doi:10.1016/j.jmb.2006.10.033 J. Mol. Biol. (2007) 365, 1033–1046

The Thioredoxin Specificity of Drosophila GPx:A Paradigm for a Peroxiredoxin-like Mechanism of manyGlutathione Peroxidases

Matilde Maiorino1⁎, Fulvio Ursini1, Valentina Bosello1, Stefano Toppo1

Silvio C. E. Tosatto2, Pierluigi Mauri3, Katja Becker4, Antonella Roveri1

Cristiana Bulato1, Louise Benazzi3, Antonella De Palma3

and Leopold Flohé5

1Department of BiologicalChemistry Viale G. Colombo, 3,University of Padova,I-35121 Padova, Italy2Department of Biology andCRIBI Biotechnology Centre,Viale G. Colombo, 3,University of Padova,I-35121 Padova, Italy3Institute for BiomedicalTechnologies, National ResearchCouncil, Viale Fratelli Cervi 93,I-2090 Segrate (Milano), Italy4Nutritional Biochemistry,Interdisciplinary ResearchCenter, Justus-Liebig UniversityGiessen, Heinrich-Buff-Ring26-32 D-35392 GiessenGermany5MOLISA GmbH,Universitätsplatz 2,D-39106 Magdeburg,Germany

Abbreviations used: BtGPx-1, bovcontaining Cys at the active site; CP,melanogaster; DmGPx, glutathione peGSH, glutathione; HsGPx-3, humanperoxiredoxin; RrGPx-4, rat glutathicontaining Sec at the active site; TrxLC-ESI-MS/MS, liquid chromatograE-mail address of the correspondi

0022-2836/$ - see front matter © 2006 E

Some members of the glutathione peroxidase (GPx) family have beenreported to accept thioredoxin as reducing substrate. However, theselenocysteine-containing ones oxidise thioredoxin (Trx), if at all, atextremely slow rates. In contrast, the Cys homolog of Drosophilamelanogaster exhibits a clear preference for Trx, the net forward rateconstant, k′+2, for reduction by Trx being 1.5×106 M−1 s−1, but only 5.4 M−1

s−1 for glutathione. Like other CysGPxs with thioredoxin peroxidaseactivity, Drosophila melanogaster (Dm)GPx oxidized by H2O2 contained anintra-molecular disulfide bridge between the active-site cysteine (C45; CP)and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trxperoxidase activity, but increased the rate constant for glutathione by twoorders of magnitude. In contrast, a replacement of C74 by Ser or Ala onlymarginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidizedDmGPx exposed to a reduced Trx C35S mutantyielded a dead-end intermediate containing a disulfide between Trx C32and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that ofselenocysteine (Sec)GPxs, involves formation of an internal disulfide that ispivotal to the interaction with Trx. Hereby C91, like the analogous secondcysteine in 2-cysteine peroxiredoxins, adopts the role of a “resolving”cysteine (CR). Molecular modeling and homology considerations based on450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as CR; (ii) deletions ofthe subunit interfaces typical of tetrameric GPxs leading to flexibility of theCR-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: glutathione peroxidase; thioredoxin peroxidase; selenocysteine;peroxiredoxin; mechanism of action

*Corresponding author

ine glutathione peroxidase-1 (EC 1.11.1.9); CysGPx, glutathione peroxidaseperoxidatic cysteine; CR, resolving cysteine; DmTrx, thioredoxin-2 of Drosophilaroxidase of D. melanogaster; GPx, glutathione peroxidase; GR, glutathione reductase;plasma glutathione peroxidase; PCOOH, phosphatidylcholine hydroperoxide; Prx,one peroxidase-4 (EC 1.11.1.12); Sec, selenocysteine; SecGPx, glutathione peroxidase, thioredoxin; TrxR, thioredoxin reductase; TrxPx, thioredoxin peroxidase;phy-electrospray-tandem mass spectrometry.ng author: matilde.maiorino@unipd.it

lsevier Ltd. All rights reserved.

Figure 1 (legend on opposite page)

1034 Thioredoxin Specificity of Glutathione Peroxidases

1035Thioredoxin Specificity of Glutathione Peroxidases

Introduction

The first glutathione peroxidase-1 (GPx-1) wasdiscovered as a glutathione-dependent antioxidantactivity that protected hemoglobin from oxidativedenaturation.1 Fifteen years later GPx activity in ratswas discovered to depend on selenium2 and thebovine enzyme proved to contain one seleniumatom per subunit.3 The nature of its redox centeremerged from X-ray crystallography showing aselenocysteine residue to be at hydrogen-bondingdistance to a Trp and a Gln residue.4 The seleno-cysteine (Sec) residue embedded in this catalytictriad is completely dissociated and polarized to reactwith any accessible hydroperoxide to a selenenicacid derivative and was demonstrated to representthe pivotal catalytic moiety by site-directed muta-genesis of GPx-1 and GPx-4,5,6 while a Lys and fourArg residues surrounding the active site of GPx-17

were suggested to determine the pronouncedspecificity for glutathione (GSH).8

Over the past two decades, however, more than450 homologous sequences have been identified thatpartially lack the characteristics implicated in thereaction and substrate specificity of GPx-1.9 Sec isconsistently replaced by Cys in GPx homologs ofterrestrial plants, insects, bacteria, fungi andprotozoa,10 and even the human selenoproteomecomprises only five of the six GPxs.11 In view of thegenerally lower reactivity of sulfur versus selenium,the Cys homologs may be assumed rather inefficientperoxidases. This statement is not meant to questionthe biological relevance of “CysGPxs”. Their con-servation in the entire living domain suggests animportant role, which, however, has to be searchedfor in a context distinct from a GSH-mediatedantioxidant defense. A first, well-documented exam-ple is the peroxide-dependent activation of thetranscription factor Yap-1 by yeast GPx-3, a homo-logue of mammalian GPx-4, via formation of amixed disulfide.12 A specific reaction with proteinthiols is not uncommon in the GPx superfamily. Infact, the basic residues that are implicated in GSHbinding7 are largely restricted to the GPx-1subfamily.9,10 Accordingly, reactions of GPxs withprotein thiols are increasingly observed, be theyselenoproteins or Cys homologues. A most unusualcase is the transformation of mammalian GPx-4 into

Figure 1. Multiple sequence alignment of 35 representatshows a representative subset of glutathione peroxidase protBtGPx-1 and pertinent secondary structure elements (PDB codeshown and numbering refers to the full length of the BtGPxreported to focus on the alignment used to construct the finnumber, species and taxon are reported. The catalytic triatryptophan is pointed out by black triangles. Some characterisand Cys active site based enzymes have been colored differalignment. Sequences are tentatively grouped, see light red bomonomers (M). Both the dimer and tetramer interfaces have bthe monomeric enzymes. The Cys-block is a delimited region wa cysteine residue, whereas Sec based proteins usually do not (in this region). Sequences, whose Trx peroxidase activity has exlight blue.

a structural protein during spermmaturation, whichmechanistically corresponds to the formation ofdead-end intermediates due to the reaction of theactive site selenenic acid with exposed thiols ofGPx-4 itself and of other proteins.13–15 In gener-al, the specificity of mammalian GPx-type peroxi-dases for GSH appears to decrease in the followingorder: from GPx-1, GPx-2, and GPx-3 to GPx-4.9,10

More recently, thioredoxins and related proteinscharacterized by a CXXC motif, which typicallyreduce peroxiredoxins,16 have been identified asspecific substrates of GPx-type peroxidases inPlasmodium falciparum,17 Saccharomyces cerevisiae,12,18

terrestrial plants,19,20 Trypanosoma brucei,21 andDrosophila melanogaster.22

Intrigued by these differences in donor substratespecificity within the GPx family23 we here try todefine the molecular basis for the preference ofcertain GPx-type proteins for thioredoxins takingDrosophila GPx (accession no. NP 647807) as aparadigm. The emerging common denominators ofthe GPx-type thioredoxin peroxidases appear to be:(i) deletions of the subunit interfaces typical oftetrameric mammalian GPxs9; (ii) a second Cysresidue in a poorly conserved region downstream ofthe second helix ; and (iii) a mechanism that isanalogous to that of the unrelated 2-Cys peroxir-edoxins, wherein the second cysteine residue formsa disulfide bridge with the peroxidatic cysteine (CP)and acts as a resolving cysteine (CR) in beingindispensable for the reduction by thioredoxin.

Results

Similarities of DmGPx with GPx-type proteinsdisplaying thioredoxin peroxidase activity

The DmGPx sequence shares with all glutathioneperoxidases the catalytic triad composed of Sec orCys coordinated to Trp and Gln. In this environ-ment, the SH (SeH) groups of the (selenol)cysteineresidues may generally be assumed to be dissociatedand thus are readily oxidized to sulfenic (selenenic)acid derivatives by the hydroperoxide substrates.10

The residues of the catalytic triad are located indistant parts of the sequence which are highlyconserved in the entire GPx superfamily, the

ive glutathione peroxidase proteins. The final alignmenteins (see Materials and Methods for details) compared to: 1GP1) as reference. Unconserved C and N termini are not-1 whereas only the crystallized portion of the protein isal model of DmGPx. For each protein UniProt accessiond composed of cysteine/seleno-cysteine, glutamine andtics of this protein superfamily have been highlighted. Secently in red and blue, respectively, in position 52 of thexes, for their propensity either to work as tetramers (T) oreen highlighted in light red showing selective deletions inhere most of the Cys-based enzymes show the presence ofsee Table 5; BtGPx-1 is an exception showing a Cys residueperimentally been demonstrated, have been highlighted in

1036 Thioredoxin Specificity of Glutathione Peroxidases

characteristic signatures typically being NVA(S/T)X(U/C)GXT between the end of the β1 sheet and thestart of the α3 helix, FPCNQFXXQEP downstreamof β2, andWNFXKFLV preceding β3 (catalytic triadresidues in bold). According to this homology, C45of DmGPx is undoubtedly the peroxidatic cysteine(CP). In mammalian Sec-containing GPxs the oxi-dized selenium (corresponding to CP) is alsoconsidered to be the site of attack by the reducingsubstrate, and a Se-glutathionylated catalytic inter-mediate of porcine GPx-4 could unequivocally bedemonstrated.15 DmGPx, however, differs from themammalian SecGPxs sufficiently enough to envi-sage variations of the catalytic scheme.When aligned to the SecGPx-1, -2 and -3 sub-

families, the DmGPx sequence displays a majordeletion corresponding to positions 137–161 and asmaller one corresponding to the six residues down-stream of the α4 helix of the bovine GPx-1 sequence(Figure 1). In the X-ray structure of bovine GPx-1,4 aswell as in human GPx-3,24 the stretch of about 20residues that is deleted in DmGPx forms the subunitinterface (designated "tetramer interface" in Figure1), while positions 89–109 comprising the α4 helixrepresent a second contact area which is equallyindispensable for subunit interaction ("dimer inter-face" in Figure 1).9

The deletions in DmGPx, which are similarly seenin monomeric mammalian GPx-4, therefore, suggestthe Drosophila enzyme to be monomeric and thisprediction was verified by gel chromatography ofthe native protein (data not shown). Interestingly, alleight GPx homologues that so far have been shownexperimentally to be specifically reduced by thior-edoxin or related proteins (horizontally shaded inFigure 1), display homologous deletions, whichsuggests that the monomeric nature of the proteinsis a prerequisite for interaction with thioredoxin.Another corollary of the DmGPx sequence is the

existence of a cysteine residue within the poorlyconserved sequence of the α4 helix, which isdesignated "Cys block" in Figure 1. Two distinctGPx homologues of Saccharomyces cerevisiae that, infunctional terms, are thioredoxin peroxidases, alsohave a cysteine residue in this area that is reported toform a disulfide bond to the typical active sitecysteine upon oxidation.18,25 Similarly, Cys95 of aGPx homologue of Trypanosoma brucei that acts onthe thioredoxin homologue tryparedoxin hasrecently been reported to form a disulfide bridgewith the peroxidatic cysteine (Cys47) and to beessential for activity.26 Moreover, all known GPxhomologues with established specificity for thior-edoxin have a cysteine in their α4 helix, theconsensus sequence being FPcnQFgxQepx3e(E/D)x0–1i C-block x1–2 Δ6x4fpi(F/M)xK(i/V)dVNG,where Δ indicates a deletion in comparison to thebovine reference sequence and "Cys block" a stretchof five residues comprising one cysteine in anyposition except the third (Figure 1).A structural model of DmGPx was built from the

homologous BtGPx-1 crystal structure (Figure 2)and visually inspected. The DmGPx model suggests

a remarkable number of stabilizing salt bridges,effectively immobilizing most of the secondarystructure elements surrounding the active site.Most of these salt bridges result from correlatedvariations unique to DmGPx, where lysine andglutamate residues appear in structurally adjacentpositions. Only the loop region surrounding C91appears to be quite flexible. This loop also contains anumber of charged residues that, however, requirestructural rearrangements to form salt bridgestowards the protein core. The distance between thesulfur gamma atoms of the active site cysteine andC91 is 13.8 Å (see Figure 2(a)), but taking intoaccount the flexibility of the loop region, a disulfidebridge between C45 and C91 can be constructed forthe oxidized form, if major structural rearrange-ments are assumed (Figure 2(b)). Such structuralchanges are known to occur when peroxiredoxin-type peroxidases become oxidized to the disulfideform and, in several examples, were demonstratedto be a prerequisite for the reduction by physiolo-gical substrates, such as thioredoxins or relatedCXXC proteins.16 In peroxiredoxins the essentialcysteine residue that is not directly reacting withthe peroxide substrate, but indispensable for effi-cient regeneration of the ground state enzyme, hasbeen termed the resolving cysteine (CR). By analogy,C91 ofDmGPx may adopt the peroxiredoxins role ofthe CR, as has similarly been discussed for yeastGPx-2.18

The striking homologies with some CysGPxs withthioredoxin peroxidase activity and the possibleanalogy to the mechanism of peroxiredoxin-typethioredoxin peroxidases prompted us to investigatethe potential role of the cysteine block of DmGPx incatalysis, in particular its relevance to donor sub-strate specificity.

Substrate specificity and kinetics of DmGPx

DmGPx activity is slightly higher with a lipophilicsubstrate such as phosphatidylcholine hydroperox-ide than with H2O2 (Table 3, below). This preferencefor hydroperoxides of complex lipids, which isshared with mammalian GPx-4, but not with anyof the types of SecGPxs, is likely explained by themonomeric nature of the enzyme which may resultin a more freely exposed CP.

9

As reported,22 DmGPx proved to be active withTrx as the reducing substrate, while its activity wastwo orders of magnitude less when tested with GSHat an almost 1000-fold higher concentration (Table1). Three different types of mammalian GPxs werecomparatively tested for Trx peroxidase activity.Expectedly, the activity of bovine GPx-1 with Trxwas close to zero. Yet, human GPx-3, which hadpreviously been reported to accept glutaredoxin andthioredoxin as reductants,27 also showed a clearpreference for GSH under the experimental condi-tions. Rat GPx-4, although being the least specific ofthe mammalian SecGPxs, was absolutely inactivewhen tested with thioredoxin (Table 1). Theseobservations reveal that neither the monomeric

Figure 2. Molecular model of DmGPx. The modelled DmGPx structure is shown in cartoons representation for boththe canonical reduced (a) and the hypothetical oxidized (b) form. The catalytic triad, as well as the presumed resolvingCys91 (right side), are shown as spheres and colored according to type. The other highly conserved Cys74 is also shown inspheres behind the catalytic triad. The backbone is coloured from the N (blue) to the C terminus (red). The topology of thevisible colored secondary structure elements is as follows: β1 (blue, center), α3 (cyan, bottom), β2 (green), α4 and α5(green, right), α6 (yellow, left), β3 (dark orange, center), β4 (red, left), α7 (red, bottom). The remaining secondary structureelements are omitted for clarity. An identical representation of the accessible surface for the reduced (c) and oxidized (d)form is also shown. The pictures were drawn using PyMol [http://www.pymol.org/].

1037Thioredoxin Specificity of Glutathione Peroxidases

nature of a GPx, as in RrGPx-4, nor a second cysteinein the cysteine block region, as is present in BtGPx-1,is by itself sufficient to permit an efficient interactionof a GPx-type protein with thioredoxin.The Trx specificity of DmGPx becomes even more

obvious when appropriate kinetic parameters areconsidered. As has consistently been observed withselenium-containing GPx-type peroxidases,9,10

DmGPx displays ping-pong kinetics with infiniteKm and infinite Vmax values, i.e. the term Φ0 of thegeneral Dalziel equation for two-substrate enzymesubstitutionmechanisms (equation (1)), proved to be

zero. This simply implies that the reactions withinany of the enzyme substrate complexes in equations(2) (3) (4) (see Materials andMethods) are faster thanformation of the latter.28 Due to the lower reactivityof sulfur versus selenium the latter condition is notalways fulfilled by CysGPxs; in consequence, satura-tion kinetics are sometimes observed.17 Rate con-stants for oxidation (k+1) or reduction (k′+2) of suchenzymes, as are here determined by steady-statekinetic analysis at variable substrate and differentfixed co-substrate concentrations, are thereforeadequate parameters to reliably describe efficiencies

Table 1. GPx and Trx peroxidase activity of someglutathione peroxidases

Enzyme

Specific rate (μmol/minm per mg)

GSH Trx Ratio of activity (Trx/GSH)

DmGPxwt* 0.12 7.55 62.9BtGPx-1 80 0.15 0.002HsGPx-3 65 0.31 0.005RrGPx-4 87 Undetectable –

Activity was measured with H2O2 as oxidizing substrate, asdescribed in. Materials and Methods. GSH and Trx concentrationwere 3.0 mM and 0.004 mM, respectively. Results represent themean of at least three experiments, with a standard deviation ofless than 5%.

Table 3.GSH and Trx peroxidase activity of theDmGPxwt

mutants

Enzyme

Specific rate (μmoles/min per mg)

GSH Trx

PCOOH H2O2 PCOOH

DmGPx wt 0.17 0.12 14.38DmGPx Cys91/Ala 0.46 0.24 0.01DmGPx Cys91/Ser 0.52 n.d. 0.02DmGPx Cys91/Lys 0.57 0.54 0DmGPx Cys74/Ala 0.07 n.d 5.26DmGPx Cys74/Ser 0.10 n.d 10.33

n.d., not determined.In the mutants, DmGPxwt Cys91 or Cys74 were replaced by Ala,Ser and Lys or Ala and Ser residues, respectively. Conditions foractivity determinations were as for Table 1. Results represent themean of at least three experiments, with a standard deviation ofless than 5%.

Table 4. Kinetic coefficients and apparent rate constantsof DmGPxwt and mutants for the reduction of PCOOH by

1038 Thioredoxin Specificity of Glutathione Peroxidases

and specificities. Expectedly, the rate constant for thereaction of DmGPx with PCOOH was found to behigh irrespective of the nature of the reductant(compare k+1 for DmGPxwt in Tables 2 and 4). Inessence, therefore, DmGPxwt displays the kineticpattern that is characteristic for typical glutathioneperoxidases when analyzed with thioredoxin assubstrate.

Cys91 in DmGPx is essential for Trx peroxidasebut not for GSH peroxidase activity

When Cys91 of DmGPxwt was mutated to Ala, Seror Lys, the Trx peroxidase activity dropped toinsignificant levels. A detailed kinetic analysis ofthe Trx peroxidase activity was therefore notfeasible. However, the low GSH peroxidase activ-ities tended to increase in the mutant proteins (Table3). With GSH as the substrate, an evaluation of thekinetic parameters was therefore possible: The k+1values were marginally, if at all, affected by themutation (Table 4). The mutant proteins thus stillefficiently react with PCOOH with impressive rateconstants near 106 M−1 s−1, while their k'+2 value forthioredoxin must have dropped from 1.5×106 M−1

s− 1 (Table 2) to practically zero. Surprisingly,however, the k'+2 values for the reduction of themutant protein with GSH were consistently foundto be increased by two orders of magnitude (Table4). These findings underscore that C91 might beconsidered as a CR that, like in 2-Cys peroxiredox-ins, is required for the Trx-sustained catalytic cycle,while being dispensable for the GSH-driven reac-tion. Furthermore, the higher reactivity with GSH ofthe mutants suggests that C91 in the wild-type

Table 2. Kinetic coefficients and apparent rate constantsof DmGPxwt for the reduction of PCOOH by Trx

Enzyme Φ1 (μM s) k+1 (M−1 s−1) Φ2 (μM s) k′+2 (M

−1 s−1)

DmGPxwt 0.75±0.4 1.3×106 0.66±0.2 1.5×106

Kinetic coefficients were calculated from a single progressioncurve from al least three independent experiments, where the Trx-dependent reduction of PCOOH by DmGPxwt was analyzed asreported in Materials and Methods.

enzyme competes with GSH for the reduction of theoxidized CP.An analogous role of C74 was convincingly ruled

out, although its exchange against Ala or Ser slightlydecreased the specific activity (Table 3). However,an activity reduction by only 30% or 70%, as isobserved with DmGPxCys74/Ser or DmGPxCys74/Ala,respectively, is certainly too small to postulate apivotal role of C74 in the catalytic mechanism. Also,the molecular model of DmGPx predicts that C74 isnot surface exposed and thus has not an obviouschance to directly interact with substrates or withCP. The model, however, suggests C74 to beintegrated in a network of hydrogen bonds in thecore of the protein that might influence the reactivityof CP. In line with this hypothesis, serine should bethe more adequate substitute for cysteine thanalanine and indeed proves to be better tolerated inthis position.

Mass spectroscopical verification of the role ofC91 in catalysis

Reduced and substrate-oxidized DmGPx wassubjected to peptic cleavage and the resultingfragments were analyzed by LC-MS/MS with

GSH

EnzymeΦ1

(μM s)k+1

(M−1 s−1)Φ2

(μM s)k′+2

(M−1 s−1)

DmGPxwt 5.5±2.3 0.2×106 185,873±10,200 5.4DmGPxCys91/Ala 1.0±0.5 1.0×106 2053±380 487DmGPxCys91/Ser 1.5±0.6 0.7×106 1808±350 553DmGPx Cys91/Lys 1.2±0.5 0.8×106 1883±310 531

Kinetic coefficients were calculated from a single progressioncurve from at least three independent experiments, where theGSH-dependent reduction of PCOOH by DmGPxwt or themutants thereof, was analyzed as reported in Materials andMethods.

1039Thioredoxin Specificity of Glutathione Peroxidases

respect to their thiol/disulfide status. Due to theacidic conditions of the peptic digestion a rearrange-ment of disulfide bridges is reliably prevented.15

By LC-MS/MS analysis of the reduced enzymethe entire sequence could be recovered with allcysteine residues being in the reduced state (data notshown).Upon oxidation by 0.1 mM H2O2 for 10 min, C74

remained in the SH form, as did the C-1 that wasintroduced by the expression system. On the otherhand, Cys45 and Cys91 were partially foundoxidized to the sulfinic form, indicating that thesetwo residues are surface-exposed in the nativeprotein. More interestingly, a variety of disulfide-bridged double peptides were detected that com-prised peptic cleavage products that were linked bya disulfide bridge between the active site C45 andthe presumed CR at position 91: (38–52) + (89–104);(38–52) + (89–103); (38–52) + (88–104); (38–52) +(90–102); (38–52) + (89–102); (38–55) + (89–104);(38–55) + (89–102); (38–55) + (88–104); (38–55) +(90–102). Figure 3 shows an example of how thedouble peptide (38–52) + (89–104) was unambigu-ously demonstrated by MS/MS to contain thesequence (38)VVN to NNY(52) that comprises CPand the sequence (89)MVC to EVF(104) with the CR.

Figure 3. LC-MS/MS analysis of peptic digestion of oxiddouble peptide; identified as two-to sevenfold charged molecuspectrum (parent ion m/z 861.5=[M+4H+]4+) represents the Dmby sequencing shown in magenta and green, respectively.

Since the mass spectrometry data do not reveal ifthe C45–C91 bond is formed within the enzyme orbetween two molecules, the molecular mass of thereduced and H2O2-oxidized enzyme was subjectedto SDS–electrophoresis each under oxidizing andreducing conditions. Irrespective of the redox status,the enzyme migrated as a double band withapparent molecular masses slightly above 21,000,the molecular basis for the inhomogeneity remain-ing unclear (Figure 4). In the electrophoresisperformed in the absence of mercaptoethanol atrace of dimerization was observed which, however,was not affected at all by pretreatment of theenzyme by 50 μM H2O2.This dimer has thus to beinterpreted as a preparation artifact. Since themolecular mass of the bulk of enzyme, irrespectiveof its redox state, corresponds to the monomericenzyme, the disulfide bond between C45 and C91must be intramolecular. The reaction mechanism ofDmGPx is thus homologous to that of 2-Cys-peroxiredoxins, in which also the oxidation equiva-lents of the primary reaction product, i.e. the sulfenicacid form of CP,

29 is also conserved as a less reactivedisulfide bond to CR.

30 Like in many peroxi-redoxins,31 over-oxidation of CP to a sulfinic acidderivative is also observed in DmGPx, when the

ized DmGPx. (a) Multi-charge MS spectrum of a 3442 Dalar ions ([M+2/7 H+]2/7+) are depicted in blue. (b) MS/MSGPx peptides 38–52 and 89–104 with residues confirmed

Figure 4. DmGPxwt maintains its monomeric charac-ter upon oxidation, as shown by SDS–PAGE underreducing (a) or non-reducing conditions (b). Lanes are asfollows: 1, molecular mass markers; 2, purified andreduced DmGPxwt, 3, purified and reduced DmGPxwt

treated with 50 μM H2O2 for 5 min. See Materials andMethods for details.

1040 Thioredoxin Specificity of Glutathione Peroxidases

peroxidase is exposed to hydroperoxide in theabsence of a reducing substrate.To further study the analogy of DmGPx and 2-

Cys-peroxiredoxins, oxidized DmGPx was incu-

Figure 5. LC-MS/MS analysis of peptic digestion of the2Cys35/Ser. (a) Multi-charge MS spectrum of a 2573 Da double p([M+2/6H+]2/6+) depicted in blue. (b)MS/MSspectrum (parentand and DmTrx28–37 peptides as confirmed by partial sequefragment with an SS group resulting from asymmetric cleavag

bated with a thioredoxin mutant with a conservedexposed active-site cysteine, C32, but the co-reactingcysteine exchanged with serine (DmTrx-2Cys35/Ser).Such Trx mutants are known to make dead-endintermediates with peroxiredoxins and other Trxsubstrates, and can be used to identify Trx-targetedcysteine residues.32–35 Several fragments that corre-sponded to masses of peptic cleavage productslinked by a disulfide bridge between the Trx activesite C32 and the CR at position 91 of DmGPx wereevidenced asmulti-charged ions (2 to 6), e.g. (28–37) +(89–104); (28–37) + (90–102); (28–37) + (89–102);(29–37) + (89–102), and some of them were con-firmed by partial MS/MS sequencing. Figure 5shows an example of the full MS (Figure 5(a)) andMS/MS (Figure 5(b)) spectra as obtained with thedouble peptide DmTrx28–37+DmGPx90–102.Thisdemonstrates that DmGPx C91, again like CR in the2-Cys peroxiredoxins, is indeed the target residuethat first participates in the thiol-disulfide exchangereaction with Trx.

Discussion

The surprising observation that quite a number ofGPx homologues preferentially use thioredoxin as

reaction between oxidized DmGPx and reduced DmTrx-eptide; identified as two-to sixfold charged molecular ionsionm/z 1285.45=[M+2H+]2+) representing theDmGPx90–102ncing shown in magenta and green, respectively. A Trxe of the disulfide bound to C91 of DmGPx is shown in red.

1041Thioredoxin Specificity of Glutathione Peroxidases

the reductant instead of GSH, as is typical for allmammalian SecGPxs, prompted us to investigatethe molecular basis of the altered substrate specifi-city. DmGPx was chosen as a particularly appealingparadigm, since it had been reported to be devoidof any GSH peroxidase activity.22

We here confirm that DmGPx is indeed a Trxperoxidase, although a marginal GPx activity couldbe detected. The rate constant k'+2 (5.4 M−1 s−1) forthe reduction of oxidized DmGPx by GSH is,however, so low compared to that with Trx(1.5×106 M−1 s−1) that the GPx activity can berated as practically irrelevant and certainly notphysiological. This conclusion is further corrobo-rated by an infinite Km value for GSH, which incombination with the low rate constant, classify thereaction with GSH as unspecific. Infinite Km andVmax values are, however, also extrapolated for thereaction of DmGPx with Trx (previously reportedKm and Vmax values determined at the highestexperimentally possible co-substrate concentra-tion22 have therefore to be viewed as “apparent”).The observed lack of saturation kinetics by itselfdoes not argue against a specific enzymatic reac-tion. It is commonly observed with peroxidasesof the GPx family and often also with peroxiredox-ins, as well with heme peroxidases.17 The phe-nomenon results from the extreme reactivity of thecatalytic centers, where reaction with the com-plexed substrate is achieved almost instantly. Anaccumulation of enzyme–substrate complexes isthus precluded. Rate constants near 106 M−1 s−1

combined with the lack of saturation kinetics, asobtained for the reductive part of the Trx-drivencatalytic cycle, are best interpreted as kon values forenzyme–substrate complex formation and, there-fore, argue in favor of high specificity.Surprisingly, the substitution of a specific cysteine

residue (C91) that is not consistently conserved inthe entire GPx family completely abolished the Trxperoxidase activity. Instead, the marginal GSHperoxidase activity was significantly enhanced bythis substitution. In absolute terms the pertinent k′+2values are still low, likely because of missing resi-dues that, as in mammalian GPxs, force the GSHmolecule into an orientation that is appropriatefor instant attack of the selenenic acid.7,15 In mam-malian GPx-1, -2 and -3 these are primarily arginineresidues that bind to the carboxyl functions of GSH,7

while in GPx-4 this is achieved by lysine residues.15

The increased GSH peroxidase activity of themutants that lack a CR, however, complies withthe assumption that the catalytic mechanism oftypical mammalian SecGPxs does not require anycooperating cysteine residue. In these enzymes areactive (selenol)cysteine, which is embedded in thecharacteristic triad composed of a (selenol)cysteine,glutamine and a tryptophan residue, is oxidized byhydroperoxide to a selenenic or a sulfenic acid,which is directly reduced by GSH or other thiolsubstrates. The preferred substrates of Trx, however,are protein disulfides. Therefore, the formation of aninternal disulfide bond in a GPx is likely the

prerequisite for an efficient reaction with a Trx,while it is counterproductive for a reaction withGSH. The preference of the two reductants, GSHand Trx, for sulfenic acids and disulfides, respec-tively, may in part be due to their degree of dis-sociation at physiological pH. The thiol of GSHwith a pK of 9.236 will react with the dissociatednegatively charged sulfenic acid (pK∼537), while thethiolate of Trx (pK near 7)38–40 might be electro-statically repelled, but instead can easily attack anuncharged disulfide.A direct reaction of a thioredoxin with the sulfenic

acid form of an atypical 2-Cys peroxiredoxin(MtTPx) has recently been demonstrated35 but sofar never for any of the typical 2-Cys Prx. Mostconvincingly is the analogy between DmGPx and 2-Cys Prxs demonstrated by an attack on CR bythioredoxin. The identification of a disulfidebetween a mutant thioredoxin and C91 of DmGPx(Figure 4), thus, unequivocally reveals a mechanismthat is novel for GPx-type peroxidases but commonfor 2-Cys peroxiredoxins.A condition for this variation of GPx catalysis

appears to be the monomeric nature of theproteins. Cysteine residues within the α4 helixare sporadically also seen in mammalian SecGPxs,e.g. in bovine and pig GPx-1, which, however, arespecific for GSH. This is likely due to the fact thatthe α4 helix contributes to the dimer/dimerinterface in these species and, therefore, a cysteineresidue within this area cannot react with theoxidized CP. The “Cys block” has evidently to besurface-exposed and flexible, as is supported bymolecular models for DmGPx. The prerequisitesfor thioredoxin specificity within the GPx family,therefore, are (i) deletions of the subunit interfaceswhich determine the monomeric nature of thissubgroup, (ii) flexibility of the loop comprising theα4 helix that forms the dimer interface in thetetrameric congeners, and (iii) a cysteine in thisflexible loop that can react with CP and thus actsas a CR. Screening of the known sequences of theGPx family reveals that these conditions are metby the majority of GPxs from plants, bacteria,fungi and protozoa (Table 5) which, thus, are likelythioredoxin peroxidases. The historical term glu-tathione peroxidase may turn out, therefore, tocorrectly describe only a small subgroup of thefamily.The biological role of the GPx-type Trx perox-

idases largely remains a matter of speculation. Theidea that they might primarily be in charge of hy-droperoxide detoxification as mammalian GPx-110

cannot be uncritically accepted. Being CysGPxs,their rate constants for hydroperoxide reduction,k+1, though quite impressive in the case of DmGPx,are one to three orders of magnitude lower thanthose of SecGPxs. Also, the capacity of thethioredoxin system with a Trx concentration inthe low micromolar range falls short when com-pared to the glutathione system with a GSHconcentration of 2 mM–10 mM. Taken together,these two criteria do not qualify a Trx-fueled GPx

†http://www.protein.cribi.unipd.it/homer/

Table 5. GPx homologues suggested to be Trx peroxidases by taxa

Taxa

Number ofsequences

Similarity with Trx peroxidase by criteria (%)

A B C A+B A+B+C

Mammalia 66 36.4 3.0 0.0 (30.3) 0.0 0.0 (0.0)Vertebrata 8 100.0 0.0 0.0 (62.5) 0.0 0.0 (0.0)Arthropoda 19 94.7 42.1 68.4 (94.7) 42.1 26.3 (42.1)Nematoda 16 56.2 0.0 0.0 (25.0) 0.0 0.0 (0.0)Trematoda 2 100.0 0.0 0.0 (100) 0.0 0.0 (0.0)Euglenozoa 16 100.0 81.2 68.8 (75) 81.2 68.8 (75)Alveolata 6 100.0 66.7 16.7 (16.7) 66.7 16.7 (16.7)Fungi 27 100.0 96.3 55.6 (96.3) 96.3 55.6 (96.3)Viridiplantae 52 98.1 98.1 94.2 (96.2) 98.1 94.2 (96.2)Viruses 3 66.7 0.0 0.0 (0.0) 0.0 0.0 (0.0)Bacteria 236 100.0 94.5 25.0 (67.4) 94.5 24.6 (66.9)Total 451 88.5 72.5 32.8 (65.9) 72.1 30.8 (56.5)

A total of 451 sequences were screened for similarity with established Trx peroxidases by (A) disruption of the subunit interfaces, (B)presence of a putative CR, (C) strict or (high) conservation of the Hidden Markov Model (HMM) profile based on position 79–122 ofestablished Trx peroxidases (Figure 1, horizontally shaded), (A+B) simultaneous presence of criteria A and B, and (A+B+C)simultaneous presence of all criteria, at a strict or (high) e-value cutoff (i.e. putative Trx peroxidases). Strict and high e-value cutoff valuesin the HMM are defined 10−21, and 10−15, respectively. HMM was built using the HMMER tool [http://www.hmmer.wustl.edu].

1042 Thioredoxin Specificity of Glutathione Peroxidases

as an ideal candidate to counteract oxidative stress.In organisms devoid of GSH such as actino-mycetes,41 or lacking functional glutathione perox-idases such as kinetoplastida,42 antioxidant defensesystems supported by Trx or Trx-related proteinsmight be pivotal for survival.43–45 In general,however, the biological role of Trx-dependentCysGPxs is more promisingly searched for in thecontext of redox regulation. H2O2 and otherhydroperoxides, apart from being toxic at excessiveconcentrations, have been recognized as mediatorsor modulators of numerous signaling cascades.46–48

A critical aspect of hydroperoxide-mediated signal-ing, however, is the almost promiscuous reaction ofthese low molecular mass oxidants with exposedand deprotonated proteins thiols. A protein such asa GPx or Prx, when oxidized by hydroperoxidesand thus acting as a peroxide sensor, has thechance to render specificity to peroxide signalingby selectively targeting SH groups of definedproteins. As mentioned in the Introduction, thisconcept has so far been verified only once.12 YeastGPx-3 specifically activates the transcription factorYap-1 in a peroxide-dependent manner by target-ing a particular SH group of Yap-1 with itsoxidized CP. Yeast GPx-3 is one of the verifiedCysGPx-type Trx peroxidases that can also form aninternal disulfide bridge between CP and CR. Thisinternal disulfide bond, as well as the intermole-cular disulfide between GPx and Yap-1, is reducedby Trx. The Trx peroxidase activity of GPx-3, in thecontext of Yap regulation, has to be viewed as amechanism to either shut off or prevent Yapactivation. Thus, with an “on” signal, i.e. H2O2rendered specific for interaction with Yap-1 byGPx, and an independent “off” or “no go” signal,i.e. reduced thioredoxin, an ideal regulatorycircuit is constructed. It would be surprising ifthe other Trx-dependent CysGPxs do not have arole in analogous or even homologous regulatorycircuits.

Materials and Methods

Multiple sequence alignment

Sequences containing an annotated glutathione perox-idase domain were extracted from UniProt DatabankRelease 48.8. The total amount of 600 hits has beenreduced to 451 non-redundant full-length proteins. CLUS-TAL W 1.8349 was used to construct the multiplealignment and a final manual editing was performed todiscard uncertain hits. A representative subset of thewhole multiple alignment is shown in Figure 1 andcontains proteins reflecting the taxonomic coverage andsequence conservation ratio of the original multiplealignment. The 35 proteins of the alignment wereclustered using cd-hit50 at 50% of sequence identitythreshold and belong to the following main taxa:Mammalia, other Vertebrates, Bacteria, Fungi, Nematoda,Viridiplantae, Arthropoda, Virus, Euglenozoa and Alveo-lata. Highly abundant Bacteria sequences were furtherreduced using a cutoff of 30% sequence identity. Themultiple alignment was prepared using Espript.51

Molecular modeling

The structure of DmGPx was modeled from the crystalstructure of bovine GPx-1 (PDB code: 1GP1).4 Thealignment between both sequences is shown as part ofFigure 1. The model, covering the DmGPx sequence(residues 11 to 169) corresponding to the crystallizedBtGPx structure shown in Figure 1, was constructed fromthe alignment using the HOMER server†. The server usesthe conserved parts of the structure to generate a rawmodel, which is then completed by modeling thedivergent regions with a fast divide and conquermethod.52 Side-chains are placed with SCWRL353 andthe final energy evaluated with FRST.54 As the mutatedcysteine residue is near a loop region predicted to bestructurally divergent between DmGPx and the crystalstructure template, the surrounding region was subjected

1043Thioredoxin Specificity of Glutathione Peroxidases

to an additional round of manually evaluated divide andconquer loop modeling in order to generate the reducedand oxidized forms.

Expression and purification of DmGPxwt

The expression vector pQE30 (Qiagen, Hilden, Ger-many) containing the full length DmGPx gene was a kindgift from O. Schmidt, Department of Applied andMolecular Ecology, Adelaide University, Australia.55 Theexpression system was designed to yield a product withthe N-terminal extension MRGSHHHHHHGSACupstream of position 2 of the authentic sequence.Escherichia coli JM 109 was transformed with thisconstruct. Expression was induced with 1 mM IPTG,and cells were harvested after 4 h by centrifugation at5000 g for 30 min. Bacterial pellets obtained from one literof culture were suspended in 60 ml of cold B-Perextraction reagent (Pierce, Rockford, IL, USA) containing5 mM 2-mercaptoethanol, 0.1 mg/ml PMSF, 0.7 mg/mlpepstatin, 0.5 mg/ml leupeptin, and stirred for 10 min onice to obtain cell lysis. Any residual particulate materialwas removed by centrifugation at 20,000 g for 30 min. Thesupernatant was diluted 1:1 (v/v) with 50 mM Tris–HCl,1 M NaCl, 20% (v/v) glycerol, 0.2% (v/v) Triton X-100,10 mM 2-mercaptoethanol (pH 7.5) and loaded onto acolumn containing 2 ml Ni-NTA resin (Qiagen, Hilden,Germany), equilibrated with the above buffer diluted 1:1with water (v/v). After washing with 20 ml of same buffercontaining 20 mM imidazole the His-tagged protein waseluted with the same buffer containing 200 mM imidazole.Pooled GPx fractions were further purified and freed ofimidazole on a Superdex 75 column (26 mm×620 mm,Pharmacia, Uppsala, Sweden) equilibrated with 25 mMTris–HCl, 0.5 M NaCl, 10% glycerol (pH 7.5), andconcentrated. Protein content was quantified accordingto Bensadoun and Weinstein.56 DmGPxwt thus obtainedappeared to be 80% homogeneous on SDS–PAGE gelsstained with Coomassie blue.

Site-directed mutagenesis

DmGPxwt was mutated at residues Cys74 and Cys91 toyield DmGPxCys74/Ala, DmGPxCys74/Ser, DmGPxCys91/Ala,DmGPxCys91/Ser and DmGPxCys91/Lys, respectively, bymeans of the QuikChange site-directed mutagenesis kit(Stratagene, Cedar Creek, TX, USA) using the primers(changed codons in italics):

fw: 5′-GTGATCCTCAACTTCCCGGCCAATCAGTTTG-GGTCCCAG-3′rev: 5′-CTGGGACCCAAACTGATTGGCCGGGAAGTTGAG-GATCAC-3′;fw: 5′-GTGATCCTCAACTTCCCGTCCAATCAGTTTG-GGTCCCAG-3′rev: 5′-CTGGGACCCAAACTGATTGGACGGGAAGTTGAG-GATCAC-3′;fw: 5′-CGATGGAGAGGCCATGGTGGCCCACCTGCGC-GACTCCAAG-3′rev: 5′-CTTGGAGTCGCGCAGGTGGGCCACCATGGCCTC-TCCATCG-3′;fw: 5′-CGATGGAGAGGCCATGGTGTCCCACCTGCGC-GACTCCAAG-3′rev: 5′-CTTGGAGTCGCGCAGGTGGGACACCATGGCC-TCTCCATCG-3′;fw: 5′-CGATGGAGAGGCCATGGTGAAGCACCTGCGC-GACTCCAAG-3′

rev: 5′-CTTGGAGTCGCGCAGGTGCTTCACCATGGCC-TCTCCATCG-3′.

Mutations were verified by double strand DNAsequencing. Expression and purification of mutant pro-teins were expressed and purified to 80% homogeneity, asdescribed for DmGPxwt.

Sources of other proteins

HsGPx-3 was purified from 1200 ml of healthy blooddonor plasma according to Maddipati and Marnett57 withthe exception that the initial protein precipitation wasperformed at an ammonium sulfate saturation of 50%.RrGPx-4 was purified from the testis of adult Wistar ratsaccording to Maiorino et al.58 BtGPx-1 was from Sigma-Aldrich. Human thioredoxinCys72/Ser was purified fromthe supernatant of E. coli transformed with the pQE30plasmid containing the human Trx sequence where theCys 72 was mutated into Ser to prevent dimerization.59

MutatedD. melanogaster Trx was obtained similarly. In thiscase the plasmid used for transformation contained theDmTrx-2 sequence (acc. no. NP 511046) where Cys 35 ofthe redox center CGPC was mutated into CGPS to yieldDmTrx-2Cys35/Ser. Both human thioredoxinCys72/Ser andDmTrx-2Cys35/Ser contained the N-terminal extensionMRGSHHHHHHGS as a purification tag. Plasmodiumfalciparum thioredoxin reductase was prepared asdescribed.60

Activity measurement and kinetic analysis

The coupled assay with NADPH and glutathionereductase or TrxR was used for GSH or Trx peroxidaseactivity measurements, respectively. The absorbance at340 nm was measured with a Beckman DU7 spectro-photometer equipped with a magnetic stirrer at roomtemperature. In the GPx assay, a 2.5 ml final volume of0.1 M Tris–HCl (pH 7.4), contained 5 mM EDTA, 0.1%(v/v) Triton X-100, 0.15 mMNADPH, 3 mMGSH, 2 units/mlglutathione reductase and a variable amount of GPx. TheTrx peroxidase assay mixture was identical except that theCys72/Ser mutant of human Trx (4 μM) and 0.5 unit/ml ofP. falciparum TrxR, replaced GSH and GR, respectively.Before use, GPx was reduced by 30 mM 2-mercaptoetha-nol on ice for 30 min and freed of the reductant by twopassages through gel permeation cartridges (MicroBios-pin, Bio-Rad) equilibrated with the assay buffer. Thereaction was started with 50 μM H2O2 or 24 μMphosphatidylcholine hydroperoxide, prepared accordingto Maiorino et al.58

Enzyme kinetics were studied by the same coupled testsat different fixed co-substrate concentrations using thetime progression curve analysis as described.17,61 In short,substrate consumption (NADPH oxidation) curves weredigitalized to yield time intervals of 3–10 s that allowedcalculating the actual rates at the respective substrateconcentrations. Under the adopted conditions, sponta-neous reduction of PCOOH was close to zero and thuswas ignored. Data were fitted to the general Dalzielequation for enzyme substitution mechanisms involvingtwo substrates (type IV mechanisms, as described byDalziel62):

(1)½E0�=v ¼ U0 þ U1=½ROOH� þ U2=½GSH�equation (1) has so far proven to be applicable to allGPx-type enzymes and other peroxidases.9,10,63 In this

1044 Thioredoxin Specificity of Glutathione Peroxidases

equation [E0] is the total enzyme molarity, v the rate,[ROOH] and [GSH] the concentrations of the hydroper-oxide and GSH respectively, and the Φ values are co-efficients obtained experimentally from slopes andintercepts of double reciprocal plots.62 Rate constants forpartial reactions according to equations (2) (3) (4) werecalculated from the empirical Dalziel coefficients bymeansof the definitions k+1=1/Φ1 and k′+2=1/Φ2; k+1 being therate constant for the oxidation of the enzyme by a hydro-peroxide and k'+2 a complex rate constant describing thenet forward reaction of all reductive steps:

(2)

(3)

(4)

Mass spectrometry analyses

LC-ESI-MS/MS characterizations of the oxidizedDmGPxwt and of the dead-end intermediate betweenDmGPxwt and mutated DmTrxCys35/Ser was performed asdescribed,35 using a LTQ ion trap mass spectrometerequipped with a nanoESI interface (ThermoElectron,Milan, Italy). In particular, a Biobasic C18 column(0.180 mm–100 mm, 5 μm; ThermoElectron) and a flow-rate of 50 μl/min splitted 1:50 were used. As describedabove, mutated DmTrx-2Cys35/Ser contained the redoxcenter CGPC mutated into CGPS.

Data handling of mass spectra

Computer analysis of peptide MS/MS spectra wasperformed using Bioworks 3.1, based on SEQUESTalgorithm (University of Washington, USA; licensed toThermoFinnigan Corp.). For the peptic peptide mixturethe ‘no enzyme’ option was used due to the limitedspecificity of pepsin cleavage. As confidence of peptideidentification the minimum values of Xcorr were greaterthan 1.5, 2.0, and 2.5 for single, double and triple chargeions, respectively. Identification of disulfides was carriedout as follows: first the most frequent cysteine-containingpeptides obtained by pepsin digestion were identified inboth, DmGPx and Trx. Then these peptides were used formanual calculation of possible peptides linked by dis-ulfide bridges. The minimum consecutive multi-chargeions of a possible “hybrid peptide” were three. Whenpossible, manual evaluation of related MS/MS spectrawere performed to obtain partial amino acid sequences ofone or both peptides.

Mass estimation by SDS–electrophoresis

The intramolecular or intermolecular nature of thedisulfide formed upon DmGPxwt oxidation by H2O2,

was studied by comparing SDS–PAGE analysis eachunder reducing and non-reducing conditions. PurifiedDmGPxwt was fully reduced by incubation with 30 mM 2-mercaptoethanol for 30 min on ice and subsequently freedof the reductant by two chromatographic passages on gelpermeation cartridges (MicroBiospin, Bio-Rad) equili-brated with 0.1 M Tris–HCl (pH 7.4), 5 mM EDTA.Oxidation was obtained by treating this sample with50 μMH2O2, for 5 min. Protein analysis was performed byan SDS–polyacrylamide gel (T=14%), after a 1:1 dilutionwith twice-concentrated Laemmli sample buffer (reducingconditions). Non-reducing conditions were obtained byomitting 2-mercaptoethanol from the Laemmli samplebuffer. At the end of the electrophoresis, the gel wasstained in colloidal Coomassie blue.

Acknowledgements

This work was supported by the Italian Ministryof University and Scientific Research Grants PRIN2003038920_002 (to M.M.) and by the University ofPadova (Fondi quota ex 60% 60A06-8048/05) (toM.M.).S. C.E. T. is funded by a Rientro dei cervelli grant

from the Italian Ministry of Education, and Research(MIUR).

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Edited by P. Wright

(Received 21 July 2006; received in revised form 9 October 2006; accepted 10 October 2006)Available online 13 October 2006