments ofSsTrxR purified from S. solfataricus cells. Analysis of S. sol-fataricus DNA by Southern blot, using the 32P-labelled PCRfragment as a probe, indicated that a 4.2-kbp EcoR I/HindIIIinsert contained the entire gene. Colony hybridisationscreening of the S. solfataricus DNA library into thepUC19 vector led to the isolation of a clone containing theentire gene coding for SsTrxR. Fig. 1 shows the nucleotidesequence of this gene and its flanking regions together withthe translated amino acid sequence. The SsTrxR gene codedfor a protein composed of 323 amino acid residues account-ing for a Mr 35,303, a value close to that determined bySDS/PAGE. The unusual start codon GTG is a feature com-mon to other S. solfataricus genes [22]. The 5′-flankingregion of SsTrxR gene, included a putative archaeal promoterwith a canonical TATA box but did not contain a Shine-Dalgarno sequence, a finding already reported for other

Fig. 1. Nucleotide sequence of the SsTrxR gene and deduced amino acid sequence. The amino acid sequence, determined on tryptic fragments of SsTrxR areunderlined. Putative promoter element and termination signal are also underlined. In italics are indicated the partial nucleotide sequences whose deduced aminoacid sequence showed homology with dihydropteroate synthase and inositol-1-monophosphatase.

886 M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

genes in S. solfataricus [33]. Moreover, in this region apartial open reading frame starting from nucleotide -210 onthe complementary strand was found and was identified inthe S. solfataricus genome [34] as the gene coding for aputative dihydropteroate synthase. The 3′-flanking region ofthe gene contained a transcription termination signal andoverlapped with another partial open reading frame, startingfrom nucleotide 969, coding for a putative inositol-1-monophosphatase. The SsTrxR gene possesses a high con-tent of A and T (34.3% and 27.6%, respectively) as comparedto G and C (26.0% and 12.1%, respectively), a finding inagreement with the selective codon usage in S. solfataricusgenes [22]. The amino acid composition of SsTrxR revealedthe presence of three cysteine residues, two of them includedin a C144XXC sequence, typical motif present in enzymesbelonging to pyridine nucleotide-disulphide oxidoreductasefamily [35]. In addition, in the primary structure the consen-sus for both FAD [36] and NAD(P) [37] binding have beenidentified. The average hydrophobicity of the amino acidresidue [38] in the SsTrxR (4.95 kJ/aa residue) was slightlylower than that calculated for other S. solfataricus proteins(5.15 kJ/aa residue), still remaining higher than that of othermesophilic archaeal, eukaryal and eubacterial proteins [22].

3.2. Amino acid sequence comparison

SsTrxR shows sequence homology with eubacterial, ar-chaeal and lower eukaryal thioredoxin reductases (not

shown). The best alignment was obtained with two putativeTrxR identified in the genome of Sulfolobus tokodaii (ACQ974W4, 76% amino acid identities, 12% conservative sub-stitutions; and AC Q975H4, 39% and 10%, respectively)[39]. In addition, SsTrxR was identical to a putative TrxRpresent in the S. solfataricus genome (AC Q97W27) andsimilar (35% and 11%, respectively) with another putativethioredoxin reductase (AC Q97W69) also present in thesame genome [34]. A structurally based multiple alignmentbetween the amino acid sequence of SsTrxR and that ofE. coli and A. thaliana TrxR (EcTrxR and AtTrxR, respec-tively), whose three-dimensional structure has been deter-mined [40,41] is reported in Fig. 2. Although the overallhomology is quite low (25% amino acid identities and 24%conservative substitutions) the highest homology was foundin the regions of EcTrxR and AtTrxR involved in the interac-tion with substrate and cofactor.

3.3. Molecular and functional properties of rSsTrxRand its C147A mutant

Purified rSsTrxR and its C147A mutant appeared homo-geneous on SDS/PAGE and showed an electrophoretic mo-bility identical to that of the enzyme isolated from S. solfa-taricus cells, accounting for a size of about 35 kDa. Themolecular mass of the enzyme determined under non-denaturing conditions by gel-filtration was about 70 kDa.

Fig. 2. Structural multiple alignment of the amino acid sequence of SsTrxR, EcTrxR and AtTrxR. Residues involved in the FAD binding are black shadowed andindicated in white. The cysteine consensus sequence is grey shadowed. Residues involved in the NA(D)P binding are grey shadowed and indicated in white.Asterisks and dots indicate invariant positions and conservative substitutions, respectively.

887M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

These results confirmed the homodimeric structure rSsTrxR,as already reported for the wild-type enzyme [18]. In addi-tion, the C147A substitution did not affect the quaternarystructure of the enzyme.

rSsTrxR and its C147A mutant were purified as flavopro-teins, as already found for SsTrxR [18]. In fact, in the absorp-tion spectrum of both enzymes, besides the maximum at272 nm due to aromatic residues, the typical flavoproteinabsorption bands centred at 375 and 450 nm, were found (notshown). This behaviour was also confirmed by emissionfluorescence spectra (Fig. 3) that also allowed the determina-tion of the number of flavin molecule bound per rSsTrxRsubunit. In particular, a stoichiometry of 0.7 mol of FAD permol of subunit of rSsTrxR was determined from the emissionband at 520 nm. Concerning the C147A mutant, a lowercontent of 0.3 mol of FAD per mol of subunit was found.

We have then determined the disulphide bond content ofrSsTrxR using the Ellman method on either the oxidised orthe reduced form of the enzyme. The results clearly indicatethat two cysteine residues were involved in a disulphidebridge. In addition, SDS/PAGE under non-reducing condi-tions, showed that the disulphide bridge occurred betweenresidues belonging to the same subunit. It has to be noted thatthe fluorescence spectrum of the reduced form of rSsTrxRdid not indicate the presence of bound FAD (not shown).

Concerning the catalytic properties, rSsTrxR showed aNADH oxidase activity as the enzyme isolated from S. solfa-taricus [18], but with a slightly lower specific activity (6.4 in-stead of 8.7 U/mg, respectively). Regarding the C147A mu-tant, a much more reduced NADH oxidase activity was found(0.9 U/mg).

We have then determined the thioredoxin reductase activ-ity of rSsTrxR by both DTNB- and thioredoxin reductionmethods in the absence or in the presence of different cofac-tors. As reported in Fig. 4, the highest activity was observedusing EcTrx as substrate and both NADPH and FAD ascofactors. The same behaviour was observed in the case ofDTNB reduction, even though the specific activity waslower. Regarding the specificity for the nicotinamide di-nucleotide substrate, the enzyme cannot use NADH as elec-tron donor for the reduction of either DTNB or EcTrx. Inaddition, the C147A mutant, possessed a hardly detectablereduction activity (<0.02 U/mg), with both methods (notshown). These results clearly indicate that the enzyme previ-ously identified in S. solfataricus as a 35 kDa NADH oxidase[18] has to be re-classified as a thioredoxin reductase. Thekinetic parameters of rSsTrxR activity were reported inTable 1. In particular, the affinity for NADPH was measuredby the DTNB method, whereas the affinity for Trx wasdetermined by the reduction of heterologous EcTrx. In theDTNB-reduction method a higher catalytic efficiency wasobserved with respect to that measured in the EcTrx reduc-

Fig. 3. Fluorescence spectra in the flavin emission region of rSsTrxR and itsC147A mutant. Fluorescence spectra were recorded for rSsTrxR (upper line)and its C147A mutant (lower line) at 25 °C at a protein concentration of0.63 µM in buffer C. Excitation wavelength was 450 nm; emission andexcitation slits were set to 5 nm. Blanks were subtracted.

Fig. 4. Reduction of DTNB and EcTrx catalysed by rSsTrxR. The reactionwas carried out as described in Section 2 using the DTNB method (A–D) orthe thioredoxin method (E–F) in the presence of 20 µg/ml of rSsTrxR and:(A) NADPH alone (250 µM); (B) NADPH and FAD (50 µM); (C) NADHalone (200 µM); (D) NADH and FAD; (E) NADPH (100 µM) and FAD; (F)NADPH, FAD and E. coli Trx (200 µM).

Table 1Kinetic parameters of rSsTrxRa

V (U/mg) kcat (min–1) Km (µM) kcat/Km (min–1 µM–1)NADPH 0.5 36.7 1.7 21.6EcTrx 1.1 77.0 44.1 1.77

a The initial velocity of DTNB reduction was measured at saturating DTNB concentration (5 mM) in the presence of different NADPH concentration(1–300 µM). In the case of the EcTrx reduction, saturating concentration of NADPH (200 µM) and different concentration of EcTrx (20–200 µM) were used.The enzyme concentration was 0.29 µM. V and Km were derived from Lineweaver–Burk plots. kcat was calculated considering a Mr for SsTrxR of 70,606.

888 M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

tion method. This finding can be ascribed to a 26-fold re-duced affinity towards the substrate, in spite of the 2.1-foldincrease of the catalytic constant.

3.4. Inhibition of rSsTrxR activities

Inhibition of rSsTrxR was evaluated using two differentspecific TrxR inhibitors: auranofin and azelaic acid [25]. Asreported in Fig. 5A, auranofin inhibited rSsTrxR activity asdetermined with both the DTNB and EcTrx reduction meth-ods, with IC50 values of 13 and 54 µM (Fig. 5B), respectively.Regarding azelaic acid, almost no inhibition was detectablewith the DTNB method. In addition, no inhibition of theNADH oxidase activity of rSsTrxR was found with bothinhibitors (not shown).

The effect of reducing agents on the NADH oxidase activ-ity of rSsTrxR was also evaluated (Fig. 6). Both DTT andb-ME inhibited this activity and the IC50 values were18.8 and 19.5 mM for DTT and b-ME, respectively (Fig. 6,inset).

3.5. Stability of rSsTrxR

The denaturation of rSsTrxR and its C147A mutant wasfollowed by fluorescence measurements. Fig. 7 showed theeffect of the treatment with 6 M guanidine•HCl on thefluorescence spectrum of both enzymes in the region of thearomatic residues. The denaturation process provoked a de-crease in the fluorescence intensity at 345 nm, but not a shiftin wavelength of the maximum. However, the decrease wasmuch more pronounced in the case of rSsTrxR with respect tothat of its C147A mutant.

The fluorescence decrease in the aromatic region was alsoused to evaluate the thermal denaturation of the enzyme. Asshown in Fig. 8, the half denaturation temperature of rSsTrxR

is 84 °C, whereas for the C147A mutant, under identicalexperimental conditions, the corresponding value signifi-cantly decreased to 63 °C.

4. Discussion

The gene encoding a homodimeric 35 kDa/subunit thiore-doxin reductase from the archaeon Sulfolobus solfataricus(SsTrxR) was cloned and sequenced. The recombinant pro-tein was expressed in E. coli, purified and characterised forits molecular and biochemical properties. This enzyme waspreviously identified as a NADH oxidase [18], but in this

Fig. 5. Inhibition of rSsTrxR activity by auranofin or azelaic acid. (A)Inhibitory effect of auranofin with the DTNB method (*) or the thioredoxinmethod (C). The inhibitory effect of azelaic acid was assayed with the DTNBassay (n). For other details see Section 2. (B) Graphical method to derive theIC50 values.

Fig. 6. Inhibition of NADH oxidase activity of rSsTrxR by reducing agents.The enzymatic activity was measured as described in Section 2 in thepresence of the indicated concentration of b-ME (C) or DTT (*). Inset,graphical method to derive the IC50 values.

Fig. 7. Chemical stability of rSsTrxR and its C147A mutant. The fluores-cence spectra were recorded at 25 °C after a treatment (20 h at 25 °C) ofrSsTrxR (A) and its C147A mutant (B) in the absence (upper line) or in thepresence (lower line) of 6 M guanidine•HCl. Excitation wavelength was295 nm. Blanks were subtracted.

889M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

report we show that it possesses a TrxR activity. From theanalysis of the deduced amino acid sequence (Fig. 1), anumber of features typical of enzymes belonging to theTrxR-like proteins were found. In fact, SsTrxR possessesputative ADP-binding bab fold regions typical of dinucle-otide binding enzymes [36]. One of this region in SsTrxRcorresponds to the segment D17-E46 containing the sequenceG22LGPAA and a core of six hydrophobic residues V18, I20,A31, A35, L42, I44. Another putative bab fold region ofSsTrxR corresponded to the segment R155-D187 that con-tained V157, V159, G161GGDSA, A170, L173, V180, I183. Thisregion also contained the consensus sequence typical of theNAD(P) binding proteins GXGXXAXXXAXXXXXXG[37] that in SsTrxR corresponded to the segmentG161GGDSALEGA. An additional consensus sequence con-served among many FAD-binding proteins is TXXXXhyh-hGD, where h represents a small hydrophobic amino acidand y is an aromatic residue [42]. This sequence was identi-fied in SsTrxR as T277SVPGVFAAGD. SsTrxR containedalso the canonical redox active site CXXC common in allenzymes showing thioredoxin reductase activity [43]. In par-ticular, this CXXC motif is contained in a class II pyridinenucleotide-disulphide oxidoreductases active siteCX(2)CD(G/A)X(2/4)(F/Y)X(4)(L/I/V/M)X(L/I/V/M)(2)G(3)(D/N), as reported in the Swiss-Prot Prosite Database atthe accession number PS00573 [35]. This motif corre-sponded in SsTrxR to the segment C144SVCDAPL-FKNRVVAVIGGGD that in the primary structure over-lapped to the NAD(H)P binding region reported above. Theproximity of these two binding regions is a feature present inbacterial TrxR, whereas in mammalian TrxR, in lipoamidedehydrogenase, glutathione and trypanothione reductases,they were spatially separated [44].

SsTrxR was purified as a homodimeric flavoenzyme, andits molecular mass of 35 kDa/subunit was similar to that of

eubacterial and lower eukaryal TrxR [43]. The primary struc-ture of SsTrxR showed significant sequence homology withthioredoxin reductases from archaea, eubacteria and loweukaryotes. The highest amino acid sequence identity wasfound with the deduced amino acid sequence of a putativeTrxR present in the genome [39] of S. tokadai (Fig. 2).However, in the S. solfataricus genome [34] another geneencoding a putative TrxR is present, but it did not contain thecanonical class II pyridine nucleotide-disulphide oxi-doreductases consensus sequence reported above. This as-pect is not unique for the S. solfataricus genome becausemore than one gene coding for putative TrxR were found alsoin the phylogenetically close related S. tokodaii as well as inother archaeal genomes such as Pyrobaculum aerophilum[45] and Ferroplasma acidarmanus [46]. This behaviourindicated that the classification of putative TrxR not harbour-ing a complete class II motif deserves further analysis.

The multiple alignment between 17 archaeal TrxR se-quences, all containing the class II consensus motif, showedthat sequence identities were mainly found in the regionscontaining the binding sites for FAD and NAD(P)H (notshown). It is interesting to note that among all the archaealTrxR analysed, the first X residue in the consensus sequenceCXXC is occupied by A or S, whereas the second position isless conserved. Therefore, the first semi-invariant positionmight represent an additional distinctive property of archaealTrxR. It is worth mentioning that the two variant residues inthe CXXC motif have been correlated to the reduction poten-tial of thiol:disulphide oxidoreductase family [47].

SsTrxR isolated from S. solfataricus binds 1 mol of FADper subunit [18]. The lower content of FAD found in recom-binant SsTrxR might be ascribed to a loss of cofactor duringthe purification procedure. In the C147A mutant the FADcontent further decreased, a finding similar to that observedin EcTrxR mutated in cysteine residues present in the activesite of the enzyme [48]. The presence of two cysteines in-volved in a disulphide bridge, prompted us to measure athioredoxin reductase activity on rSsTrxR. The thioredoxinreductase activity of rSsTrxR was measured using both theDTNB and thioredoxin reduction assays (Fig. 4). The latterassay was carried out using heterologous EcTrx as substrate,as the S. solfataricus Trx, although putatively identified in theS. solfataricus genome [34], was not available yet. The re-ductase activity is strictly dependent on the presence ofNADPH as electron donor and FAD as cofactor, a propertydemonstrating that the enzyme of S. solfataricus previouslyisolated as a NADH oxidase, belongs to the thioredoxinreductase family. This observation was also confirmed by thelack of reduction activity of the C147A mutant enzyme.Concerning the kinetic parameters, the high value of Km forEcTrx reported in Table 1 (44 µM) might depend on the useof the heterologous substrate used in the assay. This behav-iour was also confirmed by the observation that wheat TrxRshowed a higher Km for EcTrx (36 µM) with respect to thatmeasured in the presence of homologous Trx (7.6 µM) [49].Regarding the electron donor, the affinity for NADPH of

Fig. 8. Thermal stability of rSsTrxR and its C147A mutant. The decrease influorescence intensity was measured in buffer C at the indicated temperatu-res for rSsTrxR (*) or its C147A mutant (C). Excitation and emissionwavelengths were 280 and 345 nm, respectively. For other details seeSection 2.

890 M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

rSsTrxR was similar to that found for other eukaryal [49,50],eubacterial [48] and archaeal [15] TrxR.

Inhibition of SsTrxR activities was studied either in thepresence of specific TrxR inhibitors or reducing agents. Con-cerning the specific TrxR inhibitors, surprisingly SsTrxRactivity was inhibited by auranofin, a specific inhibitor ofhuman [25] and mitochondrial [51] TrxR. Vice versa, azelaicacid, a typical inhibitor of eubacterial TrxR [25], was almostunable to affect the activity of SsTrxR. These findings sup-port the hypothesis that archaeal enzymes, more structurallyrelated to eubacterial ones, are functionally closer to eu-karyal enzymes [52]. In addition, these results further sup-port the hypothesis that the Sulfolobus genus is the putativeancestor of animal mitochondria [53]. It is interesting to notethat the NADH oxidase activity of rSsTrxR was inhibited byreducing agents (Fig. 6). This finding, and the very lowNADH oxidase activity of the C147A, suggests that thedisulphide bridge in SsTrxR is also involved in the NADHoxidation catalysed by the enzyme.

Regarding the stability of rSsTrxR, the finding that theC147A mutant displays a reduced thermal stability, togetherwith the reduced sensibility to chemical denaturation, indi-cates that the disulphide bridge in the active site of theenzyme, besides its involvement in the catalytic property ofSsTrxR, is also important for the stability of the enzyme.

5. Conclusion

The data reported in this paper shed light on the realfunction of the thioredoxin reductase, an enzyme that hasbeen previously identified as NADH oxidase in Sulfolobussolfataricus [18]. The finding that a NADH oxidase can elicitalso another enzymatic activity has already been reported forseveral eubacterial species [54]. Therefore, because this en-zyme belongs to a system playing an important role in severalcellular functions, such as redox control [4,5], activity oftranscription factors [6], DNA synthesis [3], protein folding[7], and apoptosis [55], it will be of great help to bettercharacterise this redox system in S. solfataricus, also bytrying to solve its 3-D structure.

Acknowledgments

This investigation was financed by Ministerodell’Istruzione, dell’Università e della Ricerca, PRIN2003 and by Consiglio Nazionale delle Ricerche, TargetProject on Biotechnology (Rome). This paper is dedicated tothe memory of Prof. Vincenzo Bocchini. The nucleotidesequence of the gene encoding SsTrxR has been deposited inthe EMBL Nucleotide Sequence Database under the Acces-sion Number AJ315737. We thank Dr. Emmanuele De Ven-dittis for useful discussion and critical reading of the manu-script.

References

[1] C.H. Williams Jr., Lipoamide dehydrogenase, glutathione reductase,thioredoxin reductase, and mercuric ion reductase—a family of fla-voenzyme transhydrogenases, in: F. Müller (Ed.), Chemistry andBiochemistry of Flavoenzymes, CRC Press, Boca Raton, FL, USA,1992, pp. 121–211.

[2] E.S.J. Arnér, A. Holmgren, Physiological functions of thioredoxin andthioredoxin reductase, Eur. J. Biochem. 267 (2000) 6102–6109.

[3] A. Holmgren, Thioredoxin and glutaredoxin systems, J. Biol. Chem.264 (1989) 13963–13966.

[4] A. Holmgren, Enzymatic reduction–oxidation of protein disulfides bythioredoxin, Methods Enzymol. 107 (1984) 295–300.

[5] E.J. Stewart, F. Aslund, J. Beckwith, Disulfide bond formation in theEscherichia coli cytoplasm: an in vivo role reversal for the thioredox-ins, EMBO J. 17 (1998) 5543–5550.

[6] H. Schenk, M. Klein, W. Erdbrugger, W. Droge, K. Schulze-Osthoff,Distinct effects of thioredoxin and antioxidants on the activation oftranscription factors NF-kappa B and AP-1, Proc. Natl. Acad. Sci.USA 91 (1994) 1672–1676.

[7] R. Kern, A. Malki, A. Holmgren, G. Richarme, Chaperone propertiesof Escherichia coli thioredoxin and thioredoxin reductase, Biochem.J. 371 (2003) 965–972.

[8] C.H. Williams, L.D. Arscott, S. Muller, B.W. Lennon, M.L. Ludwig,P.F. Wang, et al., Thioredoxin reductase two modes of catalysis haveevolved, Eur. J. Biochem. 267 (2000) 6110–6117.

[9] P.Y. Gasdaska, M.M. Berggren, M.J. Berry, G. Powis, Cloning,sequencing and functional expression of a novel human thioredoxinreductase, FEBS Lett. 442 (1999) 105–111.

[10] V.N. Gladyshev, M. Krause, X.M. Xu, K.V. Korotkov, G.V. Kryukov,Q.A. Sun, et al., Selenocysteine containing thioredoxin reductase inC. elegans, Biochem. Biophys. Res. Commun. 259 (1999) 244–249.

[11] S.M. Kanzok, A. Fechner, H. Bauer, J.K. Ulschmid, H.M. Muller,J. Botella-Munoz, S. Schneuwly, R. Schirmer, K. Becker, Substitutionof the thioredoxin system for glutathione reductase in Drosophilamelanogaster, Science 291 (2001) 643–646.

[12] H.Z. Chae, S.J. Chung, S.G. Rhee, Thioredoxin-dependent peroxidereductase from yeast, J. Biol. Chem. 269 (1994) 27670–27678.

[13] C.H. Williams Jr., Mechanism and structure of thioredoxin reductasefrom Escherichia coli, FASEB J. 9 (1995) 1267–1276.

[14] S. Ghisla, V. Massey, Mechanisms of flavoprotein-catalyzed reac-tions, Eur. J. Biochem. 181 (1989) 1–17.

[15] S. Jeon, K. Ishikawa, Identification and characterization of thiore-doxin and thioredoxin reductase from Aeropyrum pernix K1, Eur. J.Biochem. 269 (2002) 5423–5430.

[16] Y. Kashima, K. Ishikawa, A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase fromhyperthermophilic archaeon Pyrococcus horikoshii, Arch. Biochem.Biophys. 418 (2003) 179–185.

[17] E. Waters, M.J. Hohn, I. Ahel, D.E. Graham, M.D. Adams, M. Barn-stead, K.Y. Beeson, L. Bibbs, R. Bolanos, M. Keller, K. Kretz, X. Lin,E. Mathur, J. Ni, M. Podar, T. Richardson, G.G. Sutton, M. Simon,D. Soll, K.O. Stetter, J.M. Short, M. Noordewier, The genome ofNanoarchaeum equitans: insights into early archaeal evolution andderived parasitism, Proc. Natl. Acad. Sci. USA 100 (2003) 12984–12988.

[18] M. Masullo, G. Raimo, A. Dello Russo, V. Bocchini, J.V. Bannister,Purification and characterization of NADH oxidase from the archaeaSulfolobus acidocaldarius and Sulfolobus solfataricus, Biotechnol.Appl. Biochem. 23 (1996) 47–54.

[19] P. Arcari, L. Masullo, M. Masullo, F. Catanzano, V. Bocchini, ANAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricusis not homologous with another NADH oxidase present in the samemicroorganism. Biochemical characterization of the enzyme andcloning of the encoding gene, J. Biol. Chem. 14 (2000) 895–900.

891M.R. Ruocco et al. / Biochimie 86 (2004) 883–892

[20] F. Kunst, N. Ogasawara, I. Moszer, A.M. Albertini, G. Alloni, V. Aze-vedo, et al., The complete genome sequence of the Gram-positivebacterium Bacillus subtilis, Nature 390 (1997) 249–256.

[21] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Labora-tory Manual, second ed, Cold Spring Harbor Laboratory, Cold SpringHarbor, New York, 1989.

[22] E. De Vendittis, V. Bocchini, Protein-encoding genes in the sulfother-mophilic archaea Sulfolobus and Pyrococcus, Gene 176 (1996)27–33.

[23] J. Braman, C. Papworth, A. Greener, Site-directed mutagenesis usingdouble-stranded plasmid DNA templates, Methods Mol. Biol. 57(1996) 31–44.

[24] A. Holmgren, M. Björnostedt, Thioredoxin and thioredoxin reduc-tase, Methods Enzymol. 252 (1995) 199–208.

[25] S. Gromer, H. Merkle, R.H. Schirmer, K. Becker, Human placentathioredoxin reductase: preparation and inhibitor studies, Methodsenzymol. 347 (2002) 382–394.

[26] T.E. Creighton, Disulphide bonds between cysteine residues, in:T.E. Creighton (Ed.), Protein Structure a Practical Approach, OxfordUniversity press, Oxford, UK, 1989, pp. 155–167.

[27] S. Underfriend, Fluorescence Assay in Biology and Medicine, vol. 2,Academic Press, London, UK, 1969.

[28] E. Southern, Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis, J. Mol. Biol. 98 (1975) 503–517.

[29] G. Raimo, M. Masullo, G. Savino, G. Scarano, G. Ianniciello,A. Parente, et al., Archaeal elongation factor 1 beta is a dimer. Primarystructure, molecular and biochemical properties, Biochim. Biophys.Acta 1293 (1996) 106–112.

[30] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basiclocal alignment search tool, J. Mol. Biol. 215 (1990) 403–410.

[31] D.G. Higgins, P.M. Sharp, Fast and sensitive multiple sequence align-ments on a microcomputer, Comput. Appl. Biosci. 5 (1989) 151–153.

[32] M.C. Peitsch, ProMod and Swiss-Model: internet-based tools forautomated comparative protein modelling, Biochem. Soc. Trans. 24(1996) 274–279.

[33] N. Tolstrup, C.W. Sensen, R.A. Garrett, I.G. Clausen, Two differentand highly organized mechanisms of translation initiation in thearchaeon Sulfolobus solfataricus, Extremophiles 4 (2000) 175–179.

[34] Q. She, R.K. Singh, F. Confalonieri, Y. Zivanovic, G. Allard,M.J. Awayez, C.C. Chan-Weiher, I.G. Clausen, B.A. Curtis,A. De Moors, G. Erauso, C. Fletcher, P.M. Gordon, I. Heikamp-deJong, A.C. Jeffries, C.J. Kozera, N. Medina, X. Peng, H.P. Thi-Ngoc,P. Redder, M.E. Schenk, C. Theriault, N. Tolstrup, R.L. Charlebois,W.F. Doolittle, M. Duguet, T. Gaasterland, R.A. Garrett, M.A. Ragan,C.W. Sensen, J. Van der Oost, The complete genome of the crenar-chaeon Sulfolobus solfataricus P2, Proc. Natl. Acad. Sci. USA 98(2001) 7835–7840.

[35] J. Kuriyan, T.S. Krishna, L. Wong, B. Guenther, A. Pahler, C.H. Wil-liams Jr., et al., Convergent evolution of similar function in twostructurally divergent enzymes, Nature 352 (1991) 172–174.

[36] R.K. Wierenga, P. Terpstra, W.G. Hol, Prediction of the occurrence ofthe ADP-binding beta alpha beta-fold in proteins, using an amino acidsequence fingerprint, J. Mol. Biol. 187 (1986) 101–107.

[37] I. Hanukoglu, T. Gutfinger, cDNA sequence of adrenodoxin reduc-tase. Identification of NADP-binding sites in oxidoreductases, Eur. J.Biochem. 180 (1989) 479–484.

[38] C. Tanford, Contribution of hydrophobic interaction to the stabilityand the globular conformation of proteins, J. Am. Chem. Soc. 84(1962) 4240–4247.

[39] Y. Kawarabayasi, Y. Hino, H. Horikawa, K. Jin-no, M. Takahashi,M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui,Y. Nagai, K. Nishijima, R. Otsuka, H. Nakazawa, M. Takamiya,Y. Kato, T. Yoshizawa, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida,A. Oguchi, K. Aoki, S. Masuda, M. Yanagii, M. Nishimura, A. Yam-agishi, T. Oshima, H. Kikuchi, Complete genome sequence of anaerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7,DNA Res. 8 (2001) 123–140.

[40] G. Waksman, T.S. Krishna, C.H. Williams Jr., J. Kuriyan, Crystalstructure of Escherichia coli thioredoxin reductase refined at 2 Aresolution. Implications for a large conformational change duringcatalysis, J. Mol. Biol. 236 (1994) 800–816.

[41] S. Dai, M. Saarinen, S. Ramaswamy, Y. Meyer, J.P. Jacquot,H. Eklund, Crystal structure of Arabidopsis thaliana NADPH depen-dent thioredoxin reductase at 2.5 A resolution, J. Mol. Biol. 264(1996) 1044–1057.

[42] G. Eggink, H. Engel, G. Vriend, P. Terpstra, B. Witholt, Rubredoxinreductase of Pseudomonas oleovorans. Structural relationship toother flavoprotein oxidoreductases based on one NAD and two FADfingerprints, J. Mol. Biol. 212 (1990) 135–142.

[43] R.P. Hirt, S. Muller, T.M. Embley, G.H. Coombs, The diversity andevolution of thioredoxin reductase: new perspectives, Trends Parasi-tol. 18 (2002) 302–308.

[44] M. Russel, P. Model, Sequence of thioredoxin reductase fromEscherichia coli. Relationship to other flavoprotein disulfide oxi-doreductases, J. Biol. Chem. 263 (1988) 9015–9019.

[45] S.T. Fitz-Gibbon, H. Ladner, U.J. Kim, K.O. Stetter, M.I. Simon,J.H. Miller, Genome sequence of the hyperthermophilic crenarchaeonPyrobaculum aerophilum, Proc. Natl. Acad. Sci. USA 99 (2002)984–989.

[46] G.W. Tyson, J. Chapman, P. Hugenholtz, E.E. Allen, R.J. Ram,P.M. Richardson, V.V. Solovyev, E.M. Rubin, D.S. Rokhsar, J.F. Ban-field, Community structure and metabolism through reconstruction ofmicrobial genomes from the environment, Nature 428 (2004) 37–43.

[47] P.T. Chivers, K.E. Prehoda, R.T. Raines, The CXXC motif: a rheostatin the active site, Biochemistry 36 (1997) 4061–4066.

[48] A.J. Prongay, D.R. Engelke, C.H. Williams Jr., Characterization oftwo active site mutations of thioredoxin reductase from Escherichiacoli, J. Biol. Chem. 264 (1989) 2656–2664.

[49] A.J. Serrato, J.M. Perez-Ruiz, F.J. Cejudo, Cloning of thioredoxin hreductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat, Biochem. J. 367 (2002) 491–497.

[50] S. Gromer, L.D. Arscott, C.H. Williams Jr., R.H. Schirmer, K. Becker,Human placenta thioredoxin reductase. Isolation of the selenoen-zyme, steady state kinetics, and inhibition by therapeutic gold com-pounds, J. Biol. Chem. 273 (1989) 20096–20101.

[51] M.P. Rigobello, G. Scutari, A. Folda, A. Bindoli, Mitochondrialthioredoxin reductase inhibition by gold(I) compounds and concur-rent stimulation of permeability transition and release of cytochromec, Biochem. Pharmacol. 67 (2004) 689–696.

[52] W. Zillig, P. Palm, W.D. Reiter, F. Gropp, G. Puhler, H.P. Klenk,Comparative evaluation of gene expression in archaebacteria, Eur. J.Biochem. 173 (1988) 473–482.

[53] S. Karlin, A.M. Campbell, Which bacterium is the ancestor of theanimal mitochondrial genome? Proc. Natl. Acad. Sci. USA 91 (1994)12842–12846.

[54] Y. Nishiyama, V. Massey, K. Takeda, S. Kawasaki, J. Sato,T. Watanabe,Y. Niimura, Hydrogen peroxide-forming NADH oxidasebelonging to the peroxiredoxin oxidoreductase family: existence andphysiological role in bacteria, J. Bacteriol. 183 (2001) 2431–2438.

[55] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada,et al., Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, EMBO J. 17 (1998) 2596–2606.

892 M.R. Ruocco et al. / Biochimie 86 (2004) 883–892