Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1 英文参考文献.docVIP

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Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1 英文参考文献.doc

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SiteDirectedMutagenesisofAminoAcidResiduesatthe ActiveSiteofMouseAldehydeOxidaseAOX1 SilviaSchumann1,MinekoTerao2,EnricoGarattini2,MiguelSaggu3,FriedhelmLendzian3 ,Peter Hildebrandt3,SilkeLeimku¨hler1* 1Universita¨t Potsdam, Institut fu¨r Biochemie and Biologie, Potsdam, Germany, 2Department of Biochemistry and Molecular Pharmacology, Istituto de Ricerche Farmacologiche,‘‘MarioNegri’’,Milano,Italy,3TechnischeUniversita¨t Berlin,Institutfu¨rChemie,Berlin,Germany Abstract Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalianAOsarecharacterizedbybroadsubstratespecificityandanyetobscurephysiologicalfunction.Todefinethe physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificitysimilartothenativeproteinpurifiedfrommouseliver.TheEPRdataofrecombinantmAOX1weresimilartothose ofAOfromrabbitliver,butdifferedfromthehomologousxanthineoxidoreductaseenzymes.Site-directedmutagenesisof aminoacidsVal806,Met884andGlu1265attheactivesiteresultedinadrasticdecreaseintheoxidationofaldehydeswith noincreaseintheoxidationofpurinesubstrates.ThedoublemutantV806E/M884RandthesinglemutantE1265Qwere catalyticallyinactiveenzymesregardlessofthealdehydeorpurinesubstratestested.OurresultsshowthatonlyGlu1265is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterizedaminoacidsintheactivesite. Citation:SchumannS,TeraoM,GarattiniE,SagguM,LendzianF,etal.(2009)SiteDirectedMutagenesisofAminoAcidResiduesattheActiveSiteofMouse AldehydeOxidaseAOX1.PLoSONE4(4)