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Research paper

The cold-adapted g-glutamyl-cysteine ligase from the psychrophilePseudoalteromonas haloplanktis

Antonella Albino a, Amalia De Angelis b, Salvatore Marco a, Valeria Severino c,Angela Chambery c, Antimo Di Maro c, Doriana Desiderio b, Gennaro Raimo b,Mariorosario Masullo d, Emmanuele De Vendittis a, *

a Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universit�a di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italyb Dipartimento di Bioscienze e Territorio, Universit�a del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italyc Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, II Universit�a di Napoli, Via Vivaldi 43, 81100 Caserta, Italyd Dipartimento di Scienze Motorie e del Benessere, Universit�a di Napoli “Parthenope”, Via Medina 40, 80133 Napoli, Italy

a r t i c l e i n f o

Article history:Received 18 February 2014Accepted 9 May 2014Available online 24 May 2014

Keywords:Glutamyl-cysteine ligaseGlutathione biosynthesisPsychrophileCold adaptationPseudoalteromonas haloplanktis

a b s t r a c t

A recombinant g-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis(rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathionebiosynthesis by forming g-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction.The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of thebiosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was char-acterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme wasactive even at cold temperatures in a moderately alkaline buffer containing a high concentration ofMgþþ; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rateof rPhGshA II at 15 �C was higher than that reported for rPhGshB, thus suggesting that formation of g-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA IIhad different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM),glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II,probably through the binding to an enzyme pocket different from the active site. Also the oxidised formof glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared torPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed withlower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge onglutathione biosynthesis in the first cold-adapted source; however, another possible redundant g-glu-tamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of thiscellular thiol in P. haloplanktis.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The tripeptide glutathione (g-glutamyl-cysteinyl-glycine, GSH)is a regulator of the physiological redox environment acting in botheukaryotes and prokaryotes [1e10]. The main antioxidant actiondisplayed by GSH includes a defence against damages caused byreactive oxygen species (ROS) on cellular components, detoxifica-tion of foreign compounds and toxic metals, preservation of proteinsulfhydryls against irreversible oxidation. Besides these protectiveroles, this abundant biological thiol is involved in several cellularfunctions, such as amino acid transport, metabolism of therapeuticdrugs, cell cycle regulation, carcinogenesis and apoptosis [11e14].

Abbreviations: GshA, g-glutamyl cysteine ligase; GshB, glutathione synthetase;gshA, gene encoding GshA; gshB, gene encoding GshB; Ph, Pseudoalteromonas hal-oplanktis; Ec, Escherichia coli; GSH and GSSG, reduced and oxidised form of gluta-thione; g-Glu-Cys, L-g-glutamyl cysteine; IC50, concentration of inhibitor leading to50% reduction of the activity; IPTG, isopropyl-b-thiogalactopiranoside; Ea, energy ofactivation; kin, rate constant of inactivation; ROS, reactive oxygen species; TCEP,Tris-(2-carboxyethyl)-phosphine hydrochloride.* Corresponding author. Tel.: þ39 081 7463118; fax: þ39 081 7463653.

E-mail address: devendit@unina.it (E. De Vendittis).

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The mechanism of redox homoeostasis sustained by GSH is basedon the intracellular balance with its oxidised form (GSSG), obtainedthrough a disulfide bridge between two molecules of GSH. More-over, GSH, GSSG and S-nitrosoglutathione, another modified formof glutathione originating from reactive nitrogen species, areresponsible for the S-glutathionylation on specific cysteine residuesof the target protein, and eventually affecting its functioning[11,12,15e17].

The biosynthesis of GSH occurs with a mechanism conservedthroughout prokaryotes and eukaryotes. In particular, the enzy-matic assemblage of L-Glu, L-Cys and Gly into GSH occurs in twosequential steps, both coupled to the hydrolysis of one molecule ofATP [1]. As described in the following scheme, the first step iscatalysed by the enzyme g-glutamyl-cysteine ligase and leads tothe formation of L-g-glutamyl cysteine (g-Glu-Cys); the second stepproduces GSH and includes the involvement of another enzyme,called glutathione synthetase.

L-Glu þ L-Cys þ ATP / g-Glu-Cys þ ADP þ Pi (1)

g-Glu-Cys þ Gly þ ATP / GSH þ ADP þ Pi (2)

The enzymes involved in the GSH biosynthesis isolated fromprokaryotic and eukaryotic sources are somehow different in theirstructural and functional organisation. In most prokaryotes, twodistinct genes, gshA and gshB, encode g-glutamyl-cysteine ligase(GshA) and glutathione synthetase (GshB), respectively; the firstenzyme is a monomer and the second one is organised as ahomotetramer [9]. On the other hand, few prokaryotes possess asingle bifunctional enzyme organised as a homodimer, supportingboth activities required for GSH biosynthesis [18e20]. In eukary-otes, the activities of g-glutamyl-cysteine ligase and glutathionesynthetase are respectively sustained by a heterodimeric enzyme[21,22] and a homodimeric glycosylated enzyme [23e25]. Thestudies on GSH biosynthesis pointed to some features possessed byg-glutamyl-cysteine ligase in different organisms. Indeed, the re-action catalysed by this enzyme seems to be the rate limiting step ofthe overall process [26]; the activity is feedback inhibited by GSH[21,27]; the Mg2þ ions, a specific requirement for ATP-dependentenzymes, can be replaced by Mn2þ ions, although this substitu-tion reduces the activity of the enzyme and modifies its affinitytowards different substrates [28e30].

GSH has a more crucial role in microorganisms characterised byan oxidative metabolism [4]. Pseudoalteromonas haloplanktis, apsychrophile isolated from the Antarctic sea, able to grow in the4e20 �C interval [31], is a model of aerobic cold-adapted bacteria.The presence and the functional role of GSH in this microorganismwas inferred by the finding of glutathionylated adducts either in theendogenous proteins from a growing culture of this bacterium [17]or in some purified recombinant enzymes involved in the control ofthe oxidative stress of the psychrophile [17,32]. In the presence ofsufficient nutrients and high aeration, P. haloplanktis grows to veryhigh density due to its efficient respiration, a behaviour implying agood functioning of the defence mechanisms against the oxidantenvironment [31]. Indeed, P. haloplanktis is exposed to high levels ofROS for both the increased oxygen solubility in the cold sea waterand the enhanced stability of these toxic compounds at cold tem-peratures [31,33]. Previous reports on the enzyme systems involvedin the defence against ROS damages in P. haloplanktis pointed to thehigh efficiency of superoxide dismutase [17,34,35] and thioredoxinsystem [32,36]. Furthermore, the studies on GSH biosynthesis inthis microorganism already led to the characterisation of its cold-adapted glutathione synthetase (rPhGshB) [37,38].

In order to continue the characterisation of the enzyme systemfor GSH biosynthesis in P. haloplanktis, the present work regards the

enzyme g-glutamyl-cysteine ligase from this psychrophile(PhGshA). The genome of P. haloplanktis, strain TAC125 containstwo chromosomes and a gene encoding a putative PhGshA wasannotated in chromosome I (Ph-gshA I) and II (Ph-gshA II) [31]. Forthis reason, the cloning of both genes in suitable expression vectorswas considered for the heterologous production of recombinantforms of PhGshA; however, the current study was mainly focusedon the detailed biochemical characterisation of the enzyme enco-ded by the gene Ph-gshA II. Indeed, a recombinant enzyme, namedrPhGshA II, possessing the typical activity of g-glutamyl-cysteineligases, was obtained. In particular, rPhGshA II was able to catalysethe first step of GSH biosynthesis, involving the ATP-dependentformation of g-Glu-Cys from L-Glu and L-Cys. The investigationincluded the evaluation of the substrate specificity of rPhGshA II,the determination of its kinetic and inhibition parameters and anassessment on the effect of temperature, a relevant issue for a cold-adapted enzyme. Therefore, this study, together with the previousreports on glutathione synthetase [37,38], gives further informationon GSH biosynthesis to P. haloplanktis, a typical cold-adapted or-ganism which seems to be highly responsive to glutathione meta-bolism and homoeostasis.

2. Materials and methods

2.1. Materials

Restriction and modifying enzymes were from GE Healthcare orPromega. Taq DNA polymerase from Takara was used in PCR ex-periments. Plasmid pGEM-T Easy was from Promega, whereasvector pET-22b(þ) and the Escherichia coli BL21(DE3) strain werefrom Novagen. Purification of plasmids and DNA fragments wasrealised with Qiagen kits from M-Medical. Oligonucleotide syn-thesis, nucleotide sequencing and synthesis of vectors were carriedout at Primm (Italy). Ampicillin, isopropyl-b-thiogalactopiranoside(IPTG), b-mercaptoethanol, Tris-(2-carboxyethyl)-phosphine hy-drochloride (TCEP), L-Cys, L-Glu, L-2-aminobutyric acid, L-2-hydroxy-glutaric acid, N-methyl-L-glutamic acid, 2,3-naphthalenedicarboxyaldehyde, GSH and GSSG were from Sigma-eAldrich. [g-32P]ATP (2 mCi mL�1; 10 Ci mmol�1) was purchasedfrom Perkin Elmer. The chromatographic medium Ni-NTA agarosewas from Qiagen. HPLC-grade solvents were obtained from CarloErba. All other chemicals were of analytical grade.

The following buffers were used: buffer A, 20 mM Tris$HCl, pH7.8, 5 mM MgCl2, 150 mM KCl, 200 mM TCEP; buffer B, as buffer Asupplemented with 50% (v/v) glycerol; buffer C, 100 mM Tris$HCl,pH 8.0, 20 mM MgCl2, 150 mM KCl.

2.2. Heterologous expression of the gshA gene from P. haloplanktis

The genomic DNA from P. haloplanktis, strain TAC125, was pre-pared as previously described [39]. Transformation of bacterialstrains, preparation of plasmids and other details of DNA recom-binant technology were carried out according to standard pro-cedures [40]. The two genes encoding putative GshA inchromosome I and II from P. haloplanktis, strain TAC125, have beenannotated as Ph-gshA I (locus tag: PSHAa0937) and Ph-gshA II (locustag: PSHAb0107), respectively [31]. For the production of a re-combinant form of a psychrophilic GshA, the Ph-gshA II gene wasconsidered and its heterologous expression was performed essen-tially as recently reported [41]. Its complete coding sequence wasamplified by PCR, using the genomic DNA from this psychrophile asa template, and couples of oligonucleotide primers annealing to 50-or 30-untranslated region of the gene. In particular, forward andreverse primer for the amplification of Ph-gshA II were 50d-Ae15TTACAGAGGTTCATATG$ACG$TTA$T10-30 and 50d-

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T1528GCTCTTTTGCTC$GAG$CTT$ACC$TTC$T1503-30. Numbering inprimers begins from starting codon (italics), whereas underlinedletters indicate mismatches introduced to create the NdeI and XhoIrestriction sites. The amplified segment was digested with NdeI andXhoI and cloned in pET-22b(þ) previously digested with the sameendonucleases. The resulting construct was controlled by nucleo-tide sequencing to exclude any undesired mutation occurringduring the amplification/cloning procedure. The vector for theexpression of Ph-gshA II, named vPh-gshA II, allowed the produc-tion of a recombinant form of a putative GshA from P. haloplanktis,called rPhGshA II, in which the extrapeptide LE(H)6 replaced the C-terminal V 505 of PhGshA II. After transformation of the E. coliBL21(DE3) strain with vPh-gshA II, a culture of the resultingtransformant was grown at 30 �C up to 0.5 A600 and the heterolo-gous expressionwas continued for 3 hwithout or upon the additionof 0.1 mg mL�1 IPTG. Bacterial cells were collected by centrifuga-tion, resuspended in buffer A (15 mL for 1-L culture) and then lysedby a French Press (Aminco, USA) to obtain a cell homogenate. Thissample was then centrifuged at 100,000� g and the supernatantwas used as starting material for the purification of the recombi-nant product by affinity chromatography on Ni-NTA agarose. To thisaim, the supernatant was added in batch to the Ni-NTA Agaroseresin, equilibrated in buffer A. After incubation overnight at 4 �C,the slurry was poured in a column, which was extensively washedwith the same buffer supplemented with 10 mM imidazole$HCl.The bound proteins were then eluted with buffer A supplementedwith 200mM imidazole$HCl and pure protein fractions, as analysedby SDS-polyacrylamide gel electrophoresis, were pooled togetherand extensively dialysed against buffer A. Some preparations ofrPhGshA II required a further purification step on Ni-NTA agaroseresin, in order to eliminate traces of triphosphatase contaminatingactivities. The pure protein samples were finally stored at�20 �C inbuffer B. Nearly 4e5 mg of protein were obtained from 1-L cultureof the transformant.

2.3. Biochemical assays

The activity of rPhGshA II was evaluated through the ATP hy-drolysis promoted by this enzyme, coupled to the synthesis g-Glu-Cys starting from L-Glu plus L-Cys (reaction (1)). The method isalmost identical to that successfully used for measuring the activityof rPhGshB [38]. In particular, the ATPase activity was evaluatedthrough the release of radioactive inorganic phosphate (32Pi) from[g-32P]ATP, using the phosphomolybdatemethod [42]. On the otherhand, the detection of the final product g-Glu-Cys was carried outthrough a fluorimetric method, essentially as previously reported[43]. Unless otherwise indicated, the reaction was carried outkinetically at 15 �C in buffer C, and the reaction rate was obtainedfrom the slope of linear kinetics. The kinetic parameters of theATPase catalysed by the psychrophilic enzyme, including the KM

values for the three substrates L-Cys, L-Glu and ATP, and the Vmax ofthe reaction, were derived from LineweavereBurk plots of the ac-tivity data. To this aim, the reaction mixture contained a fixed andessentially saturating concentration of two out of three substrates,and various concentrations of the third substrate distributedaround the KM of the reaction. The inhibition profile on the rPhGshAII ATPase by GSH or GSSG was obtained through activity mea-surements carried out in the presence of various concentrations ofthese glutathione forms. The kinetic parameters of the rPhGshA IIATPase were determined also in the presence of a fixed concen-tration of GSH or GSSG. The energy of activation (Ea) of the ATPaseactivity of rPhGshA II and of the heat inactivation process of thispsychrophilic enzyme was derived from Arrhenius plots; the otherthermodynamic parameters of activation (DH*, DS* and DG*) werecalculated as reported elsewhere [44]. The biochemical data

presented in each of Figs. 2e6, 9 and 10 reflect one out of at leasttwo independent experiments realised with different proteinpreparations and giving almost overlapping results. Furthermore,the significance of the linear fit in kinetics and double reciprocalplots was thoroughly evaluated through the squared correlationcoefficient r2. Where appropriate, the significance level of the datapresented was evaluated by calculating the t-parameter and thecorresponding p-values; otherwise, error bars were included in thegraphical representations.

2.4. MALDI-TOF MS mapping

To identify the cysteinyl residue target of the S-gluta-thionylation reaction, a tryptic peptide mapping was performed byMALDI-TOF mass spectrometry on rPhGshA II incubated for 15 minat 15 �C in the absence or in the presence of 3.6 mM GSH or GSSG.Treated and untreated protein samples were subjected to trypticdigestion for 16 h at 37 �C in 50 mM NH4HCO3 with trypsin(1:1000). At the end of digestion, tryptic peptides were analysed ona MALDI-TOF micro MX apparatus (Waters Co., Manchester, UK)equippedwith a pulsed nitrogen laser (l¼ 337 nm). Prior to spectraacquisition, 1 mL of each peptide solution was mixed with 1 mL ofsaturated a-cyano-4-hydroxycinnamic acid matrix solution (10mg/mL in 1:1 acetonitrile:water, v/v, containing 0.1% trifluoroaceticacid) and 1 mL of the resulting mixture was placed on the massspectrometer sample target. The droplet was dried at room tem-perature. Once the liquid was completely evaporated, the samplewas loaded into the mass spectrometer and analysed. The instru-ment was externally calibrated using a tryptic alcohol dehydroge-nase digest (Waters, Manchester, UK) in reflectron mode. Allspectra were processed and analysed using the MassLynx 4.1 soft-ware (Waters, Manchester, UK).

2.5. Other methods

Protein concentration was determined by the method of Brad-ford, using bovine serum albumin as standard [45]. Purity of proteinsamples was assessed by 12% SDS-PAGE according to standardprotocols [46]. The quaternary structure of the recombinantenzymewas evaluated by gel-filtration on a Superdex™ 200 10/300GL column connected to a FPLC apparatus (GE Healthcare). Themass spectrometry analysis was performed on protein samplesdesalted by RP-HPLC, as previously reported [34,47]. The number ofcysteine residues in protein samples was determined by the Ellmanassay [36,48]. Intracellular glutathione content wasmeasured usingthe EnzyChrome GSH/GSSG assay kit essentially as previously re-ported [49]. Briefly,1million cells from P. haloplanktis cultures weresonicated and homogenised in 50mM phosphate buffer, containing1 mM EDTA. The suspension was centrifuged at 4 �C for 15 min at10,000 � g and the supernatant was deproteinized with 5% meta-phosphoric acid. After centrifugation at 14,000 rpm for 5 min,GSH and GSSG contents were measured on supernatants followingthe manufacturer's protocol. Multiple alignments were performedby using the ClustalW2 on-line tool available at http://www.ebi.ac.uk/Tools/msa/clustalw2 with the BLOSUM matrix and two opengaps settings; all the other parameters were in the default setup.Themodel of the rPhGshA II 3D structure was obtained by using theautomated I-TASSER service available at http://zhanglab.ccmb.med.umich.edu/I-TASSER. The on line procedure yielded the 3D modelon the basis of multiple-threading alignments by LOMETS anditerative TASSER simulations [50]. The molecular model of rPhGshAII was constructed using as a template the crystallographicallyderived coordinates of g-glutamyl-cysteine ligase from E. coli(EcGshA, PDB entry code 2D33) refined to 2.6 Å resolution, sharing42.4% amino acid sequence identity with rPhGshA II. The three

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dimensional model was visualised using PYMOL application soft-ware (www.pymol.org).

3. Results

3.1. Molecular properties of a recombinant form of GshA fromP. haloplanktis

The P. haloplanktis genome contains two genes encoding puta-tive g-glutamyl-cysteine ligases (Ph-gshA I in chromosome I and Ph-gshA II in chromosome II). The amino acid sequence deduced fromthese genes was pair wise-aligned either each other or with thesequence of the corresponding single enzyme from E. coli, EcGshA,whose structural and biochemical properties were extensivelyinvestigated [21,27e29,51,52]. The percentage of amino acid iden-tity between PhGshA I and PhGshA II was 43.2%; similar percent-ages of amino acid identity were obtained from the alignment ofEcGshAwith PhGshA I (44.6%) or PhGshA II (42.4%). Therefore, thesedata could suggest a possible redundancy for g-glutamyl-cysteineligase activity in P. haloplanktis. The strategy followed for studyingthis activity in the cold-adapted microorganism considered theproduction of recombinant forms of both enzymes. However, thefirst one produced through the expression system described in theExperimental section was rPhGshA II. For this reason, the presentwork was focused on the characterisation of the molecular andbiochemical properties of this enzyme, obtained as a recombinantprotein fused to His-tag. Its production was achieved through theE. coli BL21(DE3) strain transformed with the vPh-gshA II vector.The inducible promoter of this heterologous expression system istypically activated by IPTG; however, differently from what re-ported for the production of rPhGshB [37,38], no IPTG inductionwasapparently observed in this case. Indeed, the best experimentalconditions for obtaining rPhGshA II were reached when growth ofthe BL21(DE3)/vPh-gshA II transformant continued for additional 3-h at 30 �C without IPTG induction, after the culture reached thevalue of 0.5 OD600. The presence of a reducing agent, such as TCEP,during purification procedure and storage of rPhGshA II ensured theobtainment of an active form of this enzyme.

The purity of rPhGshA II was controlled by SDS/PAGE analysis,which showed that the protein sample was homogenous and thatits electrophoretic mobility corresponded to a molecular mass of58 kDa, a value coincident with that predicted for the recombinantenzyme (Fig. 1). The molecular mass of rPhGshA II was then

determined by ESI/Q-TOF mass spectrometry after protein desalt-ing by RP-HPLC. The accurate molecular mass was found to be57662.50 Da (not shown), in good agreement with the theoreticalvalue of 57662.57 calculated for the recombinant enzyme, taking inaccount the His-tail and the lack of the first methionine residue.This result excluded the presence of covalent adducts on theenzyme, as previously observed on other recombinant antioxidantenzymes from P. haloplanktis [17,32,34,38]. The molecular mass ofrPhGshA II was also determined by gel filtration chromatography ona Superdex™ 75 10/300 GL column using non-denaturing condi-tions. A single peak was obtained and its elution time, comparedwith that of protein standards, corresponded to an extrapolatedmolecular mass of 54.5 kDa (not shown). This value clearly in-dicates that rPhGshA II behaved as a monomer under non-denaturing conditions, similarly to what reported for EcGshA [51].The molecular characterisation of rPhGshA II also included deter-mination of the free cysteines in the purified protein sample, inorder to assess whether the purification procedure of rPhGshA II inthe presence of TCEP ensured maintenance of these residues in areduced state. Indeed, the Ellman assay, carried out on the purifiedrecombinant enzyme, allowed the determination of 3.0 molcysteine/mol enzyme, in perfect agreement with total three cys-teines counted in the amino acid sequence; therefore, all cysteineresidues of the rPhGshA II sample were present as free thiols in therecombinant protein sample.

3.2. Biochemical properties of rPhGshA II

As described in previous reports, the activity of GshA was usu-ally evaluated through the ADP formed during the ATP-dependentsynthesis of g-Glu-Cys from L-Glu plus L-Cys; in particular, an in-direct spectrophotometric method was employed, which deter-mined the ADP formed through the NADH oxidation in thepresence of pyruvate kinase and lactate dehydrogenase [22]. In thiscase, the direct effect of some parameters, such as temperature, pHor ions, as well as of substrates or inhibitors, on the biochemicalproperties of GshA was impaired by the presence of the other en-zymes. To overcome this difficulty, the activity of rPhGshA II wasdetermined through a direct radioisotopic assay, using the methodsuccessfully employed for measuring the activity of rPhGshB [38].In particular, the assay involved the determination of 32Pi releasedfrom radiolabelled [g-32P]ATP in the presence of the specific sub-strates of reaction (1). The effect of various substrates on the rate of32Pi release promoted by rPhGshA II is reported in Fig. 2. The datashow that the ATPase sustained by this enzyme required thepresence of both specific substrates L-Glu and L-Cys. Indeed, a linearkinetics of ATP hydrolysis was evident in the presence of thesesubstrates, whereas no activity was measured in the presence of L-Glu or L-Cys alone. It was concluded that the ATPase activity pro-moted by rPhGshA II was likely coupled to the synthesis of g-Glu-Cys, as expected from reaction (1). The results shown in Fig. 2 alsoindicate that, in the presence of L-Glu, the substrate L-Cys could bereplaced by L-2-aminobutyric acid, although the rate of ATP hy-drolysis measured in the presence of this substrate was signifi-cantly lower than that determined with L-Cys. On the other hand,other substrates, such as L-2-hydroxy-glutaric acid or N-methyl-L-glutamic acid, could not replace L-Cys. The substitution of L-2-aminobutyric acid for L-Cys was already reported in other GshAactivities, although using a different assay [21,51]. When usingsome preparations of the enzyme with a lower grade of purity, alow and non linear rate of ATP hydrolysis was measured in thepresence of L-Glu alone (not shown). It cannot be excluded that thisfinding was due to traces of triphosphatase contaminating activ-ities present in these protein samples. The activity of rPhGshA IIwas confirmed through the detection of the final product of the

Fig. 1. SDS-PAGE of rPhGshA II. Increasing amounts (1, 3 and 5 mg) of purified rPhGshAII (lanes 1e3) were analysed on a 12% polyacrylamide gel. Migration of molecular massprotein standards (lane M) is reported on the right.

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reaction g-Glu-Cys using the reagent 2,3-naphthalenedicarboxyaldehyde. Indeed, the rate of g-Glu-Cys for-mation by rPhGshA II was of the same order of that measuredthrough the release of 32Pi from [g-32P]ATP. In particular, under thesame experimental conditions the calculated rates were 40.3 molg-Glu-Cys formed mol rPhGshA II�1 min�1 and 101.2 mol ATPhydrolysed mol rPhGshA II�1 min�1. However, for the enzymecharacterisation the radioisotopic method was preferred to thefluorimetric one because of its higher sensitivity.

The pH and ionic dependence of the ATPase activity sustained byrPhGshA II at 15 �C was investigated (Fig. 3). The effect of pH wasstudied in the 6.0e8.6 pH interval, using imidazole$HCl or Tris$HClbuffers supplemented with 20 mM MgCl2 and 150 mM KCl. Thedata reported in Fig. 3A indicate that the rPhGshA II-dependentATPase activity was affected by the pH and that maximum activ-ity was reached in the 7.8e8.6 pH range, using the Tris$HCl buffer. Areaction mixture containing 100 mM Tris HCl, pH 8.0 was consid-ered as representative of optimum pH conditions and was used forthe evaluation of the ionic dependence on the ATPase activitypromoted by rPhGshA II. In order to study the requirement for aspecific divalent cation in the assay, this buffer was supplementedwith 150 mM KCl and increasing concentration of MgCl2 or MnCl2.As shown in Fig. 3B, the presence of a divalent cation in the reactionmixture was required for triggering the ATPase, because no activitywas essentially measured in the absence of Mgþþ or Mnþþ. Amongthese two cations, Mgþþ was by far the most effective one, as itcaused a strong dose-dependent enhancement of the activity up to20 mM MgCl2. Compared to that exerted by Mgþþ, the stimulationprovoked by Mnþþ, if any, was hardly detectable (Fig. 3B); forinstance, the highest level of the Mn-dependent activity, measuredin the presence of 0.5 mM MnCl2, was 34-fold lower compared tothat measuredwith 20mMMgCl2. The effect of monovalent cationson the ATPase activity sustained by rPhGshA II was investigated bysupplementing the 100 mM Tris$HCl buffer at pH 8.0 with 20 mMMgCl2 and increasing concentration of NaCl or KCl. As shown inFig. 3C, full activity was already evident in the absence of NaCl or

KCl and therefore, the presence of a monovalent cation wasapparently dispensable for triggering the ATPase of rPhGshA II.However, the presence of Naþ or Kþ up to 320mMdid not cause anyvariation of the activity. In conclusion, a buffer containing 100 mMTris$HCl, pH 8.0, supplementedwith 20mMMgCl2 and 150mMKCl(buffer C) was selected for the following studies on the ATPaseactivity of rPhGshA II, a choice allowing the comparison with thedata referred to GshAs from other organisms.

To investigate the affinity of rPhGshA II for each substrateinvolved in reaction (1), LineweavereBurk plots of the ATPase ac-tivity were obtained at 15 �C in buffer C using three differentexperimental conditions; in particular, the concentration of oneamong the three substrates ATP, L-Glu or L-Cys was varied, whereasthat of the remaining ones was kept fixed and almost saturating.Three straight lines were obtained, extrapolating to an almostidentical y-axis intercept, a finding indicating that a similarmaximum rate of ATP hydrolysis (Vmax) was reached in the threedifferent experimental conditions (Fig. 4). This kinetic parameterwas then converted into the catalytic constant (kcat) of rPhGshA IIand the values obtained for ATP, L-Glu and L-Cys were 4.1, 3.2 and3.6 s�1, respectively. The LineweavereBurk plots of Fig. 4 allowedthe determination of the different affinity of rPhGshA II for its threesubstrates and the calculated KM values for ATP, L-Glu and L-Cyswere 0.093mM, 2.8 mM and 0.050mM, respectively. Therefore, thecatalytic efficiency of rPhGshA II, evaluated through the kcat/KM

ratio referred to the substrate ATP, was 44 s�1 M�1, a value higherthan that reported for the ATPase activity of rPhGshB (7.1 s�1 M�1)[38]. A summary of the kinetic parameters of rPhGshA II and thecomparison with the corresponding data from rPhGshB is reportedin Table 1.

Previous reports indicated that glutathione, in its reduced GSHor oxidised GSSG form, was able to affect functioning of the en-zymes involved in its biosynthesis [21,38,51,53]. The level of thesecompounds in P. haloplanktis cells was measured. The amount ofGSH and GSSG in a culture of this psychrophile grown at 5 �C in theexponential phase (0.98 A600) was 11.1 ± 0.2 and 1.19 ± 0.06 nmolper million cells, respectively. Similar values, i.e. 11.8 ± 0.3 and1.37 ± 0.05 nmol per million cells, for GSH and GSSG respectively,weremeasured when the culturewas grown up to stationary phase

Fig. 3. pH and ionic requirement for triggering the ATPase activity of rPhGshA II. (A)Effect of pH. The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu and2.5 mM L-Cys in 250 mL final volume of 100 mM Tris$HCl (C) or 50 mM imidazole$HClbuffer (:), at the pH indicated, supplemented with 20 mM MgCl2 and 150 mM KCl. (B)Effect of divalent cations. The reaction mixture contained the same concentration ofrPhGshA II, L-Glu and L-Cys as in A, in 250 mL final volume of 100 mM Tris$HCl, pH 8.0buffer, supplemented with 150 mM KCl and the indicated concentration of MgCl2 (C)or MnCl2 (:). (C) Effect of monovalent cations. The reaction mixture contained thesame concentration of rPhGshA II, L-Glu and L-Cys as in A, in 250 mL final volume of100 mM Tris$HCl, pH 8.0 buffer supplemented with 20 mM MgCl2 and the indicatedconcentration of KCl (C) or NaCl (:). All the reactions were carried out at 15 �C andstarted with the addition of 2 mM [g-32P]ATP (specific radioactivity 3.5 cpm pmol�1).At selected times, 50 mL-aliquots were withdrawn from the reaction mixture andanalysed for the 32Pi released. The data were expressed as the rate of 32Pi release,evaluated from the slope of linear kinetics (r2 values ranging between 0.97 and 0.99).

Fig. 2. Effect of various substrates on the ATPase activity catalysed by rPhGshA II. Thereaction mixture, containing 0.16 mM rPhGshA II in 350 mL of buffer C, was supple-mented with the following substrates: no addition (B); 10 mM L-Glu (◊); 2.5 mM L-Cys (▫); 10 mM L-Glu þ 2.5 mM L-Cys (C); 10 mM L-Glu þ 2.5 mM L-2-aminobutyricacid (:); 10 mM L-Glu þ 2.5 mM L-2-hydroxy-glutaric acid (▵); 10 mM L-Glu þ 2.5 mM N-methyl-L-glutamic acid (7). The reaction was carried out at 15 �C andstarted with the addition of 2 mM [g-32P]ATP (specific radioactivity 3.3 cpm pmol�1).At the times indicated, aliquots were withdrawn and analysed for the 32Pi released, asdescribed in Materials and Methods. The squared correlation coefficient r2 in (C) and(:) was 0.99 and 0.92, respectively, whereas the p-values were lower than 0.0001 and0.005, respectively.

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(7.0 A600). Interestingly, the molar GSH/GSSG ratio in P. haloplanktis,ranging between 9.3 and 8.6, was roughly ten-fold lower than thatusually reported in other bacteria [9]. To investigate the possibleregulation of the biochemical properties of rPhGshA II by GSH orGSSG, the effect of increasing concentrations of these compoundson the ATPase activity of the psychrophilic enzyme was assayed(Fig. 5). A typical dose-dependent inhibition profile was observed inthe presence of GSH and the concentration leading to 50% reduc-tion of the activity (IC50) was above 10 mM. Concerning GSSG, thereduction of activity caused by this compound was not verydifferent from that of GSH, even though the inhibition profileobserved in this case was somehow biphasic. The mechanism ofinhibition by GSH and GSSG was also studied through kineticmeasurements of the activity of rPhGshA II in the absence or in thepresence of each inhibitor (Fig. 6). The effect of the inhibitors on theVmax and KM for ATP is shown in Fig. 6A. Either GSH or GSSG causeda reduction of the Vmax of the reaction, thus excluding that theseglutathione forms could act as competitive inhibitors of the sub-strate ATP;moreover, GSH and GSSG also led to a decrease of the KM

for this substrate, and therefore the binding of these compounds tothe ATP-bound form of the enzyme could be excluded. The effect ofGSH and GSSG on the Vmax and KM for L-Glu is shown in Fig. 6B andalso in this case both inhibitors caused a reduction of the kineticparameters. This behaviour seems to exclude that GSH or GSSGcould competewith L-Glu for the binding to rPhGshA II. On the basis

of the reduction of Vmax and KM observed in the presence of GSH,the average value of the calculated Ki for GSH was 10.8 mM; anapproximately two times higher value was estimated for GSSG.Table 1 also reports the comparison of the inhibition constants ofrPhGshA II and rPhGshB.

It is known that rPhGshB, the enzyme involved in the secondstep of GSH biosynthesis in P. haloplanktis, was the target of a co-valent modification by GSSG and that the S-glutathionylation re-action caused the irreversible inhibition of the enzyme [38]. Tosearch for the occurrence of a potential covalent modification ofrPhGshA II by GSH or GSSG, an aliquot of this enzyme was analysedby ESI/Q-TOF mass spectrometry before and after its incubation for

Table 1Kinetic and inhibition parameters of the ATPase catalysed by rPhGshA II; comparisonwith rPhGshB.

Enzyme Substrate Kinetic parameter at 15 �C Inhibitor Ki (mM) Reference

kcat(s�1)

KM

(mM)kcat/KM

(s�1 mM�1)

rPhGshA II ATP 4.1 0.093 44 GSSG n.d. This workL-Glu 3.2 2.8 1.1 GSH 10.8L-Cys 3.6 0.050 72

rPhGshB ATP 1.85 0.26 7.1 GSSG 10.7 [38]g-Glu-Cys 1.93 0.25 7.7 GSH n.d.Gly 2.03 0.75 2.7

Fig. 5. Inhibition profile of rPhGshA II by GSH and GSSG. The reaction mixture formeasuring the rPhGshA II ATPase in the presence of the indicated concentrations ofGSH (C) or GSSG (-) contained 0.16 mM rPhGshA II, 10 mM L-Glu and 2.5 mM L-Cys in250 mL final volume of buffer C. The reaction, carried out at 15 �C, started with theaddition of 2 mM [g-32P]ATP (specific radioactivity 2.7 cpm pmol�1) and was followedkinetically. Care was taken to add GSH or GSSG just before the addition of [g-32P]ATP.The ATPase activity, evaluated form the slope of linear kinetics (r2 values rangingbetween 0.97 and 0.99) was expressed as a percentage of that measured in the absenceof the glutathione forms.

Fig. 6. Effect of GSH and GSSG on the LineweavereBurk plots of the rPhGshA II ATPase.(A) Affinity for ATP. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys,10 mM L-Glu, without (B) or with 10 mM GSH (C) or 10 mM GSSG (-) in 250 mL finalvolume of buffer C. The reaction started with the addition of the indicated concen-tration of [g-32P]ATP (specific radioactivity 1.3e106.7 cpm pmol�1). (B) Affinity for L-Glu. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys, the indicatedconcentration of L-Glu, without (B) or with 10 mM GSH (C) or 10 mM GSSG (-) in250 mL final volume of buffer C. The reaction started with the addition of 2 mM [g-32P]ATP (specific radioactivity 1.3 cpm pmol�1). All the reactions were carried out at 15 �Cand were followed kinetically through the analysis of the amount of 32Pi released fromaliquots withdrawn at appropriate times. The values of v (mol ATP hydrolysedmol rPhGshA II�1 min�1) were calculated from the slope of linear kinetics (r2 valuesranging between 0.93 and 0.99) and then reported in the LineweavereBurk plots. Ther2 values of double reciprocal plots ranged between 0.96 and 0.99 and the p-valueswere all lower than 0.005.

Fig. 4. LineweavereBurk plots for the three substrates of rPhGshA. (A) Affinity for ATP.The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu and 2.5 mM L-Cys in250 mL final volume of buffer C. The reaction started with the addition of the indicatedconcentration of [g-32P]ATP (specific radioactivity 6.2e552.4 cpm pmol�1). (B) Affinityfor L-Glu. The reaction mixture contained 0.16 mM rPhGshA II, 2.5 mM L-Cys and theindicated concentration of L-Glu in 250 mL final volume of buffer C. The reaction startedwith the addition of 2 mM [g-32P]ATP (specific radioactivity 3.3 cpm pmol�1). (C)Affinity for L-Cys. The reaction mixture contained 0.16 mM rPhGshA II, 10 mM L-Glu andthe indicated concentration of L-Cys in 250 mL final volume of buffer C. The reactionstarted with the addition of 2 mM [g-32P]ATP (specific radioactivity 3.9 cpm pmol�1).All the reactions were carried out at 15 �C and were followed kinetically through theanalysis of the amount of 32Pi released from aliquots withdrawn at appropriate times.The values of v (mol ATP hydrolysed mol rPhGshA II�1 min�1) were calculated from theslope of linear kinetics (r2 values ranging between 0.93 and 0.99) and then reported inthe LineweavereBurk plots. The r2 values of double reciprocal plots were 0.97 (panelA), 0.94 (panel B) and 0.66 (panel C), whereas the p-values were lower than 0.0001(panels A and B) or lower than 0.001 (panel C).

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15 min at 15 �C with 3.6 mM GSH or GSSG. The molecular mass ofuntreated rPhGshA II (57662.50 Da) remained essentially un-changed after the treatment with GSH (57662.82 Da). On the con-trary, an ion signal with a molecular mass of 57967.50 Da wasdetected following the incubation of rPhGshA II with GSSG, corre-sponding to a mono-glutathionylated form of rPhGshA II. Indeed,the 305 Da extra mass was in agreement with that expected if theenzyme formed one mixed disulfide with glutathione, thus indi-cating that rPhGshA II was subjected to S-glutathionylation reactionby GSSG. Interestingly, the mono-glutathionylated form of rPhGshAII was detected evenwhen incubationwith GSSG was performed inthe presence of 10 mM L-Glu and 2.5 mM L-Cys (not shown).

3.3. Identification of the Cys residue target of the S-glutathionylation reaction in rPhGshA II

Three cysteinyl residues are present in the rPhGshA II sequenceat positions 107, 357 and 386. To identify the Cys residue target ofthe S-glutathionylation reaction, a comparative MALDI-TOF MSmapping of tryptic peptides obtained from rPhGshA II samplesincubated in the absence or in the presence of GSH or GSSG wascarried out. The two signals at m/z 689.3 and 1630.8 were clearlyidentified in spectra of the tryptic digests of untreated rPhGshA IIand rPhGshA II incubated with GSH (see solid arrows in Fig. 7).These ions correspond to tryptic peptides with sequence positions380e385 (theoretical [M þ Hþ]: 689.36) and 386e400 (theoretical[M þ Hþ]: 1630.81), respectively. Following incubation with GSSG,these peaks disappeared, whereas an additional ion signal with m/z 2606.2, was specifically detected in this spectrum (dotted arrowin Fig. 7). This ion signal corresponds to the glutathionylatedtryptic peptide 380e400 (theoretical [M þ Hþ]: 2606.15) with amissing tryptic cleavage on Arg 385, likely occurring as a conse-quence of the S-glutathionylation of the neighbouring Cys residueat position 386.

3.4. Effect of temperature on the biochemical properties of rPhGshAII

The cold adaptation of rPhGshA II was investigated by analysingthe thermodependence of activity and stability exhibited by thepsychrophilic enzyme. To this aim, the ATPase reaction catalysed byrPhGshA II was studied in the 10e35 �C interval, in order todetermine the effect of temperature on the kinetic parameters Vmaxand KM for ATP. The temperature dependence of the Vmax shown inFig. 8 indicates that rPhGshA II was already active at 10 �C; however,the Vmax moderately increased up to 28 �C, and then decreasedabove this temperature. Also the KM for ATP showed a similarmoderate increasewith temperature (not shown). To better analysethe thermodependence of the reaction velocity, the values of Vmaxwere converted into kcat and then analysed by the Arrheniusequation. As shown in the inset to Fig. 8, the plot was linear in the10e28 �C interval and the calculated low value of the energy ofactivation (Ea, 42.3 kJ mol�1) was in agreement with the modesteffect of temperature on enzyme activity.

The significant decrease of the Vmax between 30 and 35 �C, asemerged from the study of the thermodependence of the rPhGshAII activity, could suggest the onset of a heat inactivation process ofthe psychrophilic enzyme in this temperature range. A heat inac-tivation profile realised on rPhGshA II samples incubated for 10 minat different temperatures indicated that the enzyme was rathersensitive to heat, because its half-inactivation occurred at nearly35 �C (not shown). Therefore, the thermal stability of rPhGshA IIwas analysed through heat inactivation kinetics carried out attemperatures ranging between 25 and 41 �C. At each temperaturethe kinetics was linear, when analysed according to the equation ofa first-order kinetics, and this allowed the calculation of the cor-responding inactivation rate constants (kin). The values of kin werethen treated according to the Arrhenius equation to determine theenergetic parameters of the heat inactivation process. As shown in

Fig. 7. Magnification of MALDI-TOF spectra of tryptic digests of rPhGshA II incubated without or with GSH or GSSG, as described in the text. Signal ions of the unmodified trypticpeptides in the untreated or GSH-treated sample are indicated by solid arrows; sequence of these peptides is also reported. The tryptic peptide diagnostic of the S-glutathionylationon Cys 386 in the GSSG-treated sample is indicated by a dotted arrow.

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Fig. 9, the Arrhenius plot was linear in the interval of temperatureanalysed and the calculated value of Ea (296.9 kJ mol�1) was ratherhigh for a psychrophilic enzyme.

4. Discussion

This work enlarges the information on the enzyme system forGSH biosynthesis in P. haloplanktis, to investigate the role played bythis cellular thiol in a cold-adapted organism. Under this concern,the production of this thiol in P. haloplanktis cells, although alreadyhypothesised by the presence of glutathionylated proteins in agrowing culture of this bacterium [17], was directly demonstrated.Furthermore, the lower GSH/GSSG ratio found in this psychrophilewith respect to other bacterial sources could be related to theadaptation of P. haloplanktis at a constant mild oxidant growthcondition. Previous studies described the characterisation of a re-combinant form of glutathione synthetase (rPhGshB), the enzymeinvolved in reaction (2) leading to GSH formation in this psychro-phile [37,38]; now, also the enzyme activity catalysing reaction (1),g-glutamyl-cysteine ligase, has been studied in this organism. Thework reports the molecular properties and functioning of rPhGshAII, a recombinant product from the Ph-gshA II gene located inchromosome II of P. haloplanktis.

When assessing the best experimental conditions for the pro-duction of rPhGshA II, some differences were noted with the het-erologous production of rPhGshB, in spite of the usage of a similarIPTG-inducible expression system. In particular, growth of theculture for obtaining rPhGshA II was carried out at 30 �C, a lowertemperature compared to that of the rPhGshB production, and noIPTG induction was required. Furthermore, the presence of thereducing agent TCEP during purification and storage of rPhGshA IIwas essential for obtaining the enzyme in an active form. Indeed, ifTCEP was omitted during the purification procedure, the purifiedsample of rPhGshA II was poorly active; however, its activity couldbe partially restored upon the addition of a reducing agent, such asdithiothreitol, in the assay buffer. Finally, in some cases, the

preparation of the protein sample was complicated by the precip-itation of the heterologous product either during expression orpurification. By studying the molecular properties of rPhGshA II, itemerged that the enzyme had the expected size and acted as amonomer, similarly to what was reported for EcGshA [51]. Differ-ently from EcGshA, containing nine cysteines with two of themforming a disulfide bridge [52], the active form of rPhGshA II con-tains three cysteine residues, all present as free thiols. The pair-wisealignment between the amino acid sequence of PhGshA II andEcGshA revealed that only two cysteines are conserved betweenPhGshA II and EcGshA and none of them corresponded to the res-idues forming the disulfide bridge in EcGshA.

The functional properties of rPhGshA IIwere studied through theATPase reaction coupled to the synthesis of g-Glu-Cys from L-Gluplus L-Cys (reaction (1)). Indeed, the radioisotopic method chosenallowed a direct evaluation of the effect of various molecules andphysico-chemical parameters on the psychrophilic enzyme. Con-cerning the substrate requirement for triggering the ATPase ofrPhGshA II, the results showed that this activity occurred only whenboth L-Glu and L-Cyswere present in the reactionmixture; however,the substrate L-Cys could be replaced by L-2-aminobutyric acid, afinding already reported for EcGshA and the correspondingmammalian enzyme [21,51]. Concerning the best pH and ionicconditions for triggering the ATPase activity of rPhGshA II, theenzymewas full active in amoderatelyalkaline buffer and requiredadivalent cation. In particular, the study revealed the high selectivityof the psychrophilic enzyme for the divalent cation Mgþþ, added athigh concentration for reaching high levels of activity, becauseMnþþ was essentially unable to replace Mgþþ in the stimulation ofthe activity. Therefore, compared to the corresponding enzyme fromother bacteria or eukarya, where a possible Mgþþ/Mnþþ replace-ment in the activity was observed [28e30], the psychrophilicrPhGshA II appeared to be more selective for the requirement of

Fig. 9. Arrhenius plot of the heat inactivation process of rPhGshA II. The values of therate constant of heat inactivation (kin, s�1) were obtained from inactivation kineticscarried out within the 25e41 �C interval. To this aim, a 1.0 mM solution of rPhGshA II in20 mM Tris$HCl, pH 7.8 was incubated at each temperature and at selected times,aliquots were withdrawn from the incubation and immediately chilled on ice for30 min. The residual ATPase activity of these enzyme samples was measured kineti-cally at 15 �C in buffer C supplemented with 10 mM L-Glu and 2.5 mM L-Cys essentiallyas indicated in the legend to Fig. 3 (specific radioactivity [g-32P]ATP, 1.7 cpm pmol�1).After data analysis according to first-order kinetics (r2 values ranging between 0.93and 0.99), the kin values were treated according to the Arrhenius equation (r2 value0.99, p-value lower than 0.0001).

Fig. 8. Effect of temperature on the ATPase activity of rPhGshA II. At each indicatedtemperature the kinetic parameters of the ATPase were determined essentially asindicated in the legend to Fig. 4A. In particular, the reaction started with the addition offive different concentrations of [g-32P]ATP (specific radioactivity8.9e157.5 cpm pmol�1) within the 11e201 mM interval. Values of Vmax were extrap-olated from LineweavereBurk plots and reported as a function of temperature. Afterconversion of the Vmax into kcat (s�1) values, an Arrhenius plot was built and the linearfit of the data was drawn in the 10e28 �C interval (inset panel, r2 value 0.98, p-valuelower than 0.0001).

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Mgþþ. Finally, compared to that reported for rPhGshB [38], thehigher Mgþþ concentration for triggering the rPhGshA II activitycannot be required for complexing the substrate ATP. The presentresults do not exclude that Mgþþ ions could have an effect onconformation and dynamics of the enzyme.

The natural habitat of P. haloplanktis is the Antarctic coastal seawater and therefore, this microorganism is adapted to live at tem-peratures near to 0 �C. However,most of the kinetic data in thisworkwere obtained at 15 �C, a temperature commonly used formeasuring and comparing the activity of enzymes fromP. haloplanktis [32,34,36,38], because it represents the optimumgrowth temperatureof this psychrophile [54]. In anycase, rPhGshA IIdisplayed a well measurable activity even at 10 �C, a finding inaccordance with functioning of this enzyme in the specific coldhabitat of P. haloplanktis. In other organisms it was reported thatreaction (1) is the rate-limiting step of the whole process of GSHbiosynthesis [26]. However, this seems not to be the case ofP. haloplanktis, because the catalytic efficiency of reaction (1) wasnearly 6-fold higher than that calculated for reaction (2), measuredin the same experimental conditions of temperature and ATP con-centration [38]. The apparent discrepancywith the other organismscould be also related to the different method for measuring the ac-tivity of the corresponding enzymes and/or to the fact that a sub-optimal g-glutamyl-cysteine ligase activity could be measured inother organisms when using the substrate L-2-aminobutyrate inplace of L-Cys. The differences could also derive from the specificionic requirement for maximum activity of the two enzymes, as forinstance observed for the diverse Mgþþ optimum for rPhGshA II orrPhGshB. The different affinity of rPhGshA II for its three substrates

wasevaluated through theKMvalues forATP, L-Glu and L-Cys. Indeed,the psychrophilic enzyme had an affinity for L-Glu significantlylower than that for L-Cys or ATP, a finding similar to what reportedfor the corresponding enzyme from E. coli or kidney rat [22,51].

The study on the inhibitory properties of GSH and GSSG onrPhGshA II was prompted by previous reports, pointing to the effectof these glutathione forms on enzymes involved in GSH biosyn-thesis. Indeed, GSH exerted a feedback inhibition on the activity ofg-glutamyl-cysteine ligase from E. coli or kidney rat [21,27,51]; thedata also suggested the possible involvement of free cysteine res-idues in the inhibition of the eukaryal enzyme, as well as the pro-tective role displayed by L-Glu. Concerning the second enzymeinvolved in GSH biosynthesis, it was demonstrated that the activityof glutathione synthetase from E. coli or P. haloplanktis wasinhibited by GSSG [38,53]. Furthermore, in the case of that psy-chrophilic enzyme, the formation of a mixed disulfide bridge withglutathione was demonstrated [38]. The inhibition profile of theATPase activity of rPhGshA II by GSH and GSSG showed that alsothis enzyme was inhibited by both glutathione forms. However,none of them could be considered as a strong inhibitor of rPhGshAII, and among the two inhibitors, only GSH gave a typical dose-dependent inhibition profile. An insight in the mechanism of in-hibition by GSH and GSSG was realised through LineweavereBurkplots and the results obtained seemed to exclude that these com-pounds acted as competitive inhibitors of rPhGshA II. In fact, eitherGSH or GSSG caused a decrease of the Vmax of the reaction, whilereducing also the KM for ATP or L-Glu. The possible reactivity ofrPhGshA II towards these glutathione forms was considered. Underthis concern, the ESI/Q-TOF mass spectrometry data clearly indi-cated that GSSG caused the covalent modification of rPhGshA II,whereas GSH was ineffective. Interestingly, a complete mono-glutathionylation of the enzyme was obtained after its incubationwith a GSSG concentration, that otherwise caused only a lowinhibitory effect on the enzyme. A similar result was obtained evenwhen the GSSG treatment was realised in the presence of L-Glu andL-Cys, thus excluding that these substrates could protect rPhGshA IIfrom glutathionylation. The MALDI-TOF MS mapping of the cys-teinyl residue target of the covalent modification allowed itsidentification as Cys 386. To have an information on the location ofthis residue in the enzyme structure, a three-dimensional model ofrPhGshA II, obtained by homology modelling using EcGshA as atemplate, was constructed (Fig. 10). The picture shows that Cys 386is located within a loop region at the protein surface, thus sup-porting the higher accessibility of this cysteinyl residue to GSSG.Furthermore, this residue is located in a region apparently far fromthe core of the molecule containing the active site, a findingexplaining the minimum effect on enzyme activity. Consideringthat only GSSG covalently modified the enzyme and that thepresence of a mixed disulfide caused only a slight reduction of thecatalytic properties of rPhGshA II, the atypical dose-dependent in-hibition profile by GSSG could be explained by the combination oftwo aspects. In the presence of a low GSSG concentration, a dose-dependent inhibition profile was observed, because the modifica-tion of the enzyme was probably understoichiometric, alsoconsidering the minimum reaction time required for evaluating the

Fig. 10. Three-dimensional model of rPhGshA II obtained by homology modelling. Theresidue Cys 386 located at the protein surface and identified as glutathionylatedfollowing treatment with GSSG is indicated by the red arrow.

Table 2Energy of activation and related thermodynamic parameters of the enzymes involved in the biosynthesis of glutathione in Pseudoalteromonas haloplanktis.

Process Enzyme Ea (kJ mol�1) DH*a (kJ mol�1) DS*a (J mol�1 K�1) DG*a (kJ mol�1) Reference

ATPase activity rPhGshA II 42.3 39.9 �95.4 67.4 This workrPhGshB 75.0 72.6 11.3 69.3 [38]

Heat inactivation rPhGshA II 296.9 294.3 651.2 93.6 This workrPhGshB 208.0 205.3 332.1 98.0 [38]

a Values calculated at 15 �C for both enzymes for the parameters referred to ATPase activity; values calculated at 35 �C or 50 �C for rPhGshA II or rPhGshB, respectively, forparameters referred to heat inactivation.

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inhibition; at high GSSG concentration, the covalentmodification ofthe enzymewas complete and therefore, the dose-dependence waslost. On the other hand, GSH appeared to be a typical reversibleinhibitor of rPhGshA II, as it gave a normal dose-dependent inhi-bition profile. Indeed, GSH did not cause the covalent modificationof the enzyme and its inhibitory properties were due to a non co-valent enzymeeinhibitor interaction, probably regarding a bindingpocket different from the active site of the enzyme.

Cold-adapted microorganisms possess enzymes adapted tofunction at low temperatures, a fruitful property for assessing thepossible biotechnological application of these macromolecules. Forinstance, enzymes isolated from psychrophilic sources usuallyadjust their kinetic parameters to function in the cold, a findingextremely important for the optimisation of a biotechnologicalprocess and for lowering the related energetic costs [55e57].Indeed, several studies reported that the cold-adaptation of psy-chrophilic enzymes could imply an increase of the local flexibility intheir catalytic sites and/or a decrease of the overall protein stability[35,36,58e64]. The studies on the effect of temperature on therPhGshA II activity indicated that this enzyme possessed a typicalcold adaptation. Indeed, rPhGshA II was already active at 10 �C, andwhen studying the thermodependence of its kinetic parameters,only a modest effect of temperature was observed. Indeed, both theVmax and the KM of rPhGshA II moderately increased with temper-ature between 10 and 28 �C and, as a consequence, the catalyticefficiency of the psychrohilic enzyme remained approximatelyconstant up to 28 �C; above this temperature, the enzyme activitystarted to decrease, probably for the onset of a heat-inactivationprocess. The moderate thermodependence of rPhGshA II was alsoproved by the low value of Ea (42.3 kJ mol�1) determined for theenzyme activity. This behaviour is dissimilar from that reported forrPhGshB, endowed with a significantly higher value of Ea(75.0 kJ mol�1) [38]. The difference between these enzymes is alsoevident from the comparison of their thermodynamic parametersreported inTable 2. Indeed, the lower enthalpic barrier of the ATPasecatalysed by rPhGshA II was counteracted by a significantly unfav-ourable entropic factor compared to that of rPhGshB. On the otherhand, the two enzymes possess similar values of DG*. All thesefeatures make rPhGshA II more similar to a typical psychrophilicenzyme, usually endowed with a low thermophilic behaviour.

The studies on the heat inactivation of rPhGshA II confirm thecold-adaptation of this enzyme. Indeed, rPhGshA II was rathersensitive to a modest heat treatment, because 50% of enzyme ac-tivity was lost after an incubation of rPhGshA II for 12.6 min at35 �C. Conversely, rPhGshB had a significantly higher heat resis-tance, because the activity of this enzyme was halved after an in-cubation for 11.4 min at 50 �C (not shown). These results confirmthat the decrease of enzyme activity observed for rPhGshA II attemperatures higher than 28 �C could be due to the onset of a heatinactivation process. The low heat resistance of this enzyme wasalso investigated through the determination of the energetic pa-rameters of the heat inactivation process. Surprisingly, the calcu-lated value of Ea (296.9 kJ mol�1) was rather high for apsychrophilic enzyme, even when compared with the corre-sponding value of 208.0 kJ mol�1 reported for rPhGshB, endowedwith a higher heat resistance [38]. The comparison of the thermalstability between rPhGshA II and rPhGshB was extended to theother thermodynamic parameters of the heat inactivation process(Table 2). In this case, the higher enthalpic barrier of the heatinactivation process of rPhGshA II compared to that of rPhGshB wascounteracted by a more favourable entropic factor. However, inspite of the great variations observed in temperature range forobserving heat inactivation, as well as in the correspondingenthalpic and entropic factors, the two enzymes involved in GSHbiosynthesis of P. haloplanktis possessed similar values of DG*.

5. Conclusions

Glutathione biosynthesis occurs through a two-step reactionsystem conserved among bacteria and eukarya and usuallyrequiring two distinct activities, g-glutamyl-cysteine ligase andglutathione synthetase. In aerobic cold adapted microorganisms,such as P. haloplanktis, GSH plays a crucial role in the regulation ofgrowth and survival of the microorganism. Together with theprevious characterisation on glutathione synthetase (rPhGshB), thepresent study on a g-glutamyl-cysteine ligase activity fromP. haloplanktis (rPhGshA II) extends the knowledge on the firstenzyme system leading to GSH biosynthesis in a cold-adapted or-ganism. The investigation regarded the pH and ionic conditions fortriggering the activity, substrate specificity and affinity, inhibitors,thermodependence and heat stability. The comparison with thedata related to rPhGshB revealed the different features of the cold-adaptation of these enzymes. However, P. haloplanktis possessesanother putative g-glutamyl-cysteine ligase activity (PhGshA I) andit will be interesting to evaluate if the presence of a redundantactivity in a psychrophilic organism is useful for facing differentand specific requirements occurring during the growth underextreme conditions.

Conflict of interest

The Authors declare no conflict of interest.

Acknowledgements

This workwas financially supported by grants awarded byMIUR(PRIN 2009, prot. 2009P2HZZ7_001 to E. De Vendittis; PRIN 2009,prot. 2009P2HZZ7_002 to M. Masullo; PRIN 2009, prot.2009P2HZZ7_003 to G. Raimo), Rome, Italy. V. Severino was sup-ported by a post-doctoral fellowship awarded from the Institute ofBiostructures and Bioimaging, CNR, Naples, Italy.

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