MOLECULAR CHARACTERIZATION YONG-MEI QIN OF PEROXISOMAL MULTIFUNCTIONAL 2-ENOYL-COA Biocenter Oulu HYDRATASE 2/(3R)- HYDROXYACYL-COA DEHYDROGENASE (MFE TYPE 2) FROM MAMMALS AND YEAST 1999 YONG-MEI QIN MOLECULAR CHARACTERIZATION OF PEROXISOMAL MULTIFUNCTIONAL 2-ENOYL-COA HYDRATASE 2/(3R)-HYDROXYACYL- COA DEHYDROGENASE (MFE TYPE 2) FROM MAMMALS AND YEAST Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Raahensali (Auditorium L 10), Linnanmaa, on August 13th, 1999, at 12 noon. OULUN YLIOPISTO, OULU 1999 Copyright © 1999 Oulu University Library, 1999 Manuscript received 23.6.1999 Accepted 24.6.1999 Communicated by Professor Pekka Mäenpää Professor Juhani Syväoja ISBN 951-42-5337-X (URL: http://herkules.oulu.fi/isbn951425337X/) ALSO AVAILABLE IN PRINTED FORMAT ISBN 951-42-5318-3 ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/) OULU UNIVERSITY LIBRARY OULU 1999 ÿþýüûúùøýúù÷ýöúù÷û Qin,Yong-Mei,Molecularcharacterizationofperoxisomalmultifunctional 2-enoyl-CoAhydratase2/(3R)-hydroxyacyl-CoAdehydrogenase(MFEtype2) frommammalsandyeast BiocenterOuluandDepartmentsofBiochemistryandMedicalBiochemistry,University ofOulu,FIN-90570Oulu,Finland ActaUniv.Oul.D537,1999 Oulu,Finland (Manuscriptreceived23June1999) Abstract Fattyaciddegradationinlivingorganismsoccursmainlyviatheβ-oxidationpathway.When thisworkwasstarted,itwasknownthatthehydrationanddehydrogenationreactionsinmammalian peroxisomalβ-oxidationwerecatalyzedbyonlymultifunctionalenzymetype1(MFE-1;∆2-∆3- enoyl-CoAisomerase/2-enoyl-CoAhydratase1/(3S)-hydroxyacyl-CoAdehydrogenase)viatheS- specificpathway,whereasintheyeastperoxisomesviatheR-specificpathwaybymultifunctional enzymetype2(MFE-2;2-enoyl-CoAhydratase2/(3R)-hydroxyacyl-CoAdehydrogenase). Theworkstartedwiththemolecularcloningoftherat2-enoy-CoAhydratase2(hydratase2). TheisolatedcDNA(2205bp)encodesapolypeptidewithapredictedmolecularmassof79.3kDa, whichcontainsapotentialperoxisomaltargetingsignal(AKL)inthecarboxylterminus.The hydratase2isanintegralpartoftheclonedpolypeptide,whichisassignedtobeanovelmammalian peroxisomalMFE-2. Thephysiologicalroleofthemammalianhydratase2wasinvestigatedwiththerecombinant hydratase2domainderivedfromratMFE-2.Theproteinhydratesaphysiologicalintermediate (24E)-3α,7α,12α-trihydroxy-5β-cholest-24-enoyl-CoAto(24R,25R)-3α,7α,12α,24- tetrahydroxy-5β-cholestanoyl-CoAinbileacidsynthesis. ThesequencealignmentofhumanMFE-2withMFE-2(s)ofdifferentspeciesreveals12 conservedproticaminoacidresidues,whicharepotentialcandidatesforcatalysisofthehydratase 2.Eachoftheseresidueswasreplacedbyalanine.ComplementationofSaccharomycescerevisiae fox-2(devoidofendogenousMFE-2)withhumanMFE-2providedamodelsystemforexamingthe invivofunctionofthevariants.Twoproticresidues,Glu366andAsp510,ofthehydratase2domain ofhumanMFE-2havebeenidentifiedandareproposedtoactasabaseandanacidincatalysis. MammalianMFE-2hasa(3R)-hydroxyacyl-CoAdehydrogenasedomain,whereastheyeast MFE-2hastwodehydrogenasedomains,AandB.Thepresentwork,applyingsite-directed mutagenesistodissectthetwodomains,showsthatthegrowthratesoffox-2cellsexpressinga singlefunctionaldomainarelowerthanthoseofcellsexpressingS.cerevisiaeMFE-2.Kinetic experimentswiththepurifiedproteinsdemonstratethatdomainAismoreactivethandomainBin catalysisofmedium-andlong-chain(3R)-hydroxyacyl-CoA,whereasdomainBissolely responsibleformetabolismofshort-chainsubstrates.Bothdomainsarerequiredwhenyeastcells utilizefattyacidsasthecarbonsource. Keywords:β-oxidation,fattyacid,bileacidsynthesis,enzymology Acknowledgements ThepresentstudywascarriedoutatBiocenterOulu,theDepartmentofBiochemistryand DepartmentofMedicalBiochemistry,UniversityofOuluduringtheyears1993-1999.The accomplishmentofthisworkwouldbeimpossiblewithoutthehelpofsomanypeople. Iwishtoexpressmydeepestgratitudetomysupervisor,ProfessorKalervoHiltunen, forhishospitalitytogivemetheopportunitytodomythesisworkasamemberofhis researchgroup.Hiseverlastingenthusiasm,openmindandnever-failingspiritinscience haveindefinitelyencouragedmetofulfillthiswork.IalsowishtothankProfessorsTaina Pihlajaniemi,IlmoHassinen,KariKivirikko,RailiMyllylä,SeppoVainio,RikWierenga andJohanKemminkforprovidingexcellentworkingfacilitiesandcreatingastimulating atmosphereinourweeklyseminarsatthebothDepartments. IwishtoacknowledgeProfessorsJuhaniSyväojaandPekkaMänepääfortheircritical appraisalofthemanuscript,andDr.SidneyHigleyforhercarefulrevisionofthe language.DmitryNovikov,Ph.D.,deservesmywarmestthanksforhiscriticalviewonthe subjectandapleasantcollaboration.Iamindebtedtomyco-authors,UlfHellman,Ph.D., DeanCuebas,Ph.D.,WernerSchmitz,Ph.D.,MattiPoutanen,Ph.D.,TuomoGlumoff, Ph.D.,KirsiSiivari,M.Sc.,AnttiHaapalainen,M.Sc,andMariMarttila,B.Sc.fortheir tremendouscontributiontothiswork.Formerandpresentmembersof"KHteam", especially,SirpaFilppula,HeliHelander,KariKoivuranta,Tiila-RiikkaKiema,Tanja Kokko,AhmedYagi,andTiinaKottiareappreciatedfortheirfriendshipandfruitful discussionduringandafterthework. IwishtoextendmysincerethankstowholestaffoftheDepartments.Iammuch obligedtoAilaHolappa,IrmaVuoti,AnjaMattila,RaijaPietilä,TanjaKokko,Ville RatasandMarikaKampsfortheirskillfultechnicalassistance,Anna-LeenaHietajärvifor makingourprecioussubstrates,Eeva-LiisaStefaniusforsynthesizingoligonucleotides, MaireJarvafordoingtheautomaticsequencing,JaakkoKeskitaloandKyöstiKeränenfor developingthephotosandfixingSchimadzuspectrophotometer.Specialthanksalsogoto SeppoKilpeläinen,M.Sc.,forsettingupanexcellentcomputernetwork,AuliKinnunen, Marja-LeenaKivelä,AnneliKaattariandTuulaKoretfortheirkindhelpwithsecretarial andmanypracticaltasks. Iwouldliketothankmyfather,motherandbrothersforencouragingmetostudy abroadandtheirsupportineachstepofmylife.Iamalsogratefultomyparentsandsister in-lawfortheirhelpwheneverIneed.Finally,Iowemyheartfeltthankstomyhusband 7 Qiang for his support in all matters of life and our daughter Mandi who always keeps me in touch with the true life. This work was financially supported by grants from the Sigrid Jusélius foundation, Academy of Finland and University of Oulu foundation. Oulu, June 1999 Yong-Mei Qin Abbreviations ABC ATP-bindingcassette X-ALD X-linkedadrenoleukodystrophy ATP adenosinetriphosphate bp basepair(s) cDNA complementaryDNA CoA coenzymeA FAD flavineadeninedinucleotide FADH2 flavineadeninedinucleotide(reduced) LCAD long-chainacyl-CoAdehydrogenase kDa kilodalton(s) MFE multifunctionalenzyme MFE-1 peroxisomalmultifunctional∆3-∆2-enoyl-CoAisomerase/2-enoyl-CoA hydratase1/(3S)-hydroxyacyl-CoAdehydrogenase MFE-2 peroxisomal multifunctional 2-enoyl-CoA hydratase 2/(3R)- hydroxyacyl-CoAdehydrogenase mRNA messengerRNA NAD+ nicotinamideadeninedinucleotide(oxidized) NADH nicotinamideadeninedinucleotide(reduced) NADPH nicotinamideadeninedinucleotidephosphate(reduced) PCR polymerasechainreaction peroxin proteininvolvedinperoxisomebiogenesis pI isoelectricpoint PPAR peroxisomalproliferatoractivatedreceptor PPRE peroxisomalproliferatorresponsiveelement PTS peroxisomaltargetingsignal RACE rapidamplificationofcDNAends RXR retinoidXreceptor SDR short-chainalcoholdehydrogenase/reductasefamily SDS-PAGE sodiumdodecylsulphatepolyacrylamidegelelectrophoresis THCA 3α,7α,12α-trihydroxy-5β-cholestanoicacid Xaa anyaminoacid Listoforiginalarticles Thisthesisisbasedonthefollowingarticles,whicharereferredtothetextbytheirRoman numerals: I QinY-M,PoutanenMH,HelanderHM,KvistA-P,SiivariKM,SchmitzW, ConzelmannE,HellmanU&HiltunenJK(1997)Peroxisomalmultifunctionalenzymeof β-oxidationmetabolizingD-3-hydroxyacyl-CoAestersinratliver:molecularcloning, expressionandcharacterization.BiochemJ321:21-28. II QinY-M,HaapalainenAM,ConryD,CuebasDA,HiltunenJK&NovikovDK (1997)Recombinant2-enoyl-CoAhydratasederivedfromratperoxisomalmultifunctional enzyme2:roleofthehydratasereactioninbileacidsynthesis.BiochemJ328:377-382. III QinY-M,MarttilaMS,HaapalainenAM,GlumoffT,NovikovDK&Hiltunen JKHumanperoxisomalmultifunctionalenzymetype2:site-directedmutagenesisstudies showtheimportanceoftwoproticresiduesfor2-enoyl-CoAhydratase2activity. Submitted. IV QinY-M,Marttila,MS,HaapalainenAM,SiivariKM,GlumoffT&HiltunenJK Yeastperoxisomalmultifunctionalenzyme:(3R)-hydroxyacyl-CoAdehydrogenase domainsAandBarerequiredforoptimalgrowthonoleicacid.Submitted. Contents Abstract Acknowledgements Abbreviations Listoforiginalarticles 1.Introduction…………………………………………………………………………..12 2.Reviewoftheliterature………………………...…………………………………….14 2.1.Fattyacids………………………………………………………………………14 2.1.1.Uptakeandtransportoffattyacidsintothemammaliancells.…………14 2.1.2.Pathwaysforfattyacidoxidation.………………………………………15 2.2.Peroxisomes…………………………………………………………………….16 2.2.1.Peroxisomalproliferationinmammaliancell..…………………………16 2.2.2.Inductionofgenesencodingperoxisomalproteinsinyeast…………….18 2.2.3.Activationandtransportationoffattyacidsintoperoxisomes………….18 2.2.4.Theperoxisomalβ-oxidationspiral…………………………………….19 2.2.4.1.Acyl-CoAoxidation……………………………...……………19 2.2.4.2.Hydrationoftrans-2-enoyl-CoAandsubsequent dehydrogenation…………………………………………..….21 2.2.4.3.Cleavageof3-ketoacyl-CoA………………………………….23 2.2.5.Oxidationofthecholesterolsidechain…………………………………24 2.3.Auxiliaryenzymesinvolvedinβ-oxidation…..……………………………….26 2.3.1.2,4-Dienoyl-CoAreductase…………………………………………….26 2.3.2.∆3-∆2-Enoyl-CoAisomerase………………………………………...…26 2.3.3.∆3,5-∆2,4-Dienoyl-CoAisomerase……………………………………..28 2.3.4.2-Methylacyl-CoAracemase……………………………...……….……29 2.4.Multifunctionalenzymes(MFEs)ofβ-oxidation………………………………29 2.4.1.Bacterialβ-oxidationmultienzymecomplex……………….…………..30 2.4.2.Fungalmultifunctionalenzymes………………………………………..30 2.4.3.Plantmultifunctionalenzymes.…………………………………………32