来自于变性链球菌的脱氧胞嘧啶核苷酸脱氨酶的表达纯化结晶以及初步晶体学研究

1、来自于变性链球菌的脱氧胞嘧啶核苷酸脱氨酶的表达，纯化，结晶以及初步晶体学研究生物化学与生物物理进展ProgressinBiochemistryandBhysics,2006,33(7):673-,676,)ln)n.pibb.ac.caPIlaf!Expression,Purification,CrystallizationandPreliminaryXrayStudiesofaDe0xycytidylateDeaminaseFromStreptococcusmutans霉HOUHaiFeng,GAOZeng-Qiang,XUJianHua",XURuiLILiQin,LILanFe

2、n3),LLNGYu_He,SUXiaoDong3),LI【JPeng",NDing-Chang",DONGYu-Hui'"(讽SynchrotronRadiationFacility,InstituteofHighEnergyPhysics,TheChineseAcademyofSciences,Being100049,China;ZGraduateSchoolofTheChineseAcademyofSciences,Be堍100049China;3CollegeofScience,拍Univers&100871,China)Abstr

3、actDeoxycytidylate(dCMP)deaminaseisallblz'ylnebelongedtodl姗cytdeamfamily.ThedCNdeaminasefromStreptococcusma,UAl59wasclonedandexpressedin最coli.andpurifiedtohomogeneity.TheFPLCsizeexclusionchromatographyanalysisrevealsthatthes-mI/on$dCNdeaminaseformshexamerinsolution.Theproteinwascrystallizedusing

4、hangingdropvaponr-diffusionmethodanddiffractedtoaresolutionof3.1A.ThediffractiondatawerecollectedatBSRFbeamline3wlA.ThecrystalsbelongtoP2I3spacegroup,withunitcellparametersa=b=c=ll3.2A,ot=卢=7=90.Assumingtherearetwosubunitsperasymmetricunit.theMatthewsCO0clentis3.6ADa-1.Thisisthefirstcrystallizationr

5、eportofthewild-typedeoxycytidylatedeaminase.Keywordsdeoxycytidylatedeaminase,Streptococcusma,allostcricregulation,crystallizationWild-typedeoxycytidylatedeaminase,ahexamericallosterice,mebindingzincion,whichisrequiredforcatalyticactivity,belongstodC【E'cytdeamfamilyaccordingtoPfamsearcht".Th

6、eactivityoftheenzymeisalIostericallvregulatedbytheratio0fdC:TPtod1n渊.notonlyineukaryoticcellsbutalsoinT.evenphageinfectedEscherichiacoli.withdC1Pactingasanactivatorandd1门asaninhibitor.DeoxycytidylatedeaminasecatalyzesthedeaminationofdC【E'todI.providingthenucleotidesubstrateforthymidylatesynthase

7、,whichisimportantinDNAsynthesisandcancerchemotherapvM.dC【E'deaminaseexistsinmosteukaryoticandbacterialorganisms.TherearetwoclassesinthesedC【E'deaminasesC2).MammaliandC【E'deaminasecontainsasinglezincionpersubunitforcatalyticactivity.whilebacteriadC【E'deaminasecontainstwozincion

8、spermonomer,oneisrelatedtothecatalyticactionindispensablyandtheotherseemstobeimportantforthestructuralintegrityoftheenzymeandforbindingtothephosphategroupofthesubstrate.Bothclassesfunctioninhexmericform.Inthepastdecade.dC【E'deaminase丘OmT4-bacteriophagewasstudiedextensivelyisD】inenzymeactivitiesa

9、ndmutationproperties.ThedC【E'deaminasesfromT4-bacteriophageandSmutansshowsignificantsimilaritiesintwosequencesegments,whichshare31.6%identity.OneofthesegmentslocatesatthecatalyticsiteoftheT4-bacteriophagedC【E'deaminaseaccordingtothestructure【111.WepurifiedandcrystallizeddCMPdeaminasefroms.mu

10、tmtsuccessfully,andreportinthispaperthepreliminarycrystallographicstudiesofthefirstcrystallizedwild-typedC【E'deaminase.Thedetermination0fdC【E'deaminasestructuremayprovideclueofdetailedstudiesofcatalyticmechanismandsubstratebindingproperties,alsoapossiblemodelforthemechanismfortheallostericre

11、guhfion0fthedC【E'deaminase.'ThisworkwassupportedbygrantsfromTheNationalFoundationofTalentYouth(30325012),theKeyImportantProjectandotherprojectsfromTheNationalNaturalScienceFoundationofChina(30530190,10575I13)."Correspondingauthor.Tel:8.6-10-88233090,Fax:86-10-88233201,E-mail:dongyhihcpc

12、 Received:Fdmtaty5,2006Accepted:March31,2006674生物化学与生物物理进展Prog.Biochem.Biophys.1Materialsandmethodsl_IProteinexpressionandpurificationThedCMPdeamfoasegeBewasamplifiedffomStreprJ.Jf-【rfn,"¨sUAI59eenornicDNAbypolymerasechainreaction(PCR)usingprimerscomEBF15cgcggatccatgacgaatagactttcttggc3)an

13、dcomEBRl5cggctcgatactmcacctaatttca3')ThePCRamplifiedfragment,vasthenligatedtoexpressionvectorpET28a(Novagen),whicheonminsNtermjna】His6tag.TherecombinantdCMPdeamjnasek;asexpressed"nstraii1BL21(DE3)ThetrailstinedcellswerefirstcuturcdovernightiI140nt】LuriaBrothfLB)mediawith50mlkanamvcinthenlra

14、nsrefredtolLLBmediawith5011】kanamycinand盯owna【37Whenopticaldensity'(4)reachedto0.6O.8.thecellswercinducedwith1mmol/LisopropyB.Dthiogalactopyranoside(IPTG1r0r4hThecellswerellarvestedbycentrimgat'onat6000r/'rainforl0min.Bacteriaweresuspendedwithbufferof20mmol正Tris'卜IC】pHg,0,500mmol,&qu

15、ot;LNaCl(bL【n计A1.1nrmol/LphenymcthanesulbnylfluoridefPMSF)Atierdisruptionofthecellsbysonication.thecelldebriswasremovedbycentrifuationatl6000r/rain】1nr45rain.Thesolublelysisfractionwasloadednoteanickelchelationcolumn(5mlAmersham1equilibraleditlbL【胁rAandthetargetedproteinwasthenelutedbya1inergradient

16、ofl0t0500mmol,"Limidazoleinbu蕊rAThedCMPdeaminasefractionswerecollectedandconcentratedbvultrafiltration(30000MWCO1jnanAmiconceI【IMipore,CA.USA).TheproteinissensitivetoionstrengthandinsolubleuntiltheconcetrationofNaCIreaehs500mmol/Ljnthebu壤:rSowe1oadtheproteinsolutionontoaSuperdex200cl20m1Amersha

17、m)gelfiltrationcolumnequilibratedin20mJnoL"LTrisHC1bufferpH8.0.500mmoFLNaClAI1山epurificationwascarriedOUtinroomtemperatureo11anAK下APurifierSystemfAmersham11.2OligomericandhomogeneousconditionThproteinpufftywasmonitoredbySDSPAGEandshowedameIecularmassof20kuThemolecularmassestimatedfromge1.filtra

18、tionchromatographyisaboutl18ku.whichindicatesthattheHisdClPdeaminaseisahcxameticproteinTheresulIisinconsistantwithpreviousvorkl.ThedCMPdeaminasewasconcentratedto162-z.determinedbyBi0一Radproteinassaykit.andstoredasozenaliquotsatl93K.13CrvstaIljzati0nThec0ncetrati0nofthedClPdeaminaseusedforco'stal

19、lizafionscreeningwas16gdLand8L.respecti!,hebuffercontaining20mmol,'LTnsHClpH7.5.500mmol/LNaClTheinitalscreeningwerecarriedOOIbyhangingdropvaponr-diffosionmehodsusingHamptonResearchCrystalScreenland2(HamptonResearch,Riverside.CA.USA)atroomtemperature,l】ofproteinsolutionwasmixedwithl1ofres

20、ervoirsolutionandequilibratedagainst500LL1ofreservoirsolutionina24.we1cultureplate.GOOC【shapedcrvstaIswereobtainedinmare,conditionscabout301afterabout2h.butthedifiractionresolutionisvepoor.onlyabout7AtieroptimizingtheconcentrationoftheprecipitantsandionsWendh.thereso】otiOHofthedjfiractionisabout5Aun

21、dertheconditionof1.5irtol,'1-(NH4)Sn,O.1m()TrispH85,15%0.v'v)glycele1.Inordertoimprovethedifli'actionability.dehvdrationapost-crystallizationmethodwasusedWeaddglycerolin【hereselwoirsolutiontoafina【concentrationof25%.andthediffractionabilityofthecrystalwasimprovedto3IAafter24hThephotoofsi

22、nglecrystalisshownbelow(Figure1).tFig.1His-dCMPdeaminasecrystalgrownbythehartging-dropmethodin1.2molfL(NHD04,0.1mol/LTrispH8.5.15%Iglycerolafterdehydrationusingglycerolat289KII4XraydatacollectionandanalysisDiffractiondataofdCfl'deaminasccrysta1werecolleeredatBSRFfBeijingSylachrotronRadiationFaci

23、litv1.beamline3W1Aat10OKTooptimizetheZnanomaloussignalf0rZnMAD.IheSO1.treewavelengthWaSsettoI2829(forpeak).12835A(foredge)and1.200Afforremote),respectively,accordingtotheXAFSfdatawasn0Ishown).The2006:3317l侯海峰等:来自于变性表达.纯化.氯酶的diffractionimagewasshowninFigure2ThedatawerecollectedusingaMARI65CCDdetector

24、in10oscillationstepsoverarangeof1l0.Thedatawereprocessed.scaledandmergedusingDENZOandSCALEPA.CKfromHKL2000progampackage'.疆Fig.2Diffractionpatternnfthe.murans'dCB'IPdeaminasewilh3.1diffraeti0nrIuti0n2Resultsanddiscussion675Wehavesucceededi1ltheexpression0urificationandc'stallizationof

25、theS,ldCdeaminase.TheenzymeelutedoilaSuperdex200size.exclutioncolumnasahomohexamerwhichwassatisfiedbvinitialresult.Thepurifiedproteinmigratedasasingle20kuf150residualsIoill5SDSPAGEgelInitially,crystalsweregrownbyhanngdropvapourdiffasionmethodsusingHamptonResearchCo'starScreen1and2About30solulion

26、sofHamptonResearchCrystalScreenland2producedwelllookingcrystals.Afteroptimizingthecertainconditionwclldiffractedsinglec.'stalsareobtainedfrom1.2mol/L(NH4)SO_01mol/LTt'ispH8.5,15%",'1glycerolTheco,stalot'dCMPdeaminasedifli'actedto31andbelongedtospaceWoupP2.3withunit.celIparam

27、eters=b:r=_l32.=8=90.Assumingthepresenceoftwopolypeptidcsperasymmetricunit,thecalculaledMatthewscoefficientvaluexvas36Da,withasolventcontent0f66.2%(Table1)StructuredetenninationbvZn-MADisunderwa,.'.TabletCrystallographicparametersanddala?collectionsatistiesValuesinparenthesesamforthehighestresol

28、utionshell.一一3-一,.一X.twherethesummationis0veralJreflectionsMatthewsctzefficient.IAcknowledgmentWewouldliketothankProfessorWeiMinGongandDrZhi-YiWciofInstituteofBiophysicsofTheChineseAcademyofSciencesforjnvaluableadviceinstructuredeterminationReFences1BatemanComL-DurbinR,ThePlainproteinfamilies.676.生物

29、化学与生物物理进展Prog.Biochem.Biophys.2006;33(7)database.NucleicAcidsRes,2004,32(Databaseissue):D1381412FrankMIGladysFM.Mechanismsofenzymemodulioninvolvingdeoxycytidylatedeaminaseandthymidylatesynthetase.AdvEnzymeRegul,1970,8:55663CohenSS.VirusInducedEnzymes.NewYork:ColumbiaUniversityPress.1968.891474Hemand

30、ez-SantiagoB,PlacidiL'Creaon-ScoaE,etdf.PharmacologyofB?L-thymidineandBL-2'-deoxycytidineinHepO2cellsandprimaryhumanhepatucytes:relevancetochemotherapeuticefficacyagainsthepatitisBvirus.AntimicrobAgentsChemother,2002,46(6):1728l7335LiouJY'KrishnanP,HsiehCC,etAssessmentof血eeffectofphospho

31、rylatedmabolitesofantihumanimmunodeficiencyvirusandanti-hepatitisBviruspyrimidineanalogsonthebehaviorofhumandeoxycytidyhtcdeaminase.MolPharmacol,2003,63(1):lO5llO6CacciamaniT,VitaA,CfistalliG,etdf.Purificationofhumancytidinedeaminase:Molecularandenzymaticcharacterizationandinhibitionbysyntheticpyrdi

32、neanalogs.ArchBioohemBiophys,l991,290(2):2852927WentworthDF.Wolfenden&Cytidinedeaminoses(fromEscherichiacoliandhumanliver).MethodsEnzymo1.1978,51:40l4O78J0hnTM,RuthES,OladysFM,etdf.T4-phagedeoxycytidylatedeaminaseisametalloproteincontainingtwozincatomspersubunit.JBiolChem,1993,268(4):2288229

33、l9JOhnTM,JaroslawMC,GladysFM,etdf.IdentificationofasitenecessaryforallostericregulationinT4-phagedeoxycytidylatedeaminase.Biochemistry,l994,33(8):2lo42ll2lOKathleenMM,LindaJW,JohnTM,eto1.Protein-proteininteractionsinvolvingT4phage?codeddeoxycytidylatcdeaminaseandthymidylatesynthase.JBiolChem,1996,271(38):2303723O43llRamiFrankM,GladysFM,eto2.Three-dimensionalstructureoftheR115EmutantofT4-bac