Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition

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Plant

Science

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p l a n t s c i

Plastidic

P2

glucose-6P

dehydrogenase

from

poplar

is

modulated

by

thioredoxin

m-type:

Distinct

roles

of

cysteine

residues

in

redox

regulation

and

NADPH

inhibition

Manuela

Cardi

a

_,

_Mirko

_Zaffagnini

b

_,

_Alessia

_De

_Lillo

a

_,

_Daniela

_Castiglia

a

_,

Kamel

Chibani

c,d,1

_,

_José

_Manuel

_Gualberto

e

_,

_Nicolas

_Rouhier

c,d

_,

_Jean-Pierre

_Jacquot

c,d

_,

Sergio

Esposito

a,∗

a_Dipartimento_di_Biologia,_Univ._di_Napoli_“Federico_II”,_I-80126_Napoli,_Italy b_Dipartimento_di_Farmacia_e_{Biotecnologie,}_Univ._di_Bologna,_I-40126_Bologna,_Italy

c_Université_de_Lorraine,_UMR₁₁₃₆_Interactions_{Arbres/Microorganismes,}_Faculté_des_Sciences_et_{Technologies,}_54506,_{Vandoeuvre-lès-Nancy,}_France d_INRA,_UMR₁₁₃₆_Interactions_{Arbres/Microorganismes,}_Centre_INRA_Nancy_Lorraine,_54280,_Champenoux,_France

e_Université_de_Strasbourg,_Institut_de_Biologie_Moléculaire_Des_Plantes_(IBMP),_CNRS-UPR_2357,₆₇₀₈₄_Strasbourg,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received13June2016

Receivedinrevisedform1August2016 Accepted7August2016

Availableonline8August2016 Keywords:

Cysteine

Oxidativepentosephosphatepathway Populus

Thioredoxins

a

b

s

t

r

a

c

t

AcDNAcodingforaplastidicP2-typeG6PDHisoformfrompoplar(Populustremulaxtremuloides)has

beenusedtoexpressandpurifytohomogeneitythematurerecombinantproteinwithaN-terminus

His-tag.Thestudyofthekineticpropertiesoftherecombinantenzymeshowedaninvitroredoxsensing

modulationexertedbyreducedDTT.Theinteractionwiththioredoxins(TRXs)wastheninvestigated.

Fivecysteinetoserinevariants(C145S–C175S–C183S–C195S–C242S)andavariantwitha

dou-blesubstitutionforCys175_and_Cys183_{(C175S/C183S)}_have_been_generated,_puriﬁed_and_{biochemically}

characterizedinordertoinvestigatethespeciﬁcrole(s)ofcysteinesintermsofredoxregulationand

NADPH-dependentinhibition.

Threecysteineresidues(C145_,_C194_,_C242₎_are_suggested_to_have_a_role_in_controlling_the_NADP+_access

totheactivesite,andinstabilizingtheNADPHregulatorybindingsite.

OurresultsalsoindicatethattheregulatorydisulﬁdeinvolvesresiduesCys175_and_Cys183_in_a_position

similartothoseofchloroplasticP1-G6PDHs,butthemodulationisexertedprimarilybyTRXm-type,in

contrasttoP1-G6PDH,whichisregulatedbyTRXf.

Thisunexpectedspeciﬁcityindicatesdifferencesinthemechanismofregulation,andredoxsensingof

plastidicP2-G6PDHcomparedtochloroplasticP1-G6PDHinhigherplants.

1. Introduction

Glucose-6-phosphate dehydrogenase (G6PDH – EC 1.1.1.49) catalyzes the oxidation of glucose-6-phosphate (G6P) to 6-phospho-glucono-␦-lactone, with the concomitant reduction of

Abbreviations: DTT, dithiothreitol; EDTA, ethylene diaminetetraacetic acid; OPPP, oxidativepentose phosphate pathway; Tris, tris (hydroxymethyl) aminomethane;TRX,thioredoxin.

∗ Correspondingauthorat:DipartimentodiBiologia,UniversitàdiNapoli “Fed-ericoII”,ComplessoUniversitariodiMonteSant’Angelo,ViaCinthia4,80126Naples, Italy.

E-mailaddress:sergio.esposito@unina.it(S.Esposito).

1 _Present_address:_{CEITEC—Central}_European_Institute_of_{Technology, ˇZerotínovo}

nám.9,60177Brno,CzechRepublic.

NADP+_to_NADPH._This_enzyme_is_the_key_regulator_of_the Oxida-tivePentosePhosphatePathway(OPPP),aubiquitousmetabolic pathwayinEukaryathatprovidesanalternativerouteto glycoly-sisforG6Poxidation[1].TherearegenerallyfourG6PDHgenesin photosyntheticorganisms(onecodingforacytosolicproteinand thethreeothersforchloroplasticisoforms)asreportedrecentlyin Heveabrasiliensisby[2].

Inthelastyears,considerableworkhasbeenmadeto investi-gatethebiochemicalandregulatoryfeaturesofdifferentG6PDH isoforms fromhigher plants: cytosolic (Cy-G6PDH), chloroplas-tic(P1-G6PDH)andplastidicisoforms(P2-G6PDH),thelasttwo beingexpressedinphotosyntheticandnon-photosynthetictissues, respectively[1,3,4].

Cy-G6PDHwas demonstrated toberedox-insensitive, and a recent studyby[5] demonstratedthat thecytosolic Arabidopsis

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abscissicacid(ABA)respondingpathwayhasbeenrecently con-ﬁrmedintomatoplantssubjecttodrought[6].Cy-G6PDHhasalso beenreportedashavingapositiveroleinresponsetoabioticstress insugarcane[7].

Similarphosphorylationmechanism(s)and associatedkinase havenotbeenidentified,orlinked,toeitherP1-orP2-G6PDHsso far.Infact,theactivityofP1-G6PDH(chloroplastic)isredox sen-sitive,beinginhibitedinvitrobyreduceddithiothreitol(DTT)in adose-andtime-dependentmanner[8].Theredoxregulationof P1-G6PDHthroughdithiol-disulfideexchangereactionswaslater confirmedbothinplants[8,9]andalgae[10].

Redoxmodulationinvolvesthioredoxins(TRXs)asredox cata-lystsandallowsG6PDHinhibitionunderreducingconditions(i.e. underillumination),andfullactivationinthedark,whenthe pro-teinisoxidized.Morerecently,theformationofadisulﬁdebond betweenCys149_and_Cys157_in_Arabidopsis_P1-G6PDH_was demon-stratedtobecrucialinthepositioningofArg131_,_playing_a_central roleinthedockingofNADP+_in_the_binding_site_of_the_{chloroplastic} isoform[11].

P1-G6PDHistheonlyplastidicenzymeredox-regulatedinsuch awaythatitsactivityincreasesinthedark,contrarytowhathas beendescribedforseveralenzymesoftheCalvin-Bensoncycle[12]. Thisoppositeregulationisbelievedtopreventcarbohydrate break-downanddegradationofsugarsbyOPPPinthelight,whichcould competewithphotosyntheticCO2ﬁxation,thuswastingenergyin afutileway[1,13].

Thephysiological roles of chloroplasticP1-G6PDH and plas-tidicP2-G6PDHaredistinct.ThechloroplasticP1-G6PDHisoform provides NADPH in photosynthetic tissues in the dark, when theCalvin-Benson cycle is halted, thus sustaining basal carbon metabolisminthenight.Incontrast,theplastidicP2-G6DPHplays akeyrole inplantrootmetabolism,beinginvolved indifferent basalprocesses, suchas furnishingof intermediatesfor nucleic acidmetabolisminheterotrophicconditions;orduringnitrogen assimilation[14–16];orinresponsetostress[17–19].

Thegenescodingforthesechloroplasticandplastidicisoforms clusterintwodistinctclassesandthecorrespondingisoformscan bedistinguishedbytheirafﬁnitiestowardssubstrateand cofac-tor(G6PandNADP+_),_and_the_extent_of_their_inhibition_by_NADPH at least in potato and barley [3,8,13,20]. It has been generally observed that P1-G6PDH is strongly inhibited by NADPH (e.g.

lowKiNADPH),whereas P2-G6PDHisoformsaresigniﬁcantly less

sensitivetoNADPHinhibition[1,3,20,21].Despite theirmarked differencetowardsNADPHinhibition[13],theconsequenceand inﬂuenceoftheredoxstateontheirresponsetoNADPHarenot clearlydeﬁnedyet.

Inthisstudy,weinvestigatedtheredoxsensitivityofplastidic P2-G6PDHandtheinfluenceoftheredoxstateonenzymeactivity. Todo that,we have produced and purified tohomogeneity wild-typeandcysteine variantsofP2-G6PDH andcharacterized themwithrespecttodithiol/disulfideexchangereactions, NADPH-dependentinhibition and specificity towards differentplastidic TRXs.

AmodelfortheregulationoftheP2-G6PDHisoforminhigher plantsisprovided.

2. Materialsandmethods

2.1. Cloning,expressionandpuriﬁcationofpoplarP2-G6PDHand site-directedmutagenesis

Thesequence encoding the plastidicP2-G6PDH from poplar (Potri.001G059900)wasampliﬁedfromaPopulustremulax

tremu-tionsites(underlinedintheprimers)ofpET15bexpressionvector (Novagen).Thesequence encodesaproteindeprivedoftheﬁrst 66aminoacidscorrespondingtotheputativetargetingpeptide,as comparedtootherknownplastidicP2-G6PDHfromhigherplants (Fig.1A).InthepET15b/P2-G6PDHconstruct,thecDNAsequence forP2-G6PDHwasinframewithanN-terminalHis-tag.

Usingtwocomplementarymutagenicprimers,the5cysteines (Cys145_,_Cys175_,_Cys183_,_Cys194 _and_Cys242₎_of_PtP2-G6PDH_were individuallysubstitutedintoserines,andadoublemutantinwhich Cys175₋_Cys183_are_mutagenized_into_serines_was_also_generated. TheprimersusedarelistedinSupplementalTable1.Allplasmids weresequencedtochecktheintroductionofthedesiredmutations andverifythatnoadditionalmutationswereintroduced.

Forproteinexpression,E.coliBL21(DE3)pSBETstrainwas chem-icallytransformedwiththedifferentrecombinantvectorsallowing expressionofPtP2-G6PDHWTandcysteinemutants P2-G6PDH-C145S (C145S), P2-G6PDH-C175S (C175S), P2-G6PDH-C183S (C183S),P2-G6PDH-C193S(C193S),P2-G6PDH-C242S(C242S),and P2-G6PDH-C175S/C183S(C175S/C183S).Singlecoloniesresistant toampicillinandkanamycinweregrownin100mlofLBmedium inthepresenceofampicillin(50mg/ml)andkanamycin(50mg/ml) overnightat37◦Cinaerobiosis.Afterincubation,theculturewas then transferred in 2.5l of LB medium. When OD600 reached 0.6, protein expressionwas inducedby 100␮M isopropyl- ␤-d-thiogalactopyanoside.Bacteriaweregrownat20◦Covernightwith continuous gentle shaking, and collected by centrifugation for 20minat7000xg.Thepelletwasresuspendedinbuffer[50mM potassiumphosphate,0.3MNaCl,10mMimidazole,(pH8.0)and cellslysedbysonication.Bacterialysateswerecentrifuged45min at20,000xg.Allthesubsequentstepswereperformedat2–6◦C withthesupernatant.

Theextractwasﬁltered(0.22␮m)andappliedontoaGE Health-careHiTrapFFcrudecolumn(5ml)connectedtoanAKTAPrime plussystem(GEHealthcare).Proteinpuriﬁcationwasperformed accordingtothemanufacturer’sinstructions.

Elutedfractions (1ml) wereassayedfor G6PDH activityand activefractionswerecollected,pooledanddesaltedusingPD-10 columns(GeHealthcare)in30mMTris-HCl,100mMNaCl,5% glyc-erol, pH8.0.Proteinconcentrations weredeterminedusingthe Coomassieproteinassaybased(Bio-Rad),withbovineserum albu-minasthestandard.

Thepuriﬁedenzymeshoweda speciﬁcactivityof8000Umg prot−1 (Table1), andmaintained97%ofitsactivityafter7days, whenstoredat−80◦_C._It_should_be_noted_that_the_His-tag_did_not affectthekineticparametersoftherecombinantenzyme[22]. 2.2. IntracellularlocalizationofPtP2-G6PDH

TheG6PDHcodingsequencewasampliﬁedbyPCRwithprimers indicated in SupplementalTable 1 and cloned in the NcoI and BamHIsitesofpCK-GFP3vector,upstreamandinframewiththe GFPcodingsequence.YoungleavesofNicotianabenthamianawere transfectedbybombardmentwithaBiolisticPDS-1000/HeTM Sys-tem(BioRad).After18h,theﬂuorescencesofGFPandchlorophyll wereobservedat505–540nmandbeyond650nm,respectively, afterexcitationat488nmonaZeissLSM700confocalmicroscope. 2.3. Electrophoresisandwesternblottinganalysis

SDS-PAGEwasperformedusinga10%polyacrylamide resolv-inggelwitha4%stackinggel(Bio-RadMini-Protean).Forwestern blotting,separatedpolypeptidesweretransferredfromgelstoa Hybondmembrane(GeHealthcare).Themembranewasthen incu-batedwithprimarypotato antibodiesforP1-G6PDH (1:15.000),

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Fig.1.A–AminoacidsequencealignmentofdifferentplantG6PDHsequences.Mainmotifsareindicatedintheupperlineinboldblack.Highlyconservedresiduesare highlightedinlightgrey;theRossman-fold,theactivesite,andtheNADP+_binding_site_are_in_bold_white_letters,_highlighted_in_black;_cysteine_residues_are_in_bold_white_on

greyhighlighting.B–Phylogenetictreeofcytosolic,chloroplasticandplastidialG6PDHplantisoforms.C–TransientexpressionofaPtP2-G6PDH-GFPconstructintobacco leaves[29]:brightfieldimage,chlorophyllautofluorescence,GFP-fluorescence,andmergedimagesareshown.

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Cornish-Bodenplots(Cornish-Bowden,1974).Thedetailsofthedeterminationofkineticparameters(apparentKMforG6PandNADP andtheKIforNADPH)areprovided

inthetext.Valuesareaverageof3separatedeterminations±standarderror(se).

SpeciﬁcActivity(U/mg) KM,G6P(mM) KM,NADP+(␮M) KiNADPH(␮M) InhibitionbyNADPH

WT 8000 1.06±0.06 15±3 74.1±2.5 competitive C145S 5700 1.05±0.26 71±18 73.4±2* 23.0±3.6** competitive* uncompetitive** C194S 4500 1.15±0.37 53±18 --- noinhibition C242S 6000 0.70±0.12 55±9 --- noinhibition C175S 1400 2.42±0.78 30±9 15.5±2.8 uncompetitive C183S 1500 2.31±1.00 77±7 23.9±3 uncompetitive C175S-C183S 1360 2.38±0.87 103±19 27.9±3.6 uncompetitive

P2-G6PDH(1:10.000)andCy-G6PDH(1:50.000)isoforms[8],and

Anti-His6(Roche)(1:6000).Potatoantiserawerepreviouslyproven toreactwithanddiscriminatethedifferentbarleyG6PDHisoforms [3,19].Afterincubatingthemembranewithsecondaryantibodies (anti-rabbit,1:16.000),cross-reactingpolypeptideswerestained foralkalinephosphataseactivityorbyenhanced chemilumines-cence[22,23].

2.4. G6PDHactivityassayanddeterminationofthekinetic parameters

G6PDHactivitywasassayedbymonitoringNADP+_reduction_at 340nm.Theassaymixturecontained50mMTris-HClpH8.0,5mM MgCl2,150␮MNADP+and3mMG6P.Enzymeactivity measure-mentswereperformedinduplicateagainstablankwithoutG6P. TheactivitywasexpressedasnmolesofNADP+_reduced_min−1_or pers−1,andexpressedasml−1ormg−1protein.

Kineticparameters(KmG6PandKmNADP+)weredeterminedby varyingtheconcentrationsofG6Pfrom0to30mM,andNADP+ con-centrationfrom0to150␮M.Kineticparameterswerecalculatedby non-linearregressionusingtheMichaelis-Mentenequationwith GraphPadPrism4.0software.

TheinhibitionpatternandconstantsforNADPH(KiNADPH)were determinedbyDixonplot[24]andCornish-Bowdenplot[25]after assayingproteinactivityatsubsaturatingconcentrationsofNADP+ (15,30, 50␮M)in the presence of NADPHranging from15 to 150␮M[23].

2.5. RedoxtreatmentsofPtP2-G6PDH

Recombinantenzymes(bothWTandcysteine variants)were purified under non-reducingconditions allowingthe formation ofregulatorydisulfidebond(s).Toconfirmthatrecombinant pro-teinswerefullyoxidizedafterpurification,wetestedtheeffectof oxidizeddithiothreitol(DTT)onG6PDHactivity.Asexpected,no variationofmaximalproteinactivitywasobservedindicatingthat nofurtheroxidationwasrequiredtoreachitsmaximalactivation state(datanotshown).InordertoevaluateG6PDHsensitivityto reducingtreatments,PtP2-G6PDHWTandcysteinemutants(2␮M) wereincubatedinthepresenceofvaryingDTTconcentrations rang-ingfrom5to100mM.PtP2-G6PDHWTinactivationwasalsotested inthepresenceofpurifiedchloroplasticf-(spinach),m-(spinach) andz(Populustrichocarpa) _−typeTRXs (1,5, or10_␮M) supple-mentedwith5mM reducedDTTtoallowfull reduction ofTRX [26].Attheindicatedtimes,analiquotwaswithdrawntoassay forenzymeactivity.Forotherredoxtreatments,PtP2-G6PDHwas incubatedwithvaryingconcentrationsofGSHorGSSG(0.5,1or 5mM).Attheindicatedtimes,analiquotwaswithdrawntoassay enzymeactivity.Allredoxtreatmentswerecarriedoutat25◦Cin 50mMTris-HCl,1mMEDTA,pH7.9.

2.6. Midpointredoxpotential

Redoxtitration experimentswerecarriedouttomonitorthe redoxstateoftheregulatorydisulfideofPtP2-G6PDHWTinthe presenceof mixturesof oxidizedandreduced DTTtopoisethe ambientredoxpotential.RecombinantPtP2-G6PDH(1␮M)inits initialredoxstatewasincubatedfor2hat25◦Cin100mM Tris-HCl,pH7.9,1mMEDTA,20mMreduced/oxidizedDTTinvarious dithiol/disulfideratios and TRX m(spinach) (0.2␮M) [26]. Fol-lowingincubation,NADP-dependentG6PDHactivitywasassayed as described above. Results were fitted by non-linear regres-sion(CoStat,CoHortSoftware)totheNernstequation(n=2,one dithiol/disulfide[26]).Midpointredoxpotentialsarereportedas means±standarddeviationsoftwoindependentexperiments. 2.7. Replicates

Alloftheresultsreportedarerepresentativeofatleastthree independentexperimentsandexpressedasmeans±S.D.,unless otherwisestated.

3. Resultsanddiscussion

3.1. Proteinsequenceanalysisandsubcellularlocalizationof PtP2-G6PDH

AputativeplastidicG6PDH(PtP2-G6PDH)sequencehasbeen identiﬁedinthePopulustrichocarpagenome(Potri.001G059900, http://phytozome.jgi.doe.gov/). The analysis of the protein sequence reveals the presence of typical motifs belonging to dehydrogenasesfamily:aRossman-foldmotif(G57_ASGDLAKK65₎ andaNADP+_-binding_site_(N416_ELVIRV422₎₍_Fig.₁_A)._In_addition, theactivesite region(F220_RIDHYLGKE229₎_presents_a phenylala-nineresidue,typicaloftheplastidicisoforms,whichisgenerally replacedbyatyrosineinthecytosolicisoforms[6,13,22,27,28].

AcomparisonofPtP2-G6PDHwithotherplastidic, chloroplas-ticand cytosolic G6PDHsfromplantsclearly indicatesthat this sequencebelongstoplastidicP2-G6PDH isoforms(Fig.1B). Pre-dictionsperformedbytheTargetPbioinformaticsoftware(http:// www.cbs.dtu.dk/services/TargetP/)indicatedthatthePtP2-G6PDH sequence likelypossesses a 66amino acid-longtransitpeptide. Inordertoverifythesubcellularlocalization,aPtP2-G6PDH-GFP fusionproteinwasgeneratedandusedfortransientexpressionin tobaccocells[29].Theresultsindicatedthatthetransitpeptideof PtP2-G6PDHdirectedtheconstructintoplastidsasexpectedfrom thepredictions(Fig.1C).

Inthematureenzyme,thereareﬁveCysresidues(Cys145_,_Cys175_, Cys183_,_Cys194_and_Cys242_;_Fig.₁_A),_two_of_them,_Cys175_and_Cys183_, areinapositionsimilartothetworegulatorycysteinesinvolvedin theformationofthedisulﬁdebondinP1G6PDH,assuggestedfor thepotato(Cys156_-Cys161_,_[27,13]_),_barley_(Cys159_-Cys167_,_[22]_),_and Arabidopsisthaliana enzymes(Cys169_-Cys177_,_[30]₎₍_Fig.₁_A)._The

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Fig.2.A–SDSPAGEofsamplesatdifferentpuriﬁcationstepsofPtP2-G6PDH;B– WesternblottingofthepuriﬁedPtP2-G6PDHusingantiP2-G6PDH,antiCy-G6PDH antibodies;andC–anti-Hisantibodies.

Legend:M,Markers;Control,bacteriatransformedwithan“empty”plasmid;pellet ofbacterialysate;S,surpernatantofbacteriallysate;puriﬁedproteinfromsoluble fraction.Thearrowindicatesthesubunitmolecularweight(61kDa)ofthe over-expressedPtP2-G6PDH,calculatedwiththerelativemobilityfactormethod.

conservationofregulatorycysteineswasindeedsuggestivethat P2-G6PDHisoformscouldalsoberegulatedthroughdithiol/disulﬁde exchangereactionsasdescribedforchloroplasticP1-G6PDH[9]. 3.2. PuriﬁcationofrecombinantPtP2-G6PDH

TherecombinantPtP2-G6PDHwasproducedasamature pro-teinfusedwithaHis6-tag,exhibitingaMWof61kDa(Fig.2A).The constructionwasdesignedinsuchawaythattheputative tran-sitpeptidewaseliminatedandanN-terminalHis-tagwasfused totheprotein(seeMethods).Therecombinantenzymewas sol-ubleandcouldbepurifiedtohomogeneityusinganimmobilized nickelmetalaffinitychromatography(IMAC),withafinalyieldof 0.8–3mgperlitreofculture.

Thepuriﬁedproteinreactedpositivelywithpotatoplastidic P2-G6PDH[8];(Fig.2B)andantiHis-tagantibodies(Fig.2C),whileno reactionwasobservedusingapotatocy-G6PDHantiserum(Fig.2B). Moreover,ithadtheexpectedsizeforafusionproteinwithaHis-tag anddeprivedofthetransitsequence.

3.3. RedoxpropertiesofrecombinantWTPtP2-G6PDHandofCys toSermutants

PlastidicG6PDHsarestronglyinhibitedbyincreasinglevelsof artiﬁcialreductants, suchasDTT; incontrast,whentheprotein isfullyoxidized,theenzymeexhibitsitsmaximalcatalyticrate [8,27,31].

In order to test the redox sensitivity of recombinant PtP2-G6PDH, the puriﬁed enzyme was incubated with different concentrationsofDTT.Theactivitywasreducedinadose-and time-dependentmanner,retaining 50%ofitsinitialrateafter60min incubationinthepresenceof25mMDTT,orafter20minwhen exposedto100mMDTT(Fig.3A).On theotherhand,insimilar experimentalconditionsP2G6PDHactivitywasnotaffectedby themonothiolglutathioneeitherinthereduced(GSH)oroxidized (GSSG)form,upto5mM(SupplementalFig.1AandB).

Basedonthesensitivity toreduced DTT,themidpointredox potentialofthepuriﬁedrecombinantPtP2-G6PDHwasdetermined afterproteinincubationinthepresenceofdifferentDTTred/DTTox ratiospoisingvariableredoxpotentials.AsshowninFig.3B,G6PDH

activityisfullydependentontheredoxpotentialanddata interpo-lationwiththeNernstequation,includingasingledithiol/disulﬁde redoxpair(n=2),givesastandardredoxpotentialof−332.7mV (pH7.9).Thisvalueislessnegativethantheoneofbothf-and m-typeTRXs(−351and−358mV,respectively[32]),thussuggesting thatPtP2-G6PDHcouldbefavourablyreduced,butlessfavourably oxidizedbytheseTRXtypes.Asacomparison,Arabidopsisthaliana P1-G6PDH,whichisregulatedbyTRXfexhibitsanEmof329.9mV

[9].

Toinvestigatetheregulatoryfunctionsplayedbythedifferent cysteines,eachresiduewassubstitutedintoserinebysite-directed mutagenesis. These recombinant proteins were overexpressed andpurifiedfollowingthesameproceduredescribedfortheWT enzyme,andsimilaryieldswereobtained(0.5–2mg perlitreof culture−notshown).Specificactivitiesandotherkinetic proper-tiesofthepurifiedmutatedenzymesincomparisonwiththewild typePtP2-G6PDHareindicatedinTable1.

Theredoxpropertiesofthesecysteinevariantswerethen exam-inedand,basedontheirsensitivitytoDTT-dependentinhibition, theycouldbedividedintotwodistinctgroups.Theﬁrstcluster comprises C145S,C194Sand C242Smutants, exhibiting a DTT-sensitivitysimilartotheWTPtP2-G6PDH(Fig.3C),suggestingthat these residues are not involved in the redox regulationof the enzyme.

ThesecondgroupincludestheC175S,C183Svariantsandthe doublemutantC175S-C183S,whicharecompletelyinsensitiveto DTT(Fig.3C),indicatingthatthesetworesiduesareindeedthose responsiblefortheredoxsensitivityoftheenzyme,andtherefore verylikely involvedintheformationof theregulatorydisulﬁde bond.

3.4. Localizationofcysteinesinthe3DmodelofPtP2-G6PDH BasedonPtP2-G6PDHaminoacidsequence,aputative3Dmodel wasgeneratedusingthestructureofhumancytosolicG6PDHasa template(Fig.3D).Incidentally,humancytosolicG6PDHexhibitsits highestcatalyticrateasatetramer,formedbyactivehomodimers linkedtogetherbysalinebonds[33].

Duetothewell-knownregulatoryroleofthedisulfidein plas-tidic G6PDHs, includingPtP2-G6PDH[8,13,22,27],we speculate thatthereisaconformationalchangeinducedbyformationofthe Cys175_-Cys183_bond._The_regulatory_Cys175_and_Cys183_are surface-exposed,and locatedinanunstructuredloopintheN-terminal region of the protein (Fig. 3D). Intriguingly, human cytosolic G6PDH,whichisnotredox-regulated(thisisalsotrueforhigher plantcy-G6PDH,[28]),isabletoformastructuraldisulfidebond (Cys13_-Cys446₎_in_the_same_region_of_the_3D-structure_[33]_._It_has beenproposedthatthisdisulfidemaypreventtheunstructured N-terminalregiontointerferewiththecatalyticactivityoftheenzyme [34].Similarly,thedisulfideCys175_-Cys183_present_in_the_fully_active plastidicenzymewouldallowaneasieraccessofthesubstratesto theactivesite,bystabilisingtheflexibleloopattheN-terminus.

It shouldbenoted that inthis model thepositionof Cys145 isquiteclosetotheregulatorydisulﬁdewhereas theothertwo residues(Cys194_and_Cys242₎_would_be_located_in_different_parts_of thestructure,fartherfromtheactivesite.

3.5. PtP2-G6PDHispreferentiallyregulatedbym-typeTRX

Higherplantplastidscontainatleast5conventionalTRXtypes, namelyf,m,x,yandz[35,36],aswellasseveralotherTRXswith atypicalactivesitesequences[29,37].

Generally, the m- and f-type TRXs are implicated in the regulation of enzymes involved in photosynthesis and carbon metabolism,whiletheotherTRXsareassociatedtotheanti-oxidant response [35,38–40]and TRX zplaysa role linkedtoorganelle

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Fig.3. A–EffectsofDTTredonPtP2-G6PDHactivity.Thepuriﬁedenzymewasincubatedfor20min(blackbars)or60min(greybars)atdifferentconcentrationsofDTTred,

andG6PDHactivitydetermined.B–DeterminationofthemidpointredoxpotentialofthepuriﬁedrecombinantPtP2-G6PDH.C–EffectsofDTTredonPtP2-G6PDHcysteine

mutants.Puriﬁedenzymeswereincubatedfor60minintheabsence(greybars)orinthepresence(blackbars)of25mMDTTred,andG6PDHactivitydetermined.D–3D

modelofPtP2-G6PDHmonomerstructure.Theasterisksonbarsindicateap<0,05withrespecttocontrols.

ModellingwasmadeusingSwissProtsoftwarebasedontheknownstructureofahumanG6PDHisoformandvisualizedusingPyMOL[33].TheSwiss-protPDBviewer program(availableat:http://expasy.org/spdbv/)allowedthecolorationofcysteineresiduesaccordingtotheiraccessibilityfromred(lessaccessible)toyellow(more accessible).Shapedportionsoftheproteinappearincyan(␣-helices)andmagenta(␤-sheets)whileunstructuredregionsareinlightpink.

development[36].Itshouldbenotedthattheﬁrstattemptswhich proposedaspeciﬁcroleforTRXmintheregulationofplastidic G6PDHweremadewithcrudeextractofbacteriaoverexpressinga recombinantpotatoplastidicisoform[27].

Havingobtainedapurifiedandstablerecombinantenzyme,we wereabletotesttheeffectofdifferentplastidicTRXs(m-,f-and z-types)onPtP2-G6PDH(Fig.4).ThemostefficientwasTRXm, inhibitingG6PDHactivityby50%after1minat1_␮M(Fig.4A).At thesameconcentration,TRXfwaslesseffective,(45%inhibition after20min),and TRXzwasessentiallyinefficient(Fig.4A).By increasingTRXconcentrations(Fig.4BandC),theregulatoryeffects becamemoreevident.At10␮MTRXconcentration,90%inhibition wasreachedafter1minbyTRXmandafter3minbyTRXf;while TRXzonlypromoteda60%inhibitionafter5min.Ourexperiments indicatethattheeffectivenessofthedifferentTRXsonPtP2-G6PDH canbearrangedasTRXm>TRXf>TRXz.

Theseresultssuggestthatdifferentpatternsofredox regula-tionexistforcompartmentedG6PDHsinhigherplants.Speciﬁcally, TRXfisthemostefﬁcientintheregulationofchloroplasticG6PDH from both A. thaliana (P1-type AtG6PDH); [9,11] and the psy-chrophilicalgaKoliellaantarctica[41].Itshouldbeunderlinedthat f-typeTRXs are wellknowntoregulatetheactivitiesof redox-sensitiveCalvin-Bensoncycleenzymes,namelyglyceraldehyde-3P dehydrogenase (GAPDH) [42], phosphoribulokinase (PRK) [42], fructose-1,6-bisphosphatephosphatase[43]andsedoheptulose1-7 bisphosphatase[44].

Ontheotherhand,wedemonstrateherethattheactivityof plas-tidicP2-G6PDHismainlyredox-modulatedbyTRXm-type.TRX misgenerallyindicatedasaregulatorofNADP-malate dehydro-genase(EC1.1.1.82),thusplayingacentralroleintheregulation ofredoxpoisebalancingbetweenthechloroplastandthecytosol; recently,progresshasbeendone,describingatleast3–4distinct TRXminchloroplasts,andsuggestingspeciﬁcrolesindifferent metabolicprocesses[12].Thus,itcouldbesuggestedaregulation ofP2-G6PDHisoformsbasedonreductionstateoftheplastid(e.g. malatedehydrogenaseactive;TRXmreduced−P2-G6PDH inac-tivated),sensiblydifferentfromthatofP1-G6PDH(Calvin-Benson cycleactive,TRXfreduced,P1-G6PDHinactivated)[11].

ThismodelwouldimplythatP1-G6PDHmustbemostly inac-tiveinthelight,whileP2-G6PDHregulationisindependentoflight (asexpectedforanenzymeexpressedinheterotrophiccells),and appearsmore strictlyrelated totheprimary metabolismofthe organelle.

3.6. KineticpropertiesofrecombinantPtP2-G6PDHWTand cysteinemutants

To our knowledge, the activity of plastidic P2-type G6PDH isoforms hasbeen investigated in thepast only withbacterial lysates,orusingpartiallypuriﬁedpreparationsfromplantmaterial [3,8,16,20,21].Therefore,duetotheinstabilityofthisisoform,the biochemicalpropertiesofPtP2-G6PDHhavebeenproperly

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inves-Fig.4.EffectsofTRXsonPtP2-G6PDH.Activityofpuriﬁedenzymeswasdetermined afterincubationfordifferenttimesinthepresenceof5mMDTTalone(control,open circles)orsupplementedwith:A–1␮M;B–5␮M;C–10␮MofTRXm(closed squares),TRXf(opensquares),TRXz(closedcircles).

tigatedonlyrecentlywithapuriﬁed recombinantenzymefrom barleyroots,buteventhough,thispreparationwasfarlessstable thanthepoplarenzymegeneratedhereandthusthekineticdata presentedherearethemostreliable[22].

Recombinant,his-tagged PtP2-G6PDHwaspuriﬁed fully oxi-dized,andshowedaspeciﬁcactivityof8000nmolmin−1 NADP+ reducedmg prot−1 (Table1).Thekinetic analysisrevealedthat PtP2-G6PDHdisplaysaKM,G6Pof1.06±0.06mMandKM,NADP+of 15.0±3.2␮M(Table1),similarlytovaluesobservedforotherplant P2-G6PDHs[22,30].

TheinhibitionconstantforNADPH(KI,NAPDH)wasfoundtobe 74␮M(Table1),avaluecomparabletobarley[3,22],andpotato P2-G6PDHs[8].ThecomparisonofDixonandCornish-Bowdenplots [45]revealedacompetitiveinhibitionbyNADPHofPtP2-G6PDH (Fig.5A).ItisnoteworthythatNAPDHexertsasimilarinhibitory

mechanismonrecombinantbarleyCy-G6PDH[28],althoughthe KI,NAPDHisconsiderablylowerforcytosolicisoforms(10–25␮M).

Moreover, it should be underlined that this combination of kinetic properties undoubtedly categorises the enzyme as a plastidicP2-G6PDH,andcandiscriminateitfromchloroplastic P1-G6PDHs[8,18,22]andCy-G6PDHs[28,46].

Notably,itshouldbeexpectedthatthelightinactivationof P1-G6PDH shouldbeaccompaniedby ahighsensitivity toNADPH inhibition(NADPH/NADP+_ratio_are_>2_in_the_light)_–_[47]_;_while P2-G6PDHshouldbemostlyinsensitivetoNADPH,becauseofitsmain roleinthesupplyofreducingequivalents(i.e.NADPH)forplastidic biosynthesessuchasnitritereduction[14],nitrogenassimilation [15,16,21],orlipidmetabolism[48].

ThusithasbeenproposedthatpartofP2-G6PDHactivitycould beretainedevenunderlightconditionsinchloroplaststosustain activenutrientassimilation[3],evenwhenphotosynthesiscouldbe partiallyinhibitedbystomataclosureandoxidativestress;anyway, itshouldbeunderlinedthatadeﬁnitiveevidenceforthepresence ofP2-G6PDHinchloroplastsisstilllacking.

Mutant enzymes lacking non-regulatory cysteines (C145S; C194S;C242S)exhibitedspeciﬁcactivitiesandKm,G6P similarto WTPtP2-G6PDH;a3.5-4.5foldincreaseinKm,NADP+wasobserved (Table1).

Bycontrast,C175SandC183Smutantsshowedareduced spe-ciﬁc activity (<20% of WT), and a reduced afﬁnity for sugar-P

(Km,G6P=2.3–2.4mM), as expected for an enzyme mimicking a

reduced,partiallyinactivated,WTprotein(Table1).

Correspondingly,theregulatorycysteine toserinemutations decreased their afﬁnity for NADP+ ₍₃₀_␮M_<K

m,NADP+<100␮M). Theseresultswouldsupportaroleforthedisulﬁdeinthe stabil-isationoftheenzyme-substratebinding,assuggestedforhuman G6PDH[34].

Then,theNADPH-dependentinhibitionofPtP2-G6PDHactivity wasfurtherinvestigatedoncysteinemutants.

TheC145SmutantshowedamixedinhibitionbyNADPH;the competitiveinhibition(ic)constantwasidenticaltoWTenzyme, indicating that C145 _is _not _directly _involved _in _the _regulation of activity;ontheotherhand, theCornish-Bowdenplotﬁtsan uncompetitiveinhibition (iu) KiuNADPH=23␮M, suggesting that C145_might_have_a_structural_role_in_the_docking_of_NADP+_,_its_afﬁnity being3.5-foldlowerthantheonemeasuredfortheWT(Fig.5B).

Itshouldbenotedthatthemodelofthe3Dstructureindicates thatC145_is_located_close_to_NADP+_binding_site,_and_likely_to_the disulﬁdeC175_-C183₍_Fig.₃_D).

Both C194 _and _C242 _are _somehow _structurally_important _for NADP+_/NADPH_binding._{Interestingly,}_the_substitution_of_C194_and C242_to_serines_precluded_NADPH_inhibition₍_Fig.₅_B),_and_caused a 3-foldincreaseof KM,NADP+ (Table1), althoughthese residues seemtobeburiedinthe3D-modeloftheenzyme(Fig.3D).This suggeststhattheseresiduesarepossiblyinvolvedintheoptimal foldingoftheNADP+_binding_site,_improving_substrate_afﬁnity_and modulatingthebindingoftheinhibitoryNADPH.

A different scheme applies to the mutants lacking regula-torycysteines(C175S,C183S,anddoublemutantC175S-C183S), allexhibitinganuncompetitiveinhibitionbyNADPH(Fig.5D–F; KiuNADPH of15–24␮M),andastrongdecreaseintheaffinityfor G6P(Table1).Theseresultssuggestthatthereductionofthe disul-fideinducesachangeinconformation,resultingintheabilityof theenzymetobindNADPHaftertheformationofthe enzyme-substrate complex, with an increased affinity for the inhibitor NADPHandaloweraffinityforthesubstrates(Table1).

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Fig.5.DeterminationofthetypeofinhibitionandinhibitionconstantvaluesofwildtypeandmutatedPtP2-G6PDHs.Thedescriptionoftheinhibition,andKI,NAPDHare

indicated.

A–WildtypePtP2-G6PDH(WT);Dixonplot(left)andCornish-Bowdenplot(right).B–DixonPlotforC194S(up)andC242S(below).C–C145SDixonplot(left)and Cornish-Bowdenplot(right).D–C175SDixonplot(up)andCornish-Bowdenplot(below).E–C183SDixonplot(up)andCornish-Bowdenplot(below).F–C175S-C183S Dixonplot(up)andCornish-Bowdenplot(below).

4. Conclusion

Byusingacombinationofkineticanalysis,site-directed muta-genesistogetherwith 3D-structuremodelling,we wereableto proposeamodeloftheredoxmodulationofapoplarP2-G6PDH activity.

ThekineticparametersobservedforPtP2-G6PDHsuggestthat thisisoformcouldnotbeinhibitedbyNADPHlevelsunder physio-logicalconditions,especiallyinthedark;but,possiblyevenunder

lightconditions–whenphotosynthesisisactive,andproducing moietiesofreductants–theenzymewouldretainitsactivity(and function) in order to sustain nutrients’ assimilation and stress response[3,4].

Inhigherplantsandalgae,theredoxregulationof chloroplas-tic P1-G6PDHisoformshasbeen recentlylinkedtof-type TRXs [9,11,41].Nevertheless, previousreports generallyindicated m-typeTRXscouldbephysiologicallyinvolvedinthemodulationof plantcompartmentedG6PDH[27].

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TheresultsherepresentedsuggestthatPtP2-isoformwouldbe (andactivated)bym-typeTRXs, generallyintendedasthemain regulatorsofthechloroplastcatabolism,andnotbyf-typeTRXs whicharemorestrictlyrelatedtophotosynthesis[9].

Amongtheﬁvecysteinespresentintheactiveproteinsequence, C194_and_C242_play_a_role_in_the_{stabilization}_and_properties_of_NADP+ site,whileC145_,_although_very_close_to_the_regulatory_C175_-_C183 disulﬁde,seemstointerferewiththebindingofNADP+_during_the formationoftheenzyme-substratecomplex.

Here,ourresultsshowedthatthedisulﬁdeC175_-C183_present_in plastidicP2-G6PDHisbetterreducedbym-typeTRXandtoalesser extentbyf-typeTRX;inaddition,z-typeTRXdoesnotseemtoplay asigniﬁcantroleforitsreduction.

Thus,theP1andP2isoformsmaybetargetsofdifferentTRXs. Ontheonehand,TRXfpreferentiallycontrolschloroplasticG6PDH activity (i.e. P1-G6PDH), as occurring for the redox-modulated enzymesinvolvedintheCalvin-Bensoncycle[49].

Ontheotherhand,theactivityofnon-photosyntheticG6PDH (P2-G6PDH) is better controlledby m-type TRX, which is well knowntoregulateNADP+_-malate_{dehydrogenase,}_although_the_f −typeTRXwasfoundtobeabetterreductantafterall[50,51].

Finally,TRX-mdisulﬁde-dependentredoxregulationof PtP2-G6PDH would control the supply of reductants during active nutrient assimilation(e.g.nitrogen,sulfur) [21] or understress conditions(drought,salinity)[4,6,19].

Therefore,thereisanurgentneedtoget3Dstructuresofreduced and oxidized enzymesto understandtheinvolvement of these regulatorycysteinesinthestructuralchangesrequiredforthe for-mationofastableandactiveG6PDH.

Acknowledgments

ResearchsupportedbyLeggeRegionaledellaCampania5/2002 (2007),CUPE69D15000270002.

ManuelaCardiacknowledgesEGIDEfundingfromFrench Min-istryofForeignAffairs(grant672700K;years2009and2010/11); and Project FORGIARE (Formazione Giovani alla Ricerca) V-10/FORG/ST/2012/5(2012–2015)byCompagniadiSanPaolo.

UMR1136 is supported by a grant overseen by the French NationalResearch Agency(ANR)aspartofthe“Investissements d’Avenir” program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE).

TheAuthorsthankverymuchAntjevonSchaewen(Muenster– Germany)forthegenerousgiftofG6PDHantibodies.

Noconﬂictsofinterestdeclared. AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.plantsci.2016.08. 003.

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