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ChangingtheSubstrateReactivityof2-Hydroxybiphenyl3-MonooxygenasefromPseudomonasazelaicaHBP1byDirectedEvolution*Receivedforpublication,October17,2001,andinrevisedform,November27,2001Published,JBCPapersinPress,December3,2001,DOI10.1074/jbc.M110018200AndreasMeyer‡,AndreasSchmid‡,MartinHeld‡,AdrieH.Westphal§,MartinaRo¨thlisberger‡,Hans-PeterE.Kohler¶,WillemJ.H.vanBerkel§,andBernardWitholt‡Fromthe‡InstituteofBiotechnology,ETHZ,SwissFederalInstituteofTechnology,ETHHo¨nggerberg,HPT,Zu¨richCH-8093,Switzerland,the§LaboratoryofBiochemistry,DepartmentofAgrotechnologyandFoodSciences,WageningenUniversity,Dreijenlaan3,WageningenNL-6703HA,TheNetherlands,and¶EnvironmentalMicrobiologyandMolecularEcotoxicology,EAWAG,SwissFederalInstituteofEnvironmentalSciencesandTechnology,Du¨bendorfCH-8600,SwitzerlandThesubstratereactivityoftheflavoenzyme2-hy-droxybiphenyl3-monooxygenase(EC1.14.13.44,HbpA)waschangedbydirectedevolutionusingerror-pronePCR.Insituscreeningofmutantlibrariesresultedintheidentificationofproteinswithincreasedactivitytowards2-tert-butylphenolandguaiacol(2-methoxyphe-nol).Oneenzymevariantcontainedaminoacidsubsti-tutionsV368A/L417F,whichwereinsertedbytworoundsofmutagenesis.Thedoublereplacementim-provedtheefficiencyofsubstratehydroxylationbyre-ducingtheuncoupledoxidationofNADH.Withguaiacolassubstrate,thetwosubstitutionsincreasedVmaxfrom0.22to0.43unitsmg1proteinanddecreasedtheKmfrom588to143M,improvingkcat/Kmbyafactorof8.2.With2-tert-butylphenolasthesubstrate,kcatwasin-creasedmorethan5-fold.Anotherselectedenzymevar-iantcontainedaminoacidsubstitutionI244Vandhada30%higherspecificactivitywith2-sec-butylphenol,guaiacol,andthe“natural”substrate2-hydroxybiphe-nyl.TheKmforguaiacoldecreasedwiththismutant,buttheKmfor2-hydroxybiphenylincreased.Thepri-marystructureofHbpAshares20.1%sequenceidentitywithphenol2-monooxygenasefromTrichosporoncuta-neum.Structurehomologymodelingwiththisthree-do-mainenzymesuggeststhatIle244ofHbpAislocatedinthesubstratebindingpocketandisinvolvedinaccom-modatingthephenylsubstituentofthephenol.Incon-trast,Val368andLeu417arenotclosetotheactivesiteandwouldnothavebeenobviouscandidatesformodi-ficationbyrationaldesign.2-Hydroxybiphenyl3-monooxygenase(EC1.14.13.44;HbpA)belongstothefamilyofflavoproteinhydroxylases(1–3).Theseenzymesareinvolvedinmanyimportantbiologicalprocesses,suchasthebiosynthesisofcholesterolorthedegradationofxenobioticsinmammalsandinnature(4–6).HbpAwasfirstfoundinPseudomonasazelaicaHBP1,asoilbacteriumthatisabletogrowonthefungicide2-hydroxybi-phenylassolesourceofcarbonandenergy(7).HbpAcatalyzestheortho-hydroxylationof2-hydroxybiphenylto2,3-dihydroxy-biphenyl,whichisthenconvertedto2-hydroxy-6-phenyl-6-oxo-2,4-hexadienoicacidbyametaringcleavagedioxygenase(HbpC).2-Hydroxy-6-phenyl-6-oxo-2,4-hexadienoicacidishy-drolyzedbyHbpDtobenzoateand2-hydroxy-2,4-pentadienoicacid(7,8),whicharefurthermetabolizedviaintermediatesalsoformedintheanalogousbiphenyldegradationpathway(9,10).HbpAhasabroadsubstratespectrum,catalyzingtheregioselectiveortho-hydroxylationofawiderangeof2-substi-tutedphenolstothecorrespondingcatechols(Fig.1)(7,11).Recently,thehbpAgenewasclonedintoEscherichiacoli,andthisrecombinantbiocatalysthasbeenusedfortheproductionofdifferent3-substitutedcatechols(12,13).Oneofthese,3-phenylcatechol,wasproducedonakilogramscale,showingthatthebiocatalyticproductionof3-substitutedcatecholsisapossiblealternativetochemicalsynthesisroutes(14).HbpAmutantswithanalteredsubstratereactivityshouldallowthesynthesisofcatecholsthatarenotsynthesizedbywild-typeHbpA.Rationalproteindesignbasedonaknownthree-dimensionalstructurehasbeenusedforsuchpurposes(15–18),butrandomapproacheshavelatelybecomemorepop-ular(19).Directedenzymeevolution(20–23),themostoftenusedstrategy,wasappliedtoimprovesubstratespecificity,activity,enantioselectivity,orthermostability(21,24–27).HerewereportontheuseofdirectedevolutiontochangethesubstratereactivityofHbpA.WeincreasedthespecificactivityofHbpAtowards2-hydroxybiphenyl,2-sec-butylphenol,guaia-col(2-methoxyphenol),and2-tert-butylphenol.Moreover,asig-nificantincreaseintheefficiencyofNADHutilizationwasachievedwithonemutantmonooxygenase.Theseresultsareinterpretedatthestructurallevelwiththehelpofathree-dimensionalmodelofHbpA.MATERIALSANDMETHODSChemicals,BacterialStrains,andPlasmids—E.coliJM101andplas-midpAA1wereusedforcloningandgeneexpression.PlasmidpAA1isapUC18(28)derivativeharboringthehbpAgeneasaSalI/NsiIfrag-ment(29)clonedintotheSalI/PstIsitesofthepUC18polylinker.CommerciallyavailablechemicalswerepurchasedfromFlukaAG(Buchs,Switzerland).CatalasefrombeefliverwasobtainedfromRocheMolecularBiochemicals.TaqDNApolymerase,restrictionenzymes,andT4DNAligasewerepurchasedfromRocheMolecularBiochemi-cals.2,3-Dihydroxybiphenyland3-sec-butylcatecholwerepreparedbywhole-cellbiotransformations,usingarecombinantE.coliJM101con-tainingthehbpAgene(14).RandomMutagenesis—ThehbpAgene(1758bp)inpAA1wasam-plifiedusinginvitromanganesemutagenesis(30).ForthePCR,theM13/pUC-40primers(MWG-BiotechGmbH,Mu¨nchenstein,Switzer-land)wereu
本文标题:Changing the Substrate Reactivity of 2-Hydroxybiph
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