Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity 英文参考文献.docVIP

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Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity 英文参考文献.doc

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Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity 英文参考文献

SiteDirectedMutagenesisofSchizosaccharomyces pombeGlutathioneSynthetaseProducesanEnzyme withHomoglutathioneSynthetaseActivity TamaraDworeck1,MartinZimmermann2* 1DepartmentofBiology,RWTHAachenUniversity,Aachen,Germany,2InstituteofBiologieIV-AppliedMicrobiology,RWTHAachenUniversity,Aachen,Germany Abstract ThreedifferentHis-tagged,mutantformsofthefissionyeastglutathionesynthetase(GSH2)werederivedbysite-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5a and affinity purified in a two-step procedure.AnalysisofenzymeactivityshowedthatitwaspossibletoshiftthesubstratespecificityofGSH2fromGly(km 0,19;wild-type)tob-AlaorSer.Onemutation(substitutionofIle471,Cy472toMetandValandAla485andThr486toLeu andPro)increasedtheaffinityofGSH2forb-Ala(km0,07)andloweredtheaffinityforGly(km0,83),whichisacharacteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead theaffinity of GSH2for Ser (km 0,23) as asubstrate, while affinity to Gly was preserved (km 0,12). This providesanewbiosyntheticpathwayforhydroxymethylglutathione,whichisknowntobesynthesizedfromglutathione andSerinareactioncatalysedbycarboxypeptidaseY.Thereportedfindingsprovidefurtherinsightintohowspecificamino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they supporttheevolutionarytheorythathomoglutathionesynthetaseevolvedfromglutathionesynthetasebyasinglegene duplicationevent. Citation: Dworeck T, Zimmermann M (2012) Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with HomoglutathioneSynthetaseActivity.PLoSONE7(10):e46580.doi:10.1371/journal.pone.0046580 Editor:GiovanniMaga,InstituteofMolecularGeneticsIMG-CNR,Italy ReceivedJune13,2012;AcceptedAugust31,2012;PublishedOctober16,2012 Copyright: ? 2012 Dworeck, Zimmermann. This is an open-access article distributed under the terms of the Creative Co