molecular cloning molecular cloning Molecular Cloning, Biochemical Characterization_图文

PlantMolBiolRep(2013)31:547–557DOI10.1007/s11105-012-0523-6

ORIGINALPAPER

MolecularCloning,BiochemicalCharacterization,andDifferentialExpressionofanAcetyl-CoAC-AcetyltransferaseGene(AACT)ofBrahmi(Bacopamonniera)

RishiK.Vishwakarma&Ruby&SomeshSingh&PrashantD.Sonawane&SameerSrivastava&

UmaKumari&R.J.SantoshKumar&BashirM.Khan

Published#online:21November2012

SpringerScience+BusinessMediaNewYork2012

AbstractBacopamonniera(Brahmi)isanimportantIndianmedicinalherbfoundinwetdampandmarshyplaces.Itproducesmedicinallyimportantcompoundsknownasbaco-sidesalongwithalkaloidslikebrahmineandherpestine.Bacosidesaretriterpenoidsaponinsandtheirbiosynthesistakesplaceviatheisoprenoidpathwaystartingwithacetyl-CoA.Acetyl-CoAC-acetyltransferase(AACT;EC2.3.1.9),alsoknownasacetoacetyl-CoAthiolase(ThiolaseII),cata-lyzesthecondensationoftwoacetyl-CoAtoform4-Ccom-poundacetoacetyl-CoA.Acetoacetyl-CoAisanimportantstartingmoleculeforbiosynthesisofvariousmetabolites.Here,wereportthecDNAcloningandcharacterizationofacetyl-CoAC-acetyltransferasegenefromB.monniera.Thefull-lengthgenewasisolatedusingaRACEPCRprotocol.ThecDNAencodingAACTwasdesignatedasBmAACT(FJ947159)revealedanORFof1,218bpand405aminoacids,andshares80%similaritywithotherplantAACTs.PhylogeneticanalysisshowedthatBmAACTisrelatedclose-lytootherdicotplantsAACTs.TheBmAACTgenewasover-expressedinEscherichiacoliasa6XHis-tagfusionproteinandpurifiedtohomogeneitybyNi-NTAandgelfiltrationchromatography.Activityofrecombinantproteinwascon-firmedbythiolyticcleavageofacetoacetyl-CoAinthepres-enceof5mMMg2+,showingKmandVmaxof20.67μMand96.21μmol/min,respectively,1withhighcatalyticefficiency(kcat02.30×105min?).Quantitativereal-timePCRanalysisshowedthattheexpressionofBmAACTistissue-specific,and

R.K.VishwakarmaRubyS.SinghP.D.SonawaneS.Srivastava:U.Kumari:R.J.SantoshKumar:B.M.Khan(*)PlantTissueCultureDivision,NationalChemicalLaboratory,Dr.HomiBhabhaRoad,

Pune411008Maharashtra,Indiae-mail:bm.khan@ncl.res.in

accumulationoftranscriptsisgreaterinrootsandpetals,followedbysepals,stem,leafandpedicel.

KeywordsAcetyl-CoAC-acetyltransferase.Bacopamonniera.Isoprenoidpathway.TriterpenoidsaponinAbbreviationsAMV-RTAvianmyeloblastosisvirus

reversetranscriptase

cDNAComplementaryDNAAACTAcetyl-CoAC-acetyltransferaseBmAACTBacopamonnieraAcetyl-CoA

C-acetyltransferasegene

BMEβ-MercaptoethanolRACERapidamplificationofcDNAendsqRT-PCRQuantitativereal-timePCRIPTGIsopropylβ-D-thiogalactopyranosideSDS-PAGESodiumdodecylsulfate

polyacrylamidegelelectrophoresis

UTRUntranslatedregion

Introduction

Thiolasesplayanimportantroleinmanybiochemicalprocess-es,includingisoprenopid,steroidandalkaloidbiosynthesisaswellasβ-oxidationoffattyacids.Isoprenoids,alsoreferredtoasterpenoids,representavastgroupofnaturalproductswithvarietyoffunctionsintheprimaryandsecondarymetabolismofplants(Langeetal.2000).AllisoprenoidsaresynthesizedfromtwouniversalC5compoundsknownasisopentenyldi-phosphate(IPP)anditsisomerdimethylallyldiphosphate(DMAPP)(KirbyandKeasling2009).BiosynthesisofIPPtakesplaceviaeitheroftwopathways:themevalonate

548(MVA)pathwayorthenon-mevalonate/methylerythritolphos-phate(MEP)pathway(Langeetal.2000;Setoetal.1996).TheMVApathwayoperatesinthecytosolwhereastheMEPpath-wayexistsinplastidsandisconnectedthroughIPPexchangeacrossthechloroplastmembrane(Lauleetal.2003).However,nativecross-talkbetweentheMVAandMEPpathwaysisstillunclear(Suzukietal.2009).

TheMVApathwaystartswiththetwo-carboncompoundacetylCoA,whichcondensesintothe4-Ccompoundacetoacetyl-CoAcatalyzedbyacetyl-CoAacetyltransferase(AACT).Acetyl-CoAacetyltransferase(AACT)orthiolasesareclassifiedintotwotypes,degradativethiolases(E.C.2.3.1.16)andbiosyntheticthiolases(E.C.2.1.1.9)(ModisandWierenga2000).Thedegradativethiolasesareinvolvedinβ-oxidationoffattyacids(JenkinsandNunn1987),where-asthebiosyntheticthiolasescatalyzethecondensationoftwoacetyl-CoAmoleculestoformacetoacetyl-CoA.Igualetal.(1992)reportedthatacetoacetyl-CoAisanimportantstartingmoleculeinhormoneandcholesterolbiosynthesis.Inprokar-yotes,AACTcatalyzesthefirststepofpolyhydroxybutyrate(PHB)biosynthesisviathethiolaseIIpathway(SteinbuchelandHein2001).IntheMVApathway,theenzyme3-hydroxy-3-methyl-glutaryl-CoAreductase(HMGR)catalyzesthecon-versionofHMG-CoAtomevalonateandhasbeendescribedastherate-limitingstep(BuhaescuandIzzedine2007;Hunter2007).KirbyandKeasling(2009)suggestedthatHMGRactivityispost-transcriptionallyandpost-translationallyregu-latedinplants.Interestingly,thecondensationreactionofthiolasefromeukaryoteshasnotbeenrecognizedasanim-portantregulatorypointinbiosyntheticprocesses;however,itwasprovedrecentlythatAACTisaregulatoryenzymeinisoprenoidbiosynthesisandisinvolvedinabioticstressadap-tationinalfalfa(Sotoetal.2011).

ThiolasegeneshavebeenstudiedinvariousorganismsincludingClostridium(MengandLi2006),Rhizobium(KimandCopeland1997),Dictyostelium(Tanakaetal.2010),radish(VollackandBach1996),Arabidopsis(Carrieetal.2007;Ahumadaetal.2008),sunflower(Oeljeklausetal.2002;Dyeretal.2009),andHeveabrasiliensis(Sandoetal.2008).However,littleisknownabouttheroleofthisenzymeinisoprenoidbiosynthesisinplants(VollackandBach1996;Bachetal.1999).Sandoetal.(2008)functionallycharacter-izedH.brasiliensisAACT(HbAACT1)inayeastheterolo-goussystem,andreportedthatheterozygousyeastlackedanalleleofthethiolaseIIgeneinvolvedintheMVApathway.Moreover,itwasalsofoundthatexpressionofHbAACT1promotedyeastgrowthonnonselectionmedium.Recently,theAACTgenefromsunflowercotyledonswascloned,andarecombinantenzymeofspecificactivityof63nkat/mgproteinwaspurified(Dyeretal.2009).Inarecentstudy,MedicagosativathiolaseII(MsAACT1)wasover-expressedinplanta,andtransgenicplantsexhibitedsalinitytoleranceanden-hancedproductionofsqualenewithoutalteringHMGR

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activityundersaltstressconditions(Sotoetal.2011).Inotherstudies,thenumberofgenesassociatedwithsaltstressresponsesinrootsofRhizophorastylosa(Basyunietal.2011)andHibiscus(Yangetal.2011)havebeendetermined.TransgenicArabidopsisandtobaccoplantsover-expressingglycogensynthasekinaseaswellaslateembryogenesisabun-dant(LEA)andbasicleucinezipper(bZIP)genesshowedenhancedtolerancetosaltandosmoticstresses(Heetal.2012;Quetal.2012).

Inthisstudy,ageneencodingthiolasewasisolated,clonedandcharacterizedfromthemedicinalherbBacopamonniera(Brahmi).ItisanimportantAyurvedicmedicineusedasabraintonic,memoryenhancer,revitaliserofsensoryorgans,diureticandcardio-tonic(SivarajanandBalchandran1994;Chopraetal.1969).Themainactiveconstituentsofthisplantaretriter-penoidsaponinscalledbacosides;differentformsofbacosideshavebeenidentified(Chakravartyetal.2001;Rastogietal.1994;Chatterjeeetal.1963).Theyaresynthesizedfromacetyl-CoAmoleculeviatheMVApathway.Recently,itwasreportedthatAACTisregulatoryenzymeintheisoprenoidpathwayundersaltstressconditionsandenhancestheproduc-tionofsqualene(Sotoetal.2011).Therefore,studyingtheAACTgenecouldhavegreatbiotechnologicalimportance.Over-expressionofthisgeneinBacopacouldhelpabioticstresstolerance.Thetransgenicplantsdevelopedwouldhaveimprovedsqualenecontent,ultimatelyleadingtoformationofvarioustriterpenoids,andneedstobefurtherstudiedinplanta.

MaterialsandMethodsPlantMaterial

TheaerialpartofB.monnieragrowninagreenhousemaintainedattheNationalChemicalLaboratory(NCL)wasusedforisolationoftheBmAACTgene.Quantitativereal-timePCR(qRT-PCR)wasperformedusingstem,leaf,root,sepal,pedicelandpetaloffield-grownplants.TotalRNAIsolationandFirst-StrandcDNASynthesisAerialpartsofB.monnieraplantsamplesweregroundinliquidnitrogentoafinepowderusingamortarandpestle.TotalRNAwasextractedfromapproximately100mgtissueusingTRIzol-Reagent(Invitrogen,Carlsbad,CA)asperthemanufacturer’sinstructions.ThequantityandqualityofisolatedRNAwascheckedat260nmand280nmwithUV-Visiblespectropho-tometer(NanoVueplus,GEHealthcare,Uppsala,Sweden)andRNAwasstoredinDEPC-waterat?80°Cforfutureuse.First-strandcDNAsynthesiswasperformedbyreversetranscriptionPCRwithtotalRNA(1μg)andoligo(dT)18asprimerusingavianmyeloblastosisvirusreversetranscriptase(AMV-RT;Promega,Madison,WI),accordingtothemanufacturer’s

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protocol.ForqRT-PCR,totalRNAwasisolatedfromdifferenttissuesandfirststrandcDNAwassynthesizedasabove.IsolationofPartialcDNAFragmentofBmAACTGeneApartialfragmentoftheBmAACTgenewasisolatedbyaPCR-basedapproach,usingcDNAasatemplate.TheAACTsequencesfromNCBIGenBankwerealignedusingtheClustalWprogramandprimersweredesignedfromconservedregions.Sixdifferentprimersweredesigned,namelycATF1,cATF2,cATF3,cATR1,cATR2andcATR3(Table1).PCRamplificationwascarriedoutusingdifferentcombinationsofprimersetsandelecrophoreticseparationwasdoneforamplifiedPCRproductson1%agarosegel.TheamplifiedproductwaspurifiedandclonedinpGEM-TeasyPCRcloningvector(Promega).AftertransformationinEscherichiacoli(XL-10Gold)competentcells,plasmidswereisolatedfrompositiveclonesandsequenced.

IsolationandCloningofFull-LengthBmAACTcDNAbyRACEPCR

Thefull-lengthcDNAsequenceofBmAACTwasisolatedby5′and3′RACEPCR.Gene-specificforwardandreverseprimersweredesignedfromknownpartialsequenceof

Table1Primersusedforthestudy

Serialno.1.2345678910111213141516171819

a

B.monniera.For5′and3′RACE,tworoundsofPCRwereperformedusinggene-specificprimerpairsRACEATBMR1andRACEATBMNestedR1,andRACEATBMF1andRACEATBMNestedF1,respectively,alongwithprimerssuppliedinkit(Table1).SMARTRACEcDNAamplifica-tionkit(Clontech,PaloAlto,CA)wasusedfor5′RACEandfor3′RACE,GeneRacerkit(Invitrogen)wasused.ThePCRproductsobtainedwereclonedinpGEM-Teasyvectorandsequenced.Thesequenceinformationof5′and3′RACEPCRproductcloneswasusedfordesigningofprimersATBMFullF1andATBMFullR1fromthestartandstopcodon(Table1)toobtainthefull-lengthgene.PCRwasperformedwithpfxTaqDNApolymerase(Invitrogen)usingcDNAasatemplate.Thefull-lengthampliconwasclonedinpGEM-Teasyvectorandsequenced.

BioinformaticsandEvolutionaryRelatednessAnalysisTheobtainedBmAACTsequenceswereanalyzedusingonlinebioinformaticstools(http://www.ncbi.nlm.nih.gov).Thede-ductionoftheaminoacidsequences,calculationofthetheo-reticalmolecularmassandpIwereperformedwithExPASyProteomictools(http://www.expasy.ch/tools/).Globalalign-mentoftwonucleotideoraminoacidsequences,andpercent-ageofidentitywerecalculatedusingtheEMBOSSPairwiseAlignmentAlgorithms(http://www.ebi.ac.uk/emboss/).

Primersequences(5′→3′)b

CTCGAGATGTTTGTGTTGTGGAGATGTTTGYRTTGTGGGWGTAAGAGAGCMATGTTGATCCGCWCCTCCTCCTCCGTTGCAGCTTTWGGWATAGCAAGAGCGCWGCYGCACCATCACTTATGCTGCCACCACAACATCATTGATTCCCACCTGTCCCAAGTTTGCACTTAGCAGTTGCCCGGACACCTATGGGA

GCTATTGAGAGTGCTCTTAAGAGAGCAATGGCTCCAGCAGCAGCAGCG

TTAGACAAGCTCCACAACGAGAGCCGCATATGGCTCCAGCAGCAGCAGCG

CTCGAGGACAAGCTCCACAACGAGAGCGCACGCGCGCTACACCGAAGGTCTGTACAAAGGGCAGGGACGGACTACGGCATGGGAGTTTGATTCCACGCTCAAAACTTTGG

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

AAGCAGTGGTATCAACGCAGAGTGCTGTCAACGATACGCTACGTAACGCGCTATGTAACGGCATGACAGTG

PrimernamecATF1cATF2cATF3cATR1cATR2cATR3

5′RACEATBM5′RACEATBM3′RACEATBM3′RACEATBMATBMFullF1

R1

NestedR1F1

NestedF1

ATBMFullR1pETATBMF1pETATBMR1

18SF(18SrRNA)a

18SR(18SrRNA)qAACTFqAACTR

UniversalprimerAMix(UPM)NestedUniversalprimerA(NUP)GeneRacer3′primer(3′GRP)

GeneRacer3′nestedprimer(3′NGRP)

a

GenBankaccession.No.JN148054

W0AorT,S0GorC,N0AorTorGorC,Y0TorC,R0AorG,M0AorC

b

202122

550MultiplesequencealignmentsofvariousplantAACTswerecarriedoutwithClustalW1.8(http://www.ebi.ac.uk/clustalw/).ConserveddomainsinBmAACTweredetectedusingConservedDomainDatabasesearchtool(CDD)onNCBIserver(http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi).ForevolutionaryrelatednesswithotherplantAACTs,aphylogenetictreewasbuilt.TheknowndeducedaminoacidAACTsequenceswereretrievedfromGenBankdatabaseandalignedwithMEGA4.0(Tamuraetal.2007).PhylogenetictreewasconstructedbyaNeighbor-Joiningmethod(SaitouandNei1987).Thepercentageofreplicatetreesinwhichtheassociatedtaxaclusteredtogetherinthebootstraptest(500replicates)isshownnexttothebranches(Felsenstein1985).TheevolutionarydistanceswerecomputedusingthePoissoncorrectionmethod(ZuckerkandlandPauling1965)andareintheunitsofthenumberofaminoacidsubstitutionspersite.TheAACTsequencesfromprokaryotesweretakenasanoutgroup.ExpressionofBmAACTinE.coliandPurificationofRecombinantProtein

ApairofprimerspETATBMF1andpETATBMR1(Table1)wasdesignedforcloningofBmAACTintoanexpressionvector,andthecodingregionwasamplifiedbyPCRtoincor-poratesuitablerestrictionenzymesites.Aftersequencecon-firmation,theresultingrecombinantplasmid(pET30b-BmAACT)wasmobilizedintoE.coliBL21(DE3)competentcells.AsinglecolonyofE.coliharboringpET30b-BmAACTwasinoculatedintoLBmediumcontainingkanamycin(50μg/mL)andgrowninashaking(200rpm)incubatorat37°Cuntiltheopticaldensity(OD600)reachedabout0.5–0.6.Forinductionofrecombinantproteinexpression,IPTGwasaddedtoafinalconcentrationof0.08mMandthecellswerefurtherincubatedat20°Cfor14h.Thecellswereharvestedbycentrifugationfor10minat5,000rpmand4°C.Thepelletwasresuspendedinlysisbuffer[50mMTris-HCl,pH8.0;300mMNaCl;5mMβ-mercaptoethanol(BME);10%Glyceroland1%TritonX-100]andsonicatedfor3min(10son/off)at75%amplitude.Lysozymewasaddedatafinalconcentrationof0.1mg/mLandafterincubationonicefor15min,centrifugedfor10minat10,000rpmand4°C.Supernatant(lysate)andpellet(inclusionbodiesdissolvedinureabuffer)wereanalyzedbySDS-PAGEandCoomassieBrilliantBlueG(Sigma,St.Louis,MO)staining.

RecombinantBmAACTproteinwaspurifiedbyaffinitychromatographyusingNi2+-NTAagarosebeads(Qiagen,Valencia,CA)andusedforinvitroenzymeassay.Inbrief,thecolumnwasequilibratedwithequilibrationbuffer(50mMTris-HCl,pH8.0;300mMNaCland20mMimidazole).Lysatewasloadedonequilibratedcolumnandincubatedfor2hat4–7°Conadancingshaker.Thebeadswereallowedtosettledownandwashedwith6–8columnvolumesofwashbuffer(50mMTris-HCl,pH8.0;300mMNaCland30mM

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Imidazole),untilOD280reachestoabaseline.Finally,therecombinantBmAACTwaselutedwith2.5mLofelutionbuffer(50mMTris-HClpH8.0;300mMNaCland250mMimidazole)inthreefractions.ThepurifiedBmAACTwasfurthersubjectedtogelfiltrationchromatography(HiLoad16/60Superdex-75,GEHealthcare,Sweden)andelutedfrac-tionsin20mMphosphatebuffer(pH7.5)wereanalyzedonSDS-PAGEwithTris/glycinebuffer.ThegelwassilverstainedandproteinconcentrationwasdeterminedbyBradford(Bio-Rad)method(Bradford1976)usingbo-vineserumalbumin(BSA)asastandard.ThiolysisActivityofRecombinantBmAACTProtein:313nmAssay

Substrate,acetoacetyl-CoAandco-factorCoAwerepurchasedfromSigma.ThiolysisactivityofrecombinantBmAACTwasanalyzedbymonitoringthedecreaseinabsorbanceduetodisappearanceofMg2+-enolcomplexofacetoacetyl-CoAat313nm(Oeljeklausetal.2002).ThereactionwasmonitoredonLambda650spectrophotometer(PerkinElmer,Waltham,MA)over2min.Astandardassaymixturecontained150mMTris-HCl(pH8.9),30μMacetoacetyl-CoA,5mMMgCl2and100μMCoA.Thereactionwasstartedbyadding6.98nMofpurifiedBmAACTtothereactionmixture.Theapparentex-tinctioncoefficientofacetoacetyl-CoAwas14,328M?1cm?1.Oneunitofthiolaseactivitywasdefinedastheamountofthiolasesthatcatalyzethecleavageof1μmolacetoacetyl-CoAperminunderstandardconditions.EffectofMetalIonsonThiolysisActivity

ApartfromMgCl2atdifferentconcentrations(1–10mMat1mMintervaland25mM),someotherdivalentmetalions(CaCl2,ZnCl2andNiCl2)wereaddedtothestandardreac-tionmixture,andenzymeactivitywasmeasuredinthesamewayasmentionedabove.

DeterminationofKmandVmaxofRecombinantBmAACTKineticconstantsKmandkcatfortherecombinantpurifiedBmAACTweredeterminedbyusingacetoacetyl-CoA(0.001–0.1mM)andCoA(0.001–0.5mM).Theassayreac-tionswerecarriedoutintriplicatesunderstandardassayconditionsandvaluesforKmandkcatwerecalculatedfromMichaelis-Mentenplot(R200.990)byfittingthedatatothenon-linearregressionusingsoftwareSigmaPlot12.0.RelativeandTissueSpecificTranscriptQuantificationbyqRT-PCR

TheexpressionpatternofBmAACTwasanalyzedinsixdiffer-enttissuesincludingstem,leaf,root,sepal,pedicelandpetal.

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TotalRNAwasextractedfromalltissuesandcDNAwassynthesizedasmentionedabove.An18SrRNAwasusedasinternalcontrolfornormalizationofallthereactions.Primersusedfornormalization(18S-Fand18S-R)andexpressionanalysisofBmAACT(qAACTFandqAACTR)aregiveninTable1.BrilliantSYBRGreenQPCRmastermix(2XwithlowROX,Stratagene,LaJolla,CA)andStratageneMx3000Pinstrumentwereused.qRT-PCRreactionswerecarriedoutunderthefollowingconditions:1cycleof10minat94°C,followedby40cyclesof30sat94°C,30sat55°C,30sat72°C.AmplicondissociationcurvewasalsorecordedattheendofthePCRcycles.Allreactionswererunintriplicateandrepeatedtwice.RelativeexpressionofgenewasanalyzedusingcomparativeCtmethod(2?ΔΔCt).

Fig.1aSchematic

representationoffull-lengthBmAACTshowingpositionofprimersusedforPCRandexpectedampliconsize.bNu-cleotideanddeducedaminoacidsequencesofAACTfromBacopamonnierawith5′(95bp)and3′(300bp)UTRsfollowedbyapoly-Atail.Cat-alyticresidues(Cys99Cys391His361)andpoly-Asignalsite(AATAAA)arecircledandunderlined,respectively

Results

MolecularCloningandCharacterizationofFull-LengthBmAACTcDNA

Anapproximate770bppartialfragmentofBmAACTwasamplifiedbydegenerateprimerscATF2andcATR3.UpstreamanddownstreamofpartialBmAACTwereamplifiedbyRACEPCRwithgenespecificprimersandprimerssuppliedwithRACEkit,asmentionedin“Materialandmethods”.Approximately350bp5′RACEPCRproductwithprimers5′RACEATBMNstdR1andNUPwasclonedinthepGEM-Teasyvectorandsequenced.Similarly,approximately1,300bp3′RACEPCRproductwithprimers3′RACEATBMnested

F1

552PlantMolBiolRep(2013)31:547–557

and3′NGRPwasclonedandsequenced.Thefull-length(1,628bp)BmAACTgene(GenBankaccessionno.FJ947159)containedanORFof1,215bpflankedby95bp5′-UTRand300bp3′-UTR(Fig.1a).Thepoly(A)signalsite(AATAAA)wasfoundat31bpupstreamfromthepoly(A)tail.Onthebasisof5′and3′RACEsequences,extremesprimersweredesignedfromstartandstopcodonandORF(1,215bp)wasamplifiedbyPCRusingcDNAasatemplate.Theobtainedampliconwasclonedandsequenced(Fig.1b).Thededucedproteinwasfoundtobe405aawithanexpectedmolecularmassof41.32kDaandpIof6.15(ExPASyserver).ThededucedaminoacidsequenceofBmAACTwascom-paredwithdeducedaminoacidsequencesofAACTfromotherorganismsavailableintheNCBIdatabase.TheBmAACTsequenceexhibited90.6,90.0,89.1and83.9%identityand97.0,96.1,94.8and89.2%similaritywithPicrorhizakurrooaa,Camelliaoleifera,NicotianatabacumandSalviamiltiorrhiza,respectively.Twocystineandone

Fig.2aMultiplesequencealignmentsofdeducedaminoacidsequenceofbiosyntheticthiolasefromdifferentplants.GreenConservedaminoacidresidues,redthreeconservedaminoacidresidues(Cys99,Cys391andHis361)intheconserveddimerinterface.bConserveddomaindatabase(CDD)searchforBmAACTontheNCBIserver

histidineresidues,whicharehighlyconservedamongthethiolasesfromdifferentsources,areimportantforcatalyticactivity(Andersonetal.1990;Williamsetal.1992).ThesethreeresiduescorrespondtoCys99,Cys391andHis361inBmAACT(Fig.2ahighlightedinred).Abasicaminoacid(Asn,Thretc.)residuepresentatthelastpositionoftheactivesiteisspecificforAACT(Yangetal.1990).InBmAACT,Asn(N)residueispresentatthelastpositionoftheactivesite.ThroughtheNCBIconserveddomainsearch,BmAACTshowedaspecifichitagainstthiolaseandcondensingenzymesuperfamily.Thecatalyticactivesitesanddimerinterfaceregionswerealsopredicted(Fig.2b).

AphylogenetictreewasconstructedusingknownAACTsequencesfromabroadrangeoforganismsincludingdicots,monocots,yeastsandbacteria.ThedicotAACTswereclus-teredinonegroupwhereBmAACTwascloselyrelatedtothatofS.miltiorrhiza,withAACTofP.kurrooaandN.tabacumformingdistinctsubgroups(Fig.3

).

PlantMolBiolRep(2013)31:547–557Fig.3Phylogenetic

relationshipofBmAACTwithotherAACTs.Theevolutionarytreewasformedusingthe

ClustalX2programandMEGA4.0.2softwarewithneighbor-joining(NJ)method.Accessionnumbersaregiveninbrackets

553

ExpressionofBmAACTinE.coliandPurificationofRecombinantProtein

TheORF(1,215bp)ofBmAACTwascloneddirectionallyintoexpressionvectorpET30b(+)withNdeIandXhoIrestrictionsites.TherecombinantpET30b(+)plasmidhar-boringBmAACTwasover-expressedinE.coliBL21(DE3).ThelevelofexpressionandsolubilitywasoptimizedatdifferentIPTGconcentrations,temperatureanddurationofinduction,andanalyzedonSDS-PAGE(datanotshown).Itwasfoundthat0.08mMIPTGfor14hat20°Cgavemaximumover-expressedproteininthesolublefraction.Approximately43kDaproteinwaspurifiedbyaffinityandgelfiltrationchromatographyusingNi-NTAagarosebeadsandSuperdex-75,respectively.Theresultsofthepurifica-tionproceduresforrecombinantBmAACTaresummarizedinTable2.Thefinalpurificationresultedin62.22foldpurificationwithspecificactivityof56U/mgandrelativelygoodrecoveryof69.1%.Figure4showsSDS-PAGEanal-ysisofelutedfractionsateachstepwithasinglepurifiedbandofBmAACTinsilverstaining(Fig.4,lanes1and2).

ThiolysisActivity

Thiolysisactivitywasdeterminedbyusingacetoacetyl-CoAasasubstrateat313nm.TheactivityofrecombinantBmAACTwasmaximalat5mMMg2+concentrationandfurtherincreaseinconcentrationswereinhibitory.AtsameconcentrationofNi2+,Ca2+andZn2+theactivitywas69%,60%and28%,respectively,incomparisontoMg2+,whereactivitywasmaximumandconsideredas100%(Fig.5).KineticParametersofBmAACT

KineticconstantsforthethiolysisreactionsweredeterminedbyfittingtotheMichaelis-Mentenequationofinitialratedatafromexperimentsincludingvaryingconcentrationofacetoacetyl-CoA(0.001mM–0.1mM)atfixedconcentrationCoAandvaryingconcentrationofCoA(0.001mM–0.5mM)atfixedconcentrationofacetoacetyl-CoA.TheKmandVmaxforacetoacetyl-CoAwere20.67μMand96.21μmol/minrespectively(Fig.6),whiletheKmforCoAwas25.80μM.KineticparametersaresummarizedinTable3

.

Table2PurificationofrecombinantBacopamonnieraacetoacetyl-CoAthiolase(BmAACT)

PurificationstepsTotalvolume(ml)Totalprotein(mg)Totalactivity(U)Specificactivity(U/mgofprotein)FoldpurificationYield(%)CrudelysateNi2+-NTASuperdex-75

601510

9001510

810582560

0.938.856

143.1162.22

10071.8569.1

554Fig.4SDS-PAGEanalysisofcrudelysateandpurifiedrecombinantBmAACTfromEscherichiacoliBL21(DE3)cells.Lanes:MStandardmolecularweightmarker;1,2purified(elutedfractionsfromNi-NTAagarosebeadsandSuperdex-75respectively)recombinantBmAACT;3crudebacterial

lysate

Tissue-SpecificExpressionAnalysisofBmAACT

Todeterminethetissue-specificexpressionpatternofBmAACTinB.monniera,totalRNAwasextractedfromroot,stem,leaf,pedicel,sepalandpetal,andqRT-PCRwasperformed.InBacopa,BmAACTwasexpressedmoreinrootsandpetals,atlevelsapproximately5-and3-fold

Fig.5Effectofdifferentdivalentmetalionconcentrationsonthiolysis

activityPlantMolBiolRep(2013)31:547–557

Fig.6Effectofacetoacetyl-CoAconcentrationonthethiolysisactiv-ityofAACTfromB.monniera.KmandVmaxweredeterminedwithvariedconcentrationofacetoacetyl-CoA(0.001mM–0.1mM)andfixedconcentrationofCoA(0.1mM).Thedataisobtainedfromduplicateexperiments.InsetDoublereciprocalplot

higherthanpedicel,respectively.Incomparisontostem,approximately3-and2-foldhigherexpressionwasobservedinrootsandpetals,respectively.Ontheotherhand,2.7-and1.67-foldhigherexpressionwasmonitoredinrootsandpetals,respectively,comparedtosepal(Fig.7).

Discussion

Thiolasesplayanimportantroleinmanybiosyntheticanddegradativemetabolicpathways.Acetyl-CoAacetyltransferase(oracetoacetyl-CoAthiolase)catalyzestheformationofacetoacetyl-CoAandisinvolvedinbiosyntheticpath-wayssuchasPHBsynthesisinprokaryotes(Slateretal.1998)andisoprenoidbiosynthesisviatheMVApath-wayinplants(VollackandBach1996;Bachetal.1999).Bacosidesaretriterpenoidsaponins,presentasmainbioactivecompoundinthemedicinalherbB.monniera.Thisplantisusedmainlyinthetreatmentofanxietyandimprovingmemory(Singhand

Dhawan

Table3KineticconstantsofrecombinantBmAACTReactionparameterValue(mean±SE)aVmax(μmol/min)b96.21±1.90Km(μM)

Acetoacetyl-CoA20.67±0.79CoA

25.80±1.1Kcat(min?1)

2.30×105Kcat/Km(min?1μM?1)

11.13×103

a

Thevaluesshownarethemeans±SEofduplicateexperiments

b

Vmaxwascalculatedforacetoacetyl-CoAatfixedconcentrationofCoA(0.1mM)

PlantMolBiolRep(2013)31:547–557Fig.7Quantitativereal-timePCRanalysisofBmAACTmRNAtranscriptsindifferentfloralandvegetativeparts.Val-ueswerenormalizedbythe18SrRNAexpressionanddataarepresentedasmeans±SEfromthreeexperimentalreplicates

555

1997).AlthoughBacopahasbeeninvestigatedinvari-ouslaboratoriestodevelopanextractionprocedureforbacosidesandimproveitscontentbymeansofplanttissueculture,themolecularbasisofitsbiosynthesisisstillunexplored.

Inthispresentstudy,acetoacetyl-CoAthiolasefromB.monnierawasclonedandcharacterized.Full-lengthBmAACTwasisolatedbyRACE-PCRandBLASTanalysisshowedsignificanthomologywithreportedAACTsattheNCBIGenBankdatabase.Aconserveddomaindatabase(CDD)searchrevealedthatitbelongstothesuperfamilyofcondensingenzymesandshowedspecifichitswiththio-lase.TheconservedresiduesCys99,Cys391andHis361werealsofoundinBmAACTbyalignmentwithotherreportedthiolases(Fig.2a,b),whichisinagreementwithotherreports(Kursulaetal.2005;Andersonetal.1990;Williamsetal.1992;ModisandWierenga1999).PhylogeneticanalysisofBmAACTshowedthatitisrelatedcloselytoS.miltiorrhiza(Fig.3).InS.miltiorrhiza,micro-arrayanalysisidentifiedtheinvolvementofAACTgeneintanshinonebiosynthesisandexpressionprofilewascorrelatedwithsecondarymetaboliteaccumulation(Cuietal.2011).ThiolysisactivityofrecombinantBmAACTwasmaxi-mumat5mMMg2+concentrationandfurtherincreaseinconcentrationwasinhibitory,withactivityof76%at10mMMg2+(60%at25mMMg2+,datanotshown)(KimandCopeland1997).Theactivitywith5mMNi2+andCa2+wasintherangeof60–70%ofthatwith5mMMg2+.InthecaseofNi2+,adrasticdecreaseinactivitywasobserved,becomingalmostnegligibleat10mMconcentra-tion,whichmaybeduetonon-specificbindingofNi2+totheactivesitehistidineresidueofBmAACT.Inthecaseof

Ca2+andNi2+ions,approximately73%activitywasob-servedatconcentrationsof3mMand6mM,respectively.InthepresenceofZn2+at5mMconcentration,theactivitywasapproximately28%ofthatwith5mMMg2+andnegligiblewithafurtherincreaseinZn2+concentration(Fig.5).

Hyperbolicsubstratekineticswereobservedforthethi-olysisreactionofrecombinantBmAACTwheneitheracetoacetyl-CoAorCoAconcentrationswerevaried.BmAACTshowsaslightlyhighersubstrateaffinityforacetoacetyl-CoA(Km020.67μM)ascomparedtoCoA(Km025.80μM).Previousenzymekineticstudiesonthio-lasesfromvarioussourcesshowedCoAasapreferentialsubstrateoveracetoacetyl-CoA(MengandLi2006;Hedletal.2002;ModisandWierenga2000),whileafewreportssuggestedacetoacetyl-CoAasafavoredsubstrate(Suzukietal.1987;KimandCopeland1997).Inplantssuchaslikesunflower,glyoxysomalacetoacetyl-CoAthiolasefromcotyledonsdisplayedaKmof11μMandkcatof2.164×103min?1foracetoacetyl-CoAatfixedCoA(240μM)concen-tration(Oeljeklausetal.2002).However,inourcase,Kmvaluesforbothacetoacetyl-CoAandCoAareinthesameorderofmagnitudeandexhibitedhighercatalyticefficiency(kcat2.30×105min?1)aswellasspecificityconstant(kcat/Km11.13×103min?1μM?1)comparedtoearlierreports.

Tissue-specificexpressionanalysisofBmAACTbyqRT-PCRinalltestedtissuesshowedlowestexpressioninped-icel.Higherexpressionswereobservedinrootandpetals(approximatelyfive-andthree-foldhigherthanpedicel,respectively)followedbysepals,stemandleaftissues(Fig.7).InArabidopsis,theAACT2geneaccumulatesmoreinseedling,rootsandinflorescencesascomparedto

stem

556andleaves(Ahumadaetal.2008).AsimilarpatternoftranscriptaccumulationwasobservedinArabidopsisforsomeotherpathwaygenessuchasHMG-CoAreductase,MVAkinase,farnesyldiphosphatesynthaseandsqualenesynthase(Enjutoetal.1994;Cunilleraetal.1996;Kribiietal.1997;Lluchetal.2000).ThissuggeststhatBmAACTisinvolvedinbiosyntheticpathways.

Inconclusion,thebiosyntheticthiolasegenefromB.mon-niera(BmAACT)hasbeencloned,over-expressedinE.coliandpurifiedbydifferentchromatographictechniques.ThecatalyticefficiencyofBmAACTwasfoundtobeexception-allyhighascomparedtoAACTsfromothersources.qRT-PCRanalysisrevealedthattheexpressionprofileofBmAACTisdistributedmoreorlessthroughoutallplantparts,withhighertranscriptaccumulationinrootsandinflorescenceascomparedtoothertissues.TheroleofAACTsinisoprenoidbiosynthesisinplantsisstillnotveryclear.InitiallyAACTwasnotconsideredasaregulatoryenzymeintheisoprenoidpathway,butrecentreportsfromArabidopsis,SalviaandMedicagoshowedthatthisgeneplaysanimportantroleinisoprenoidbiosynthesis.Furthermore,theexpressionprofilewascorrelatedwithsecondarymetaboliteaccumulationandabioticstressresistance.ThewiderangeofpharmaceuticalandmedicinalapplicationsoftriterpenoidsandalkaloidsitselfexplainsimportanceofBacopainmedicinalchemistry.FurtherstudiesareplannedtoinvestigatethefunctionalroleofBmACCTinplantsecondarymetabolismbydevelopingtransgeniclineswithimprovedtriterpenoidcontentanditsadaptabilitytothriveindifferentabioticstressconditions.

AcknowledgmentThisworkwassupportedbyCouncilofScientificandIndustrialResearch-NetworkProject(CSIR-NWP),NewDelhi,India.

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