极限环境微生物学会志

Vol.4,20051ISSN1348-5474July2005JournalofJapaneseSocietyforExtremophilesVol.4,20052Vol.4,200531010100o40100o100o100o100oVol.4,20054199412Hsp70PCRD-JBCD-Sulfolobustokodaiistrain7J.TrentSulfolobusshibatea(JAMSTEC)JAMSTECThermococcussp.strainKS-1(T.KS-1)BreakThroughT.KS-1ATPPointmutationVol.4,200551PyrococcushorikoshiiT.KS-1T.KS-1GroELIIGroELITRiCIITRiC8IIGroEL2ATPGroESIIGroESL.DitzelThermoplasmaacidophilusXIIGroESBuilt-inLidBuilt-inLidBuilt-inLidOpenT.KS-1IIAdvantageBuilt-inLidADPOpenATPBuilt-inLidBuilt-inLidATPII6IIAdvantage10Vol.4,2005645.5384020ThermoplasmaThermoplasmaVol.4,20057200466119781988199132Vol.4,2005820022635000200422101610403635381019542200232005522200347122332240121251Vol.4,20059pH12pH13NaK706548947312200420053322373568pHpH2pHVol.4,20051015200491010%1001027100pH3110.511.50.337101657510502238200448712113Vol.4,20051148JSEC200491125Vol.4,20051220055197019961997”Extremophiles”(Springer-VerlagTokyo)6001099.995556NHKBooks9923.814.640.833720152025Vol.4,200513382025203563-6762-7620152521OD21120055p.488-489URL(2005),Vol.4,14-24HirotaN,MatsuoT,IkedaA,YatsunamiR,FukuiT,NakamuraSRoleofanN-terminaldomainfoundintheferredoxinfromextremelyhalophilicarchaeonHaloarculajaponicaDepartmentofBioengineering,TokyoInstituteofTechnology,4259Nagatsuta,Midori-ku,Yokohama226-8501,JapanCorrespondingauthor:NakamuraS,[e-mail]:[phone].Fax:[phone].Received:December.10,2004/Accepted:January.11,2005AbstractTheferredoxin(Fd)fromHaloarculajaponicapossessesaplant-type[2Fe-2S]clusterandisstableathighsaltconcentrations.Ha.japonicaFd(HjFd)includesanN-terminaladditionaldomainrichinacidicaminoacids,aswellasacommoncoredomainthatcontainstheFe-Scluster.TheN-terminallyHAT-taggedintactHjFd(HAT/HjFd)andspinach/Ha.japonicachimericFd(HAT/Sp/HjFd)werepreparedandcharacterized.Escherichiacoli-producedHAT/Sp/HjFdandHa.japonica-producedHAT/HjFdwereproducedasholoproteins.Ontheotherhand,E.coli-producedHAT/HjFddidnotincorporatetheFe-Scluster.TheseresultssuggestedthattheN-terminaldomainofHjFdcontributedtothepolypeptidefoldingandsuccessiveFe-Sclusterincorporationunderhighsaltconditions.BothHa.japonica-producedHAT/HjFdandE.coli-producedHAT/Sp/HjFdwerestableathighsaltconcentrations(≥1.5MNaCl),althoughareductioninstabilitywasobservedatlowerconcentrations.LackoftheN-terminaldomaindidnotaffectthestabilityofHjFd,indicatingthatthecoredomainmainlycontributedtothestabilityofHjFdathighsaltconcentrations.SolubilityofE.coli-producedHAT/Sp/HjFdunderhighsaltconditionswassignificantlylowerthanthatofHa.japonica-producedHAT/HjFd.ItwasrevealedthatsubstitutionoftheN-terminaldomainofHjFdtothatofspinachFdinjuredthesolubilityofHjFd.Thus,itwasconcludedthattheN-terminaldomainofHjFdshouldperformtheessentialfunctionsforhalophilicadaptationfromthefoldingprocessthroughthefoldedstate.Keywords:extremelyhalophilicarchaeon,ferredoxin,Haloarculajaponica,iron-sulfurclusterIntroductionExtremelyhalophilicarchaea(haloarchaea)areextremophiles,thatcangrowinhypersalineenvironmentsevenintheNaCl-saturatedconcentration1).Sinceintracellularsaltconcentrationsarealsoveryhighinthesemicroorganisms2),theirentirebiochemicalsystemsadapttofunctionatsuchhighsaltconcentrations.Indeed,someproteinsfromhaloarchaeahavebeeninvestigatedtobefunctionallyactiveonlyunderhighsaltconditions3-7).Halophilicproteins,ingeneral,haveahighermolarexcessofacidicaminoacidresidues,fewerbasicresidues,andahighernumberofsmallhydrophobicresiduesthanthenonhalophiliccounterparts8,9).Ferredoxins(Fds)areubiquitousiron-sulfur(Fe-S)proteinsinvolvedinelectrontransferreactionsinavarietyofmetabolicpathways10).AsFe-Sclusters,[2Fe-2S]and[4Fe-4S]([3Fe-4S])clustershavebeenfoundinFdsfrommanyorganisms.Todate,[2Fe-2S]Fdshavebeenisolatedfromplants,algaeandcyanobacteria;othersmainlyfrombacteria11,12).HaloarchaealFdshavebeenpurifiedandcharacterizedfromextremelyhalophilicarchaeaHalobacteriumsalinarum(formerlyHalobacteriumhalobium)13),Haloarculamarismortui(formerlyHalobacteriumoftheDeadSea)14)andHaloarculajaponica15).TheseFdscontainedaplant-type[2Fe-2S]clusterineachproteinmolecule.TheFdgenesareclonedfromHb.salinarum16),Haloferaxvolcanii17)Vol.4,200515andHa.japonica18).HaloarchaealFdsarefairlyhomologoustoplantFds,buttheywere30residueslonger.AminoacidsequencealignmentofhaloarchaealandplantFdssuggestedthathaloarchaealFdsconsistedoftwodomains:anN-terminaladditionaldomain[correspondingtoPro1-Glu46inHa.japonicaFd(HjFd)]andacommoncoredomain(correspondingtoTyr47-Ile128inHjFd)18).Cysteineresiduesthatserveasligandstoironatomsarelocatedinthecoredomain.AcidicaminoacidcontentsintheN-terminaldomainsofhaloarchaealFdsareabout40%,whilecontentsinthecoredomainsareabout20%.ThecrystalstructureofHa.marismortuiFdhasbeensolved19).ManycarboxylgroupscoveredtheproteinsurfaceexceptforthevicinityoftheFe-Scluster.Furthermore,theN-terminaldomain,locatedfarfromtheFe-Scluster,contributedtoincreasethesolvent-accessiblesurfaceareasbyprovidingmoresurfacecarboxylates.Thus,itwass