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GeorgiaStateUniversityGeorgiaStateUniversityScholarWorks@GeorgiaStateUniversityScholarWorks@GeorgiaStateUniversityChemistryThesesDepartmentofChemistry4-22-2008BiochemicalCharacterizationof2-NitropropaneDioxygenaseBiochemicalCharacterizationof2-NitropropaneDioxygenasefromHansenulaMRAKIIfromHansenulaMRAKIISlavicaMijatovicFollowthisandadditionalworksat:https://scholarworks.
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edu/chemistry_thesesRecommendedCitationRecommendedCitationMijatovic,Slavica,"BiochemicalCharacterizationof2-NitropropaneDioxygenasefromHansenulaMRAKII.
"Thesis,GeorgiaStateUniversity,2008.
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BIOCHEMICALCHARACTERIZATIONOF2-NITROPROPANEDIOXYGENASEFROMHANSENULAMRAKIIbySLAVICAMIJATOVICUndertheDirectionofDr.
GiovanniGaddaABSTRACT2-NitropropanedioxygenasefromHansenulamrakiiisaflavin-dependentenzymethatcatalyzestheoxidationofanionicnitroalkanesintothecorrespondingcarbonylcompoundsandnitrite,withoxygenastheelectronacceptor.
Althoughnitroalkanesareanticipatedtobetoxicandcarcinogenic,theyareusedwidelyinchemicalindustryforaquickandeffectivewayofsynthesizingcommonreagents.
Consequently,thebiochemicalandbiophysicalanalysisof2-nitropropanedioxyganasehasapotentialforbioremediationpurposes.
Inthisstudy,recombinantenzymeispurifiedtohighlevels,allowingfordetailedcharacterization.
Thebiochemicalanalysisof2-nitropropanedioxygenasepresentedinthisstudyhasestablishedthatenzymeutilizesalkylnitronatesassubstratesbyformingananionicflavosemiquinoneincatalysis.
Theenzymeisinhibitedbyhalideions,doesnotcontainironandhasapositivechargelocatedclosetotheN(1)-C(2)=OlocusoftheisoalloxazinemoietyoftheFMNcofactor.
INDEXWORDS:2-NitropropaneDioxygenase,Nitronate,Sulfite,Flavoprotein,FMN,Enzymekinetics,Flavinsemiquinone,Hansenulamrakii.
iBIOCHEMICALCHARACTERIZATIONOF2-NITROPROPANEDIOXYGENASEFROMHANSENULAMRAKIIbySLAVICAMIJATOVICAThesisSubmittedinPartialFulfillmentoftheRequirementsfortheDegreeofMasterofScienceintheCollegeofArtsandSciencesGeorgiaStateUniversity2008iiCopyrightbySlavicaMijatovic2008iiiBIOCHEMICALCHARACTERIZATIONOF2-NITROPROPANEDIOXYGENASEFROMHANSENULAMRAKIIBySLAVICAMIJATOVICCommitteeChair:Dr.
GiovanniGaddaCommittee:Dr.
JennyYangDr.
AlfonsBaumstarkElectronicVersionApprovedby:OfficeofGraduateStudiesCollegeofArtsandSciencesGeorgiaStateUniversityFebruary2008ivDEDICATIONTomylategrandfatherTomislavVidakovicvACKNOWLEDGEMENTSTostandwhereIamtodaywouldnotbepossiblewithoutmyparentsSlavkoandEdisMijatovic.
Thereareneverenoughadequatewordstoexpressmygratitudeforyoursupportandencouragementinpursuitofmyeducation.
My"rock",MuamerRustempasic,thankyouforthesupport,listeningandunderstanding.
WithoutyouwherewouldIfindthecouragetogothroughlifeandasafeplaceattheendoftheday.
IamthankfultomybrotherTomislav,nieceAdriana,nephewDarioandgrandmotherKataforprovidingtheemotionalsupportandlaughter.
Iwouldnotbeheretodaywithoutmyadvisor,Dr.
GiovanniGadda.
Firstofall,thankyouforgivingmetheopportunitytoworkinthelab.
Thankyouforpayingtheattentiontomyweaknesses.
WithoutyouIstillwouldnotknowhowmuchscientificworkmeanstome.
Iamalsoverythankfultomycommitteemembers,Dr.
YangandDr.
Baumstark,foryourguidanceandsuggestions.
Lastbutnotleast,IamthankfultohaveDr.
AndreaPennati,Kunchala,Hongling,Trang,Osbourne,Kevin,SteffanandTranaslabmates.
Joiningthelabwithoutanyexperiencewasscary,buthavingthelabmateslikeyoumadeitsomucheasier.
Itishardtoimaginethatthereisanotherlabinthisworldthatisasmuchfunasours.
Andrea,thankyouforbeingatruefriend.
KunchalaandHongling,thankyouforyoucompassionandunderstanding.
Ozzy,thanksforsharing"my"bench.
Thankyouallsomuchforallyourencouragementandsupport.
viTABLEOFCONTENTSDEDICATION…ivACKNOWLEDGEMENTS…vLISTOFTABLES…xiLISTOFFIGURES…xiiLISTOFSCHEMES…xiiiChapterIINTRODUCTION…11.
Nitroalkanes…11.
1.
Physicalproperties…11.
2.
Toxicityandcarcinogenity.
42.
Previousstudiesof2-nitropropanedioxygenasefromHansenulamrakii…62.
1.
Purificationfromtheoriginalsource…62.
2.
Biochemicalcharacterization…62.
3.
Kineticstudies…82.
4.
Inhibitionof2-nitropropanedioxygenasebysuperoxidescavengers……….
.
.
92.
5.
Stoichiometryofthereaction…103.
Flavindependentnitroalkane-oxidizingenzyme…103.
1.
Nitroalkaneoxidase.
123.
2.
2-NitropropanedioxygenasefromNeurosporacrassa.
153.
3.
Glucoseoxidase.
183.
4.
D-Aminoacidoxidase…203.
5.
Propionate-3-nitronateoxidase…224.
Goals…23viiReferences…25ChapterIIOXIDATIONOFALKYLNITRONATESCATALYZEDBY2-NITROPROPANEDIOXYGENASE39Abstract…39Introduction…41Materialsandmethods…43Materials…43Instruments…43Cloningof2-NpdintopET20b(44Expressionandpurificationof2-nitropropanedioxygenase…44Biochemicalmethods…45Enzymekineticassays…46Dataanalysis…47Results…48Cloning,expressionandpurificationof2-nitropropanedioxygenase………….
.
.
48Cofactorcontent…48Alkylnitronatesassubstratesfor2-nitropropanedioxygenase………………….
50Effectofsuperoxidedismutaseandcatalaseontheenzymaticactivity…………53Substratespecificity…54FormationofanN(5)-flavinadductwithsulfite…55Discussion…58References…63ChapterIIISTOICHIOMETRYOFTHEREACTIONCATALYZEDBY2-NITROPROPANEviiiDIOXYGENASE…68Abstract…68Introduction…69ExperimentalProcedures…71Materials,enzymeandorganicsubstratepreparations…71Instruments…71Determinationoforganicsubstratetooxygenratio…71Determinationoftheorganicsubstratetonitriteratio…72Determinationoforganicsubstratetocarbonylcompoundratio…72QuantificationoftheproductsbyMALDI-TOF…73DataAnalysis…73Results…74Discussion…78References…80ChapterIVINHIBITIONOF2-NITROPROPANEDIOXYGENASEWITHCHLORIDEIONS83Abstract…83Introduction…84ExperimentalProcedure…87Materials…87Methods…87Dataanalysis…87ResultsandDiscussion…89ixReferences…94ChapterVpHSTUDIESON2-NITROPROPANEDIOXYGENASE…96Abstract…96Introduction…97Experimentalprocedure…99Materials…99Methods…99Preparationofsubstrates…99Dataanalysis…100Resultsanddiscussion…102References…107ChapterVIGROWINGTHECRYSTALSOFRECOMBINANT2-NITROPROPANEDIOXYGENASE…109Abstract…109Introduction…110ExperimentalProcedures…114MaterialsandMethods…114Crystallization…114Resultsanddiscussion…115References…119ChapterVIIGENERALDISCUSSION…120References…125Appendix1…127xAppendix2…131Appendix3…135xiLISTOFTABLESTable1.
1.
pKavaluesofnitroalkanes…3Table2.
1.
InductivelycoupledplasmamassspectroscopicanalysisofH.
mrakii2-nitropropanedioxygenase…50Table2.
2.
EnzymaticactivityofH.
mrakii2-nitropropanedioxygenasewith1mMethylnitronateasasubstrateintheabsenceandpresenceofnitroethanein50mMpotassiumphosphatepH7.
4and30oC…53Table2.
3.
EffectofsuperoxidedismutaseandcatalaseontheenzymaticactivityofH.
mrakii2-nitropropanedioxygenase…54Table2.
4.
Apparentsteadystatesecondorderrateconstants,kcat/KM,foralkylnitronatesassubstratesforH.
mrakii2-nitropropanedioxygenasein50mMpotassiumphosphatepH6and30oC…55Table3.
1.
Oxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwithbutyl-1-nitronateassubstrates…74Table3.
2.
Oxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwith100Mpentyl-1-nitronateassubstrates…75Table3.
3.
Effectofsuperoxidedismutaseandcatalaseonoxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwith10mMalkylnitronatesassubstrates…76Table4.
1.
Apparentsteadystatesecondorderrateconstants,kcat/KM,inabsenceandpresenceofpotassiumchloridein50mMpotassiumphosphatepH6and30oC……………………90Table6.
1.
CrystalscreenHR2-110reagentformulation…117Table6.
2.
CrystalscreenliteHR2-128reagentformulation…118xiiLISTOFFIGURESFigure2.
1.
Purificationofrecombinant2-nitropropanedioxygenasefromHansenulamrakii….
48Figure2.
2.
UV-visibleabsorbancespectrumof2-nitropropanedioxygenaseaspurified……….
49Figure2.
3.
Anaerobicsubstratereductionof2-nitropropanedioxygenasewithalkylnitronates.
52Figure2.
4.
Apparentsteadystatekineticsof2-nitropropanedioxygenasewithethylnitronateorpropyl-1-nitronateasdeterminedin50mMpotassiumphosphate,pH6and30oC…….
55Figure2.
5.
Reactionof2-nitropropanedioxygenasewithsodiumsulfite…56Figure3.
1.
Betaine-aldehydeandhexanalstandardcurve77Figure4.
1.
Oxygenconsumptionof2-nitropropanedioxygenasewith20mMethylnitronateduringturnover…88Figure4.
2.
EffectofKClonkcat/KMof2-nitropropanedioxygenase…90Figure4.
3.
Apparentsteadystatekineticsof2-nitropropanedioxygenasewithethylnitronateinpresenceof1mMpotassiumiodide,potassiumbromideorpotassiumfluorideasdeterminedin50mMpotassiumphosphate,pH6and30oC…92Figure5.
1.
pHdependenceofthekcat/Kmvalueof2-nitropropanedioxygenasefromNeurosporacrassawithnitroalkanes…98Figure5.
2.
pHdependenceofthekcatvalueof2-nitropropanedioxygenasefromNeurosporacrassawithalkylnitronates…98Figure5.
3.
Steadystatekineticwithethylnitronateandoxygenassubstratesin50mMpotassiumphosphatepH6…102Figure5.
4.
Steadystatekineticwithethylnitronateandoxygenassubstratesin50mMpotassiumphosphatepH8…103xiiiFigure5.
5.
Steadystatekineticwithethylnitronateandoxygenassubstratesin50mMpotassiumpyrophosphatepH10.
5…103Figure5.
6.
pHdependenceonkcat/Kmwithethylnitronateandmixtureofethylnitronateandnitroethane…104Figure5.
7.
pHdependenceonkcat/Kmwithbutyl-1-nitronateandmixtureofbutyl-1-nitronateandnitrobutane…105Figure6.
1.
CrystalstructureofnitroalkaneoxidasefromFusariumoxysporum………………110Figure6.
2.
ActivesiteofnitroalkaneoxidaseillustratinganalternativeorientationofAsp402andSer276…111Figure6.
3.
Overallstructureof2-nitropropanedioxygenasefromPseudomonasaeruginosa.
.
.
112Figure6.
4.
Activesiteof2-nitropropanedioxygenasefromPseudomonasaeruginosa……….
113xivLISTOFSCHEMESScheme1.
1Nitroalkanesinaqueoussolution…5Scheme1.
2.
Theionizationofnitroethane(left)toyieldethylnitronate(right)insolution………7Scheme1.
3.
Denitrificationreactioncatalyzedbyoxidaseanddioxygenase…12Scheme1.
4.
Kineticmechanismofnitroalkaneoxidase…13Scheme1.
5.
Proposedmechanismfornitroalkaneoxidase…14Scheme1.
6.
Proposedmechanismfor2-nitropropanedioxygenasefromNeurosporacrassa….
17Scheme1.
7.
Theenzymaticreactioncatalyzedbyglucoseoxidase…18Scheme1.
8.
ReactioncatalyzedbyflavindependentD-aminoacidoxidase…20Scheme2.
1.
Theionizationofnitroethane(left)toyieldethylnitronate(right)insolution…….
.
41Scheme3.
1.
Reactioncatalyzedby2-nitropropanedioxygenase…69Scheme4.
1.
Reactioncatalyzedby2-nitropropanedioxygenase…831ChapterIINTRODUCTION1.
Nitroalkanes1.
1.
Physicalproperties.
Oneofthefundamentalclassesofsubstancesinorganicchemistry,nitroalkanesarepolarandacidiccompounds[1].
Theyexistastheneutralforminorganicsolvents,whereasinaqueoussolutionstheyareinequilibriumbetweenprotonated,non-protonatednitronicacidandtheanionofthenitronate(Scheme1.
1)[2].
CHRNOOHCHRNOOCHRNOOHScheme1.
1Nitroalkanesinaqueoussolution.
Nitroalkanescanbegeneratedfromawidevarietyoforganiccompounds.
Directnitrationofaliphatichydrocarbonsviaanionicintermediates,alkenesorketones(-nitration)yieldsnitroalkanes[3;4].
Inaddition,nitroalkanescanbegeneratedbyconversionofotherfunctionalities,suchas:carbonyls,oximesandazides[5;6;7]orbynitrationofthealkylhalideswithmetalnitrites,suchassilvernitriteindiethylether(Victor-Meyerreaction),potassiumnitrite,orsodiumnitriteinN,N-dimethylformamideorindimethylsulfoxide(Kornblumreaction)[8;9].
Themostcommonmethodusedinpreparationofnitroalkanesistheconversionofalkylhalidestonitrocompounds.
Alkylnitronatesoranionicnitroalkanescanbegeneratedfromnitroalkanesbyaddinganyofthebasesthatactascarbonnucleophilesincludinghaloalkanes[10],aldehydes[11;12]andMichaelacceptors[13].
2ThenitrogroupcanbeconvertedintoacarbonylgroupbytreatingconjugatebaseswithsulfuricacidviaareactionknownastheNefreaction,whichinvolveshydrolysisoftheC=Ndoublebond[14;15].
Also,thereareseveralothermethodsforconvertingnitroalkanestocarbonylcompounds,suchasreactionofaliphaticnitrocompoundswithaqueousTiCl3,cethyltrimethylammoniumpermanganate,tincomplexesandNaHSO3,activateddrysilicagelor30%H2O2-K2CO3[16].
Inaddition,conversionofnitroalkanestocarbonylcompoundscanbeachievedbytreatmentofthenitrocompoundwithKMnO4,t-BuOOHandacatalyst,cericammoniumnitrate,MoO5-pyridine-HMPA,ozone,orsingletoxygen[16].
CHH3CNOOpKa=8.
5H+CHH3CNOOHCHH3CNOOScheme1.
2.
Theionizationofnitroethane(left)toyieldethylnitronate(right)insolution.
Sincenitroalkanesundergoavarietyofcarbon-carbonbond-formingprocessesandthenitrogroupcanbeconvertedintoseveralotherfunctionalgroups,nitroalkaneshavebeenimportantforproductionofexplosivesandasprecursorsforazodyes[14][4].
Duetothehighelectron-withdrawingpowerofthenitrogroupthatprovidesanenhancementofthehydrogenacidityattheα-position,nitroalkanesaresourceofstabilizedcarbanions[5;17;18].
Today,becauseofthelowpKavaluesfordeprotonationoftheα-carbon(Scheme1.
2)[19],nitroalkanesplayanimportantroleassyntheticintermediatesinthepreparationofperfumes,pharmaceuticals,dyes,plasticsandmanynaturalproducts[5;20;21;22;23].
Inadditiontobeingsynthesizedinthechemicalindustry,leguminousplantsproducenitrotoxinssuchas3-nitro-1-propionicacidand3-nitro-1-propanol[24;25;26].
3Nitroalkanesareunusualacidsbecausetheprotonationoftheα-carbonismuchslowerthanexpectedforthemajorityoftheacids.
Consequently,protontransferreactionsinvolvingnitroalkaneshavegeneratedinterestformanyyears[27;28;29;30;31;32;33;34;35;36].
Usually,increasedcarbanionbasicityisgenerallyobservedwitheachadditionofthesubstituentthatincreasesnucleophilicity[28;37].
Incontrast(Table1.
1),thepKavalueofRCH2NO2decreasesintheorderCH3NO2>CH3CH2NO2>(CH3)2CHNO2inwater,buttherateofprotonabstractionbyhydroxideiondecreasesinthesameorder[38].
Therefore,thereactionisslowerforthemoreacidicsubstrate.
NitroalkanepKaNitromethane10.
21Nitroethane8.
51-Nitropropane8.
982-Nitropropane7.
7Table1.
1.
pKavaluesofnitroalkanesinwaterat25oC[19].
Bernasconiattributestheslowdeprotonationofthenitroalkanestotheimbalanceofthetransitionstate,wherethedegreeofchargedelocalizationintotheπ-acceptorgrouplagsbehindprotontransfer[39].
Therefore,therateconstantisdecreasedwhenproduct-stabilizingfactorslagbehindthetransitionstate.
Becauseofthatimbalance,asBernasconifurtherrationalizes,thetransitionstatecannottakeadvantageoftheresonancestabilizationofthecarbanionbecausestabilizationisbarelydevelopedduringthetransitionstate.
Thisunusualrelationshipbetweenratesandequilibriafordeprotonationofnitroalkanesisknownasthenitroalkaneanomaly[38].
Theanomalyhasbeenanalyzedintermsoffourinteractions:electrostaticandconjugativeeffectsofthesubstituentwhichoperatebothattheproductandtransitionstate,andinteractionsbetweenthesubstituentandthebase,andbetweenthesubstituentandapartialnegativechargelocalizedatCαwhichareeffectiveonlyatthetransitionstate[38].
41.
2.
Toxicityandcarcinogenity.
Nitroalkaneshavebeenanticipatedtobetoxic[40]andsometobecarcinogenic[41;42;43;44;45;46;47].
Nitromethanewasshowntohavecarcinogenicactivityinmicebasedona2-yearinhalationstudythatshowedincreasedincidencesofmammaryglandfibroadenomasandcarcinomas[48].
Additionally,1-nitropropanehasbeenshowntobeamutagenincells,sinceitinducesunscheduledDNAsynthesisinratsbutdoesnotinducetumorsinratsfollowingchronicexposure[49].
Thesecondarynitroalkanes,2-nitrobutaneand3-nitropentane,produceahighlysignificantincidenceofhepatocarcinomawithmetastasestothelungs,whereastheprimarynitroalkane1-nitrobutanewasnotfoundtobecarcinogenic[50].
Thenitronatesofthesecondarynitroalkanes2-nitropropane,2-nitrobutane,3-nitropentane,2-nitroheptane,nitrocyclopentaneandnitrocyclohexanewerefoundtobesubstratesforthearylsulfotransferase-catalyzedproductionof8-aminoguanosineand8-oxoguanosinefromguanosineinvitro[50].
Noneoftheprimarynitronatesof1-nitropropane,1-nitrobutane,1-nitropentaneand1-nitroheptanewerefoundtobesubstratesforthearylsulfotransferaseinvitro[50].
AnumberofcompoundshavebeenusedtoinduceoxidativeDNAdamageinexperimentalanimals[51].
Asapotenthepatocarcinogen,2-nitropropaneanditsanionicformpropyl-2-nitronatehavebeenusedinmanystudiesforinductionofcarcinogeniccellsinrats[52;53;54;55;56;57;58;59;60;61;62].
Inaddition,2-nitropropanehasbeenfoundtobemutagenicinanumberofshort-termmutagenicityassaysbothinvitroandinvivo[63]andtocausepointmutationsinamicrobialtestsystem[64].
Allthoughmanystudieshavebeenfocusedonthemetabolicpathwayof2-nitropropaneandpropyl-2-nitronateinthecell,nomechanismforDNAandRNAmodificationby2-nitropropanehasbeengenerallyaccepted.
Ithasbeenhypothesizedthatthegenotoxicityof5propyl-2-nitronatemaybeduetothegenerationofDNA-damagingreactiveformsofoxygenorfreeradicals[65].
Thenitriteradicals,formedafterthenonenzymaticdegradationof2-nitropropaneorpropyl-2-nitronatethroughperoxidativechainreaction,reactappreciablyfastwithribonucleosides,deoxyribonucleosides,andguanosinenucleotides,inducingDNAdamage[66].
Onthecontrary,itwasalsoproposedthatmetabolicsequencefrom2-nitropropanetothereactivespeciescausingDNAandRNAmodificationsdoesnotinvolvetheremovalofthenitrogroup[67].
Additionally,itwasfoundthattheDNAdamagewasalsocausedbyradicalsgeneratedviahydrogenperoxidespeciesinmetabolismof2-nitropropane[68].
Secondly,thestimulatoryeffectof2-nitropropaneonthecellularproliferationandtherateofDNAsynthesisintheliverhasbeenhypothesizedasthemechanismbywhichthecarcinogenicactionisinduced[63].
Thegenotoxicityof2-nitropropaneinratshasbeenattributedtosulfotransferase-mediatedformationofDNA-reactivenitreniumionsfromtheanionicformof2-nitropropane[69].
Thesulfotransferase-mediatedpathway,inthecaseoftheformationof8-aminoguanineinbothDNAandRNA,includedconvertingthecarcinogenintospeciescapableofaminatingnucleicacidsandproteinsinvolvingoxime-andhydroxylamine-O-sulfonatesasintermediates[70].
Neutralformof2-nitropropaneismetabolizedtohydroxylamine-O-sulfonateoracetate,whichyieldsthereactivenitreniumionaswell[71].
2-Nitropropanehasbeenfoundtoinducearylsulfotransferase-mediatedliverDNAandRNAbasemodificationsidentifiedas8-aminoguanine,8-oxoguanine,8-hydroxyguanine,8-oxo-2'-deoxyguanosine[72].
Tworatsulfotransferaseswereidentifiedofbeingcapableofcatalyzingthemetabolicactivationstepofpropyl-2-nitronate[73].
Inaddition,thehumanphenolsulfotransferaseswerecapableofmetabolicallyactivatingpropyl-2-nitronateandareapparentlyidenticaltosulfotransferasesinrat6liver,where2-nitropropanecausescarcinomas[74].
Inanotherstudyauthorsconcludedthatspeciesotherthantheratsandorgansotherthanthelivercanbetargetsforthegenotoxicityandcarcinogenicityofsecondarynitroalkanes[75].
Inconclusion,2-nitropropaneinneutralandanionicformshouldberegardedasapotentialhumancarcinogen,eitherbecauseofradicalformationortheformationofintermediatesinsulfotransferase-mediatedpathways.
2.
Previousstudiesof2-nitropropanedioxygenasefromHansenulamrakii2.
1.
Purificationfromtheoriginalsource.
In1975,KidoandSodafoundthatsomeintercellularenzymesoftheyeastHansenulamrakiicanoxidativelydenitrify2-nitropropane,1-nitropropaneandnitroethane[76].
Ayearlater,2-nitropropanedioxygenasewasisolatedforthefirsttimefromHansenulamrakiibythesamescientificteam[77].
Theyeastextractwasgrowninamediumcontainingnitroethaneasthesolenitrogensource.
Thepurificationprocedureinvolvedfractionationwithammoniumsulfate,followedbycolumnchromatographyusingtheionicexchangerdiethylamminoethylcelloulose(DEAE)andhydroxyapatite(HA),aswellasgelfiltrationthroughaBio-GelP-150column.
Thisprocedureallowedfora1300-foldpurificationoftheenzyme,withanoverallyieldof22%.
Theresultingenzymeoftwonon-identicalsubunitsshowedtobehomogeneousbydisc-gelelectrophoresisandultracentrifugationtechniques[77;78;79].
Subsequently,thesamegrouppurifiedtheenzymeconsistingofasinglepolypeptidebyutilizingthesameprocedureinadditiontoanotherDEAE-cellulosechromatographystep[80].
Overall,41mgofpurifiedenzymecouldbeobtainedfrom18kgwetcellpasteoftheoriginalhostHansenulamrakii.
2.
2.
Biochemicalcharacterization.
ThepurifiedH.
mrakiienzymewascharacterizedasanon-hemeironflavoproteinbasedontheobservationthattheUV-visibleabsorbance7spectrummaximaat370,415and440nmresembledthespectraofdihydrooroticaciddehydrogenaseandxanthineoxidase[77;79].
Themolecularmassoftheisolatedenzymewas62,000Da,comprisedoftwonon-identicalsubunitsof39,000and25,000Da,asdeterminedbygelfiltration,sedimentationequilibriummethodsanddiscgelelectrophoresis.
Theflavinspeciesin2-nitropropanedioxygenasewasidentifiedasFADratherthanFMNduetotheobservationthatapo-D-aminoacidoxidasecanbeactivatedwiththeflavinisolatedfrom2-nitropropanedioxygenasebyaciddenaturationandcentrifugationoftheprotein[77].
Inaddition,a1:1ratioofflavintoproteinwasdeterminedbycomparingthefluorescenceintensityoftheflavinisolatedfrom2-nitropropanedioxygenasetothefluorescenceoffreeFAD[79].
A1:1ratioofirontoproteinwasdeterminedbyatomicabsorptionmethod[77].
Basedonthisdata,theauthorsinitiallyproposedthat2-nitropropanedioxygenasecontainstwonon-identicalsubunitswithFADandironforcofactors.
However,subsequentpurificationmethods,whichincludedadditionalDEAE-cellulosechromatography,yieldedanenzymeformthatconsistedofasinglepolypeptidewithamolecularweightof42,000[80].
Inadditiontoperformingthedisc-gelelectrophoresisandsedimentationequilibriumanalysis,theatomicabsorptionanalysisandUV-visibleabsorbancespectraconfirmedtheabsenceofthepreviouslyreportedsmallersubunitandiron.
Withthisstudy,theauthorsconcludedthataniron-containingprotein,identifiedasasmaller25,000Daunit,rapidlyboundtothe2-nitropropanedioxygenase.
Thereforethetwo-proteincomplex,exhibitedcharacteristicsofiron-containingproteininallofthepreviousstudies.
Bycomparingtheaminoacidsequenceofoldandnewpurifications,theauthorsidentifiedadditional200aminoacidsintheinitialtwopurifications[80].
Inaddition,aminoacidanalysisof2-nitropropanedioxygenaserevealedthattheenzymehasflavinandNADHbindingsites[81].
82.
3.
Kineticstudies.
Theoxidationofnitroalkanesby2-nitropropanedioxygenasewasobserveduponinitialpurificationoftheenzyme[77].
Theenzymaticassayswereperformedbyincubatingthenitroalkanesubstratesin100molpotassiumphosphatepH7or8withtheenzymeaerobicallyat30oCandmeasuringtheoxygenconsumptionornitriteproductionafter60minutesofincubation[77].
Theenzymewasfoundtobecatalyticallyactivewith2-nitropropane,1-nitropropaneandnitroethanebutnotwithnitromethane[77].
Additionally,theenzymewasfoundtoutilizealcoholsassubstrates,suchas3-nitro-2-pentanol,3-nitro-2-butanoland2-nitro-1-butanol,byconvertingthemintonitriteandthecorrespondingcarbonylcompounds[77].
Spectrophotometricstudiesontheenzymeincubatedwith2-nitropropaneconfirmedthattheenzymeiscapableofoxidizingnitroalkanes[77;79].
Inlaterstudiesbythesamegroup,theenzymewasfoundtobemoreefficientwiththeanionicformofnitroalkanes[80].
Thekineticparametersoftheenzymewith1-nitropropane,2-nitropropane,nitroethane,3-nitro-2-butanol,3-nitro-2-pentanolandtheiranioniccounterpartsshowedthatthesecond-orderrateconstantkcat/KMissignificantlyhigherandkcatvalueswere20to70foldlargerforanionicsubstratescomparedtotheirneutralcounterparts.
TheenzymewasmostefficientatpH8withneutralandpH6.
5withanionicspecies,assupportedbynitriteformationoroxygenconsumptionstudies.
TheauthorsdidnotlistalloftheconditionsneededtoexamineenzymaticactivityatvariouspHwithanionicandneutralnitroalkanesassubstrates,butjuststatedtheoptimumconditionsfordenitrificationofthesesubstrates[80].
Basedonthesedata,theauthorsproposedthattheneutralnitroalkanesareenzymaticallyconvertedintotheanionicformpriortotheoxidationandthatthedeprotonationistherate-limitingstepindenitrificationofneutralnitroalkanes[80].
Abibiorderedkineticmechanismwasdeterminedfor2-nitropropanedioxygenase,where2-nitropropanebindstotheenzymefirst,followedbytheoxygentoforman9oxygenatedternarycomplex.
Thesubsequentreleaseofthenitriteandacetoneoccurswiththeconcomitantreoxidationofthereducedflavin.
Inaddition,theconclusionofthekineticmechanismwasfurthersupportedbyproductinhibitionstudies.
Acetoneandnitritewerefoundtobecompetitiveandnoncompetitiveinhibitorsoftheenzymewithrespectto2-nitropropane,whereasbothproductsinhibitedtheenzymenon-competitivelywithrespecttomolecularoxygenwhenenzymewasnotsaturatedby2-nitropropane,resultsconsistentwithsequentialkineticmechanism[78].
2.
4.
Inhibitionof2-nitropropanedioxygenasebysuperoxidescavengers.
Sincetheinitialpurificationscontainedtheproteinwithiron,theinhibitionstudieswerepreformedwithchelatingagentsaswellasseveralothercompound[77].
Tiron,cysteineandglutathionewerefoundtoinhibit2-nitropropanedioxygenasecompletelywhileHgCl2andoxinewerefoundtobeinhibitors.
Incontrast,chelatingagentsfordivalentandtrivalentmetalssuchasα,α'-dipyridylando-phenanthrolineactedasweakactivators.
Basedonthesestudiesauthorsconcludedthatinhibitionofenzymaticactivityisnotduetochelatingoftheiron.
Sincethecompoundssuchastiron,oxineandvariousthiolswereknowntotrapsuperoxideanionseffectively,formationofthesuperoxideintermediatewashypothesized.
Furtherstudiesonenzymaticactivityinthepresenceofsuperoxidedismutaseandvarioussuperoxidescavengerssuchascytochromec,epinephrine,NADH,thiolsandpolyhydricphenolsconfirmedtheformationofsuperoxideduringthedenitrificationof2-nitropropane[78].
Theobservationthatadditionofsuperoxidetotheenzymaticreactioninducedtheoxidationofnitroalkanesfurther,confirmedtheformationofsuperoxideduringcatalysis.
Thesestudiesledtothehypothesisofsuperoxidefunctionasanessentialintermediateintheoxygenation,whichisformedfromorganicsubstrateandoxygen[78;82].
102.
5.
Stoichiometryofthereaction.
Todeterminewhether2-nitropropanedioxygenasecatalyzesthedenitrificationofnitroalkanesviaoxidaseoroxygenasemechanism,formationofhydrogenperoxidewasmonitoredduetoabilityofoxidasesandnotoxygenasestoproducehydrogenperoxide[77].
Thelackofhydrogenperoxideformationduringcatalysisindicatedthatthenitroalkane-oxidizingenzymeactsasanoxygenaseratherthananoxidase.
Additionally,incorporationof18O2intoacetoneduringtheoxidationof2-nitropropaneconfirmedthatbothatomsofmolecularoxygenareincorporatedintoacetone[79].
Incontrast,replacingthe2-nitropropanewith1-nitropropaneandnitroethaneassubstrates,noincorporationof18O2wasobservedinfinalproductspropionaldehydeandacetaldehyde,respectively.
Basedonthesestudies,theauthorshypothesizedthatexchangeofoxygenbetweenaldehydesandwaterisfarmorerapidcomparedtotheexchangebetweenacetoneandwater.
Therefore,2-nitropropanedioxygenasewasclassifiedasoxygenase.
Furthermore,oxygenconsumptionaswellasnitriteandacetoneformationwasdeterminedinordertoelucidatethestoichiometryoftheenzymaticreaction.
Reactionmixturewasconstructedbyincubating2-nitropropanein13mMpotassiumphosphatepH8withtheenzymeat30oCfor60minutes.
Thisstudysuggeststhat2-nitropropanedioxygenaseisanintermoleculardioxygenasecapableofdenitrifying2moleculesof2-nitropropanebyformingtwomoleculesofnitritewhileincorporatingtwoatomsofoxygenintotwomoleculesoftheacetone.
3.
Flavindependentnitroalkane-oxidizingenzymes.
SinceLittle'sdiscoveryofoxidativedegradationofnitroethaneand2-nitropropanebyextractsofNeurosporacrassaandpeaseedling,respectively,manyenzymeshavebeencharacterizedtohavetheabilitytoutilizenitrocompoundsassubstrates[83;84].
Several11Streptomycesstrainshavebeenidentifiedtocatalyzetheoxidationof2-nitropropanetoacetoneandnitrite[85].
AnenzymefoundinAspergillusflavusiscapableofnitriteformationfromβ-nitropropionicacid[86].
Degradationof3-nitropropanolbyunidentifiedbacteriafromtheruminalfluidofcattlehavebeenreported[87].
Inaddition,theflavoenzymepropionate-3-nitronateoxidasecanoxidativelydenitrifybranchedanionicnitroalkanes[88].
Otherflavin-dependentenzymes,suchasglucoseoxidase[89]andD-aminoacidoxidase[90],havebeenfoundtooxidativelydenitrifynitroalkaneanionsinadditiontotheirphysiologicalsubstrates.
2-NitropropanedioxygenasefromH.
mrakiicomparedtoD-aminoacidoxidaseandglucoseoxidasewasfoundtobe1200and1800timesmoreeffectivewithanionicnitroalkanes,respectively[91].
Intheattempttoelucidatethemechanismofflavoproteinoxidaseswiththephysiologicalsubstrates,theanalogswereusedinmechanisticstudies.
Sucesfully,studiesonD-aminoacidoxidasewithanionicnitroalkanesassubstrateshaveprovidedevidenceforN-5flavin-carbanionadducttobeobligatoryintermediateinoxidationreactionsofD-aminoacidoxidasewithnitroalkanes[92].
Incontrast,thereationofDAAOwiththephysiologicalsubstratesproceedsviahydridetransfermechanism[120].
Todate,twoflavin-dependentenzymes,nitroalkaneoxidasefromFusariumoxysporumand2-nitropropnanedioxygenasefromNeurosporacrassa,havebeenidentifiedtoutilizenitroalkanesastheirsubstrates[93;94].
Thetwoenzymesdiffergreatlyintheirproposedmechanism,oneisanoxidaseandsecondisdioxygenase(Scheme3.
1.
).
CH3CH3N+OO-CH3CH3Ooxidase+OH+O2+H2O2+NO-212CH3CH3N+OO-CH3CH3Odioxygenase+O2+2HNO2Scheme1.
3.
Denitrificationreactionscatalyzedbyoxidasesanddioxygenases.
3.
1.
Nitroalkaneoxidase.
Amongallthenitroalkaneoxidizingenzymes,nitroalkaneoxidase(NAO,EC1.
7.
3.
1.
)isthemostextensivelycharacterizedenzymeinitsbiochemical,structural,kineticandmechanisticproperties.
Nitroalkaneoxidaseisaflavindependentenzyme[95]thatcatalyzestheoxidationofnitroalkanestoaldehydeswiththeproductionofhydrogenperoxideandnitrite,withoxygenasanelectronacceptor[93].
In1978,aninactiveformofnitroalkaneoxidasewasisolatedfromthefungusFusariumoxysporumbyKido,HashizumeandSoda[96].
Uponadditionofexogenousflavinadeninedinucleotide(FAD),theactiveformoftheenzymewascapableofoxidizingprimaryandsecondarynitroalkanes.
SubsequentworkbyFitzpatrick'sgroupprovidedadetailedbiochemicalcharacterizationoftheenzymeaswellasadetailedmechanism.
Nitroalkaneoxidaseconsistsoffoursubunitsofidenticalmolecularweight(47,000)[95].
EventhoughtheenzymerequiresFADforactivity,upondenaturationoftheproteinattheneutralpHastoichiometricamountofoxidizedflavinwasobservedspectroscopically[97].
Thespectroscopicandchromatographicstudiesontheflavincofactorrevealedthattheinactiveformoftheenzymecontainsa5-nitrobutyl-FADadductasacofactor[97].
Theauthorsproposedthatformationoftheadductoccursinthefungalcellduringtheincubationwithnitroethane,whereformationoftheadductrequiresreactionoftheflavinwithaneutralnitroethanefollowedbythenucleophilicattackbytheanionicnitroethane[97].
13Moreover,thecarbanionintermediateisinvolvedinbothcatalysisandinactivationoftheenzyme[97].
Nitroalkaneoxidaseutilizesabroadrangeofnon-aromaticnitroalkanesassubstrates,butithasapreferenceforprimaryunsubstitutednitroalkanes[98].
Monotonicincreaseofthesecond-orderrateconstantkcat/KMwithincreasinglengthofthealkylchainuptofourcarbonwasobserved.
Thetwoordersofmagnitudesmallerspecificityconstantforsubstratescontaininghydroxylgroupsuggestedthattheenzymehasahydrophobicactivesite[98].
Inthiscontext,nitroalkaneoxidaseutilizeshydrophobicinteractionstostabilizeboththebindingofthesubstrateandthetransitionstatedevelopedduringcatalysis[99].
Scheme1.
4.
Kineticmechanismofnitroalkaneoxidase.
Takenwithoutpermission[93].
Aping-pongkineticmechanismwasdeterminedfornitroalkaneoxidase[93],inwhichfollowingsubstrateoxidationandflavinreduction,thealdehydeproductisreleasedandthereducedflavinreactswithoxygen(Scheme1.
4.
).
ThepHdependencestudiesonthekineticparameters(kcatandkcat/KM)ofnitroalkaneoxidasewithnitroethaneshowedtherequirementfortwoionizablegroups:onemustbeunprotonatedandthesecondprotonatedforcatalysis[100].
Thetyrosine398,whichparticipatesinbindingofthesubstratebyformingthehydrogenbondto14thenitrogroup,isanaminoacidthatneedstobeprotonatedforcatalysis,basedontheinactivationstudieswithtyrosinedirectedreagent,tetranitromethane[101].
Theoxidationofnitroalkanesbynitroalkaneoxidaseisinitiatedbyabase-catalyzedprotonabstractionfromtheα-carbonofthesubstratebyAsp402,asindicatedbythecrystallographicandmutagenesisstudies,asseeninScheme1.
5[102;103;104].
Scheme1.
5.
Proposedmechanismfornitroalkaneoxidase.
Takenwithoutpermission[104].
Protonabstractionisirreversibleandfullyrate-limitingforreductionasindicatedbythekineticisotopestudiesonkcat/KMwithnitroethaneandreductive-halfreactionstudies[104].
Upongenerationoftheanionicsubstrate,theresultingcarbonanioncanattacktheN(5)-positionoftheFADandformacovalentadduct,assuggestedbytrappingexperimentswithcyanide[105].
Thecarbon-hydrogenbondcleavageonthesubstrateistheratelimitingstepincatalysis,15asindicatedbykineticisotopeeffectsstudies[100].
Thecleavageofcarbon-nitrogenbondresultsineliminationofnitriteandformationofacationicelectrophiliciminethatcanbeattackedbyhydroxide[100].
Theactivesitebaseisrequiredfordeprotonationofthewatermoleculetoformthehydroxide,whichreactswiththecationicimine.
Theiminecanbetrappedbyanionicnitroethanetoformthestableandinactiveformoftheflavin,5-nitrobutyl-flavinadduct[97],orbyeliminationofaldehydecanyieldreducedFAD[100].
Theinhibitionstudiesrevealedthatbothoxidizedandreducedflavincanformthedead-endcomplexesbyeitherbindingtothealdehydeasaproductorthenitroalkaneasthesubstrate,respectively[106].
3.
2.
2-NitropropanedioxygenasefromNeurosporacrassa.
2-nitropropanedioxygenase(EC1.
13.
11.
32)fromNeurosporacrassaisaflavin-dependentenzymethatcatalyzestheoxidationofbothanionicandneutralnitroalkanestothecorrespondingcarbonylcompoundsandnitrite,withoxygenasanelectronacceptor[94].
In1951,2-nitropropanedioxygenasewasisolatedfromNeurosporacrassabyLittleandcharacterizedasanoxidase[83].
Atthelaterdatetheenzymewasreclassifiedasadioxygenasebasedon18O2isotopestudyobservationthattheoxygenatomoforganicproductformedduringtheoxidationofpropyl-2-nitronatewasderivedfrommolecularoxygenandnotfromwater[107].
Biochemicalandmechanisticstudieswerepossibleuponobtaininglargequantitiesofpureandstableenzymeusingrecombinanttechnology[94].
ThepurificationprocedureinvolvedfractionationwithammoniumsulfatefollowedbycolumnchromatographyusingtheanionexchangerDEAE-sepharoseandoctyl-sepharosecolumns.
Therecombinantenzymeisahomodimerof80kDa,witheachmonomercontainingonetightlybutnotcovalentlyboundFMN.
Theenzymehasbroadsubstratespecificity,inthat2-nitropropanedioxygenasecanoxidativelydenitrify2-nitropropaneinadditiontoanumberofprimarynitroalkanesinanionicorneutralforms.
16Furthermore,thesizeofthealkylchaindoesnotaffecttheoverallenzymaticrateofturnover,withtheenzymebeingmorespecificfornitronatesascomparedtonitroalkanes[94].
Scheme1.
6.
Proposedmechanismfor2-nitropropanedioxygansefromNeurosporacrassa.
Takenwithoutpermission[108].
17Asequentialsteadystatekineticmechanismwasdeterminedfortherecombinantenzyme,asindicatedbysteadystatekineticstudieswitheitherneutraloranionicformof2-nitropropane,nitroethane,nitrobutane,andnitrohexane[94].
Oxidationofbothneutralandanionicnitroalkanesproceedsinthesamemanner,inadditiontotheinitialabstractionoftheprotonfromtheα-carbonoftheneutralnitroalkanes(Scheme1.
6)[94].
Mechanistically,theoxidationcatalyzedby2-nitropropanedioxygenasecanbesummarizedasfollows.
First,duringthereductivehalf-reaction,aftertheorganicsubstratebindstothefreeenzyme,theenzyme-boundflavinisreducedthroughasingleelectrontransferfromtheorganicsubstrate,formingananionicsemiquinone[94].
Second,duringtheoxidativehalf-reaction,theanionicsemiquinonereactswithmolecularoxygentoformasuperoxideanion,whichinturnreactswiththenitroradicaltoyieldanitroperoxideanionspeciesthatisreleasedfromtheactivesite.
Itwasproposedthatthenitroperoxideanionspeciesundergoesnon-enzymaticnucleophilicattacktoyieldnitriteandcarbonylproduct.
Anon-oxidativedeprotonation/protonationpathway,inwhichtheenzymecatalyzestheinterconversionofnitroalkanesbetweentheiranionicandneutralformswasproposedbasedontheinverseα-secondarykineticisotopeeffectsonkcat/KMwithethylnitronateand1-[2H]-ethylnitronateassubstrates[108].
pHdependencestudiesonthekineticparameters(kcatandkcat/KM)of2-nitropropanedioxyganseshowedtherequirementofanacidandbothacidandabaseforcatalysis,foranionicandneutralnitroalkanes,respectively[94].
Alikelyroleforthecatalyticbase,proposedtobeHis196,istoabstracttheprotonfromtheα-carbonoftheneutralnitroalkanesubstrate.
Incontrast,nosolventviscosityeffecthasbeendetectedonthekineticparameters(kcatandkcat/KM)withnitroethaneorethylnitronateassubstrates,suggestingthatthesubstrateandproductbindtotheenzymeinrapidequilibrium[108].
Overall,mechanisticstudiessuggestthatenzymaticturnoverwithneutralsubstratesislimitedbyproton18abstractionatlowpHandformationoftheflavosemiquinoneatthehighpH.
Incontrast,theturnoverwithanionicsubstratesislimitedbythenon-oxidativetautomerizationofethylnitronatetonitroethaneathighpH[108].
3.
3.
Glucoseoxidase.
AnFAD-dependentglucoseoxidase(E.
C.
1.
1.
3.
4)catalyzestheoxidationofβ-D-glucosetoδ-gluconolactone(Scheme1.
7)[109].
OHOHOHHOOOHOHHOHOOHOOH2O2O2Scheme1.
7.
Theenzymaticreactioncatalyzedbyglucoseoxidase.
Glucoseoxidasehasbeenusedasabiosensorforthequantitativedeterminationofglucoseinbodilyfluids,beverages,foodandfermentationliquor[110;111],aswellasfortheproductionofgluconicacidthatservesasafoodpreservative[112].
Theactiveformofglucoseoxidasehasbeenisolatedfromseveralmoldsaswellasfungi,suchasPenicilliumamagasakienase,Aspergillusniger,PhanerochaetechrosporiumandTalaromycesflavus[109;113;114;115].
ThemajorityofbiochemicalandmechanisticstudieshavebeenperformedonglucoseoxidasefromAspergillusniger;thereforebiochemicalcharacteristicsoftheenzymeinthischapterfocusonthisenzyme.
Thedimerhasthemolecularmassbetween150to180kDadependendingonthedegreeofglycosylation;witheachmonomercontainingtightlyattachedFADneartheinterface[109;114].
ApresenceofthenegativechargeattheN(1)-C(2)=OlocusoftheisoalloxazinemoietyhasbeenidentifiedbasedonNMRstudiesontheanaerobicallyreducedspeciesofglucoseoxidaseatpH5.
6[116].
Thecrystallographicdatashowedthe19presenceofHis516Nε2located~3.
8fromN(1)locusoftheflavin,thereforestabilizinginitsprotonatedstatethenegativechargeattheN(1)-C(2)=Olocusoftheflavin[110].
Theoxidationofglucosebyglucoseoxidaseproceedsviaapingpongkineticmechanism[117];whereuponformationofenzymesubstratecomplex,glucoseisoxidizedtogluconolactoneandsubsequentreductionofflavinoccurs.
Theoxidativehalf-reactionproceedswiththereleaseoftheproductwiththesubsequentoxidationofthereducedFADbymolecularoxygenandproductionofthehydrogenperoxide[117].
Productdissociationisafirstorderrate-limitingstep,withglucoseassubstrate.
However,substrateoxidationbecomespartiallyratelimitinginsteadystate,whereastheproductreleasedoesnotlimittheenzymeturnoverwith2-deoxyglucoseassubstrate[117].
Whiletheoxidationofglucosebytheenzymehasbeenextensivelystudied,denitrificationofnitroalkanesbyglucoseoxidasehasbeenonlyreportedfornitroethaneasasubstratebyPorterandBrightin1976[89].
Priortoanykineticstudies,substratespecificitystudyrevealedthatglucoseoxidaseismostefficientwithnitroethane,followedbynitromethane,1-nitropropaneand2-nitropropane.
Inaddition,anionicformswere6x103timesmorereactivethantheirneutralcounterparts.
Aping-pongkineticmechanismwasdeterminedforglucoseoxidasewithnitroalkanesassubstrates[89].
Theoxidationofthenitroalkanesbyglucoseoxidaseinvolvesformationofthesemiquinoneformoftheflavin.
Foreachequivalentethylnitronateandoxygenconsumed,formationofanequivalentofacetaldehyde,nitrateandhydrogenperoxidewerenotobserved.
Inaddition,formationofdinitroethaneandnitritewereobservedasproductsoftheenzymaticreactionwithnitroethaneassubstrate.
Formationofthedinitroethanewasfoundtobeinverselyproportionaltotheoxygenconsumedwhenconcentrationofoxygenwasbelow240M.
Theauthorsproposedthattheratioof1:1of20productstoreactantswasnotmeasuredduetotheinvolvementofthesemiquinonespeciesoftheflavinintheoxidationofthenitroalkanes,thereforeglucoseoxidaseisincapableofstabilizingtheradicalpairssufficientlyinordertotransferthesecondelectrontotheflavinandtherebycompleteoveralloxidationofthenitroalkanes[89].
3.
4.
D-Aminoacidoxidase.
AnFAD-dependentD-aminoacidoxidase(E.
C.
1.
4.
3.
3,DAAO)catalyzesthedehydrogenationofD-aminoacidstothecorrespondingα-iminoacidsthataresubsequentlyhydrolyzedtoα-ketoacidsandammonia[118;119](Scheme1.
8).
Scheme1.
8.
ReactioncatalyzedbyflavindependentD-aminoacidoxidase.
TheoxidationofD-aminoacidsbyDAAOproceedsviaasequentialkineticmechanism[120];duringthereductivehalf-reaction,theaminoacidisoxidizedwithconcomitantreductionoftheboundflavin,followedbytheoxidativehalf-reactioninwhichreducedflavinisoxidizedbymolecularoxygenwithsubsequentreleaseoftheproducts[118;121].
Inanattempttobetterunderstandthereactivityofenzyme-boundFAD,studiesontheoxidationofalternativesubstratewereperformed[90;92].
DAAOwasfoundtobeirreversiblyinactivatedbynitromethane[90].
Duetotheinactivationoftheenzymebeingdependentontheconcentrationofnitromethane,theauthors21concludedthatnitromethaneisasubstrateforDAAOonlyatlowconcentrationswhereasatconcentrationhigherthan5mMnitroethaneinactivestheenzyme[90;92].
Consequently,thesecondstudyinvolvingoxidationofnitroalkanesbyDAAOwasperformedwithanionicnitroethaneasasubstrate[92].
TheoxidationofnitroethanebyDAAOinvolvestheformationofacovalentadductbetweenthesubstratecarbanionandtheN-5positionoftheflavin,whichrapidlyeliminatesnitrite,becomeshydrated,andfinallyrearrangestoexpelacetylaldehydeandfullyreducedflavin.
Foreachequivalentethylnitronateandoxygenconsumed,oneequivalentofacetaldehyde,nitriteandhydrogenperoxideisformed[92].
TheoxidationofnitroethaneanionbyD-aminoacidoxidaseproceedsviaping-pongkineticmechanismassupportedbythesteadystatekineticdata.
Itwasalsonotedthatenzymeisinactivatedbythesubstratewhentheconcentrationofthesubstrateexceeded5mM.
Theinhibition,asauthorsproposed,isduetotheinteractionsofasecondanionofnitroethanebindingtotheenzyme[92].
Theformationofthemodifiedcoenzymeoftheinactivatedenzyme,N5-acetyl-1,5-dihydroFAD,didnotrequireoxygenassupportedbysimilarresultsobtainedinanaerobicandaerobicconditions[123].
TheinhibitionoftheDAAObytheanionspecieswasfurtherexaminedinthepresenceof1-chloro-1-nitroethane,wherethechloroandnitrogroupswererecoveredasfreeCl-andNO2-uponcompleteinactivationoftheenzymeby1.
5flavinequivalentsof1-chloro-1-nitroethane[123].
Furthermore,theinhibitionoftheDAAObyN-chloro-D-leucinewasstudiedwithrespecttothesiteandmechanismofchlorination[124].
Inthatstudyitwasshownthatflavinreductionslowsdownbythefactorof2x103duetochlorinationofthetyrosinethatisconvertedto3,5-dichlorotyrosine,assupportedbyaminoacidanalysisandspectraltitrations.
Thechlorinationreactionishighlyspecific,asonlychloroderivativesofD-leucine,D-isoleucine,andD-22norvalineareabletoinactivateDAAO,whilechloroderivativesofL-aminoacidscantightlybindandperturbthespectrumofenzyme-boundFAD[124].
FurtherstudieswithN-chloro-D-leucineconfirmedthatthechlorinationoftheDAAOintheactivesiteregionisconsistentwithconsecutivechlorinationofanaminoacidresiduebythefirst2moleculesofN-chloro-D-leucine[125].
AnaerobicandaerobicspectralstudiesofDAAOwithD-alanineand-chloro-D-alanineassubstratesfurthersupportedthatthechlorinatedderivativesinhibittheenzymereactionwithD-alaninecompetitively.
Furthermorethekineticstudiesrevealedthattheenzymereactswith-chloro-D-alaninefourtimeslessefficientlythanwithD-alanine[126].
3.
5.
Propionate-3-nitronateoxidase.
AnFMN-dependentpropionate-3-nitronateoxidase(EC1.
7.
3.
5)fromPenicilliumatrovenetumcatalyzesoxidationof3-nitropropionatetomalonatesemialdehyde,nitriteandhydrogenperoxide[88].
Todate,noin-depthbiochemical,structuralorkineticcharacterizationhasbeenperformedonpropionate-3-nitronateoxidase.
Onlyonestudyonthepurification,cofactorandsubstratespecificityoftheenzymehasbeenpublishedin1987byPorterandBright[88].
Inthatstudy,propionate-3-nitronateoxidasehasbeenpurifiedfromtheoriginalsource,i.
e.
fungusPenicilliumatrovenetum,inactiveandstableformusingthreechromatographicstepsontoanionexchange,hydroxyapatiteandblue-agarosecolumns.
Thehomogeneousenzymeisadimerwithmolecularmassof73,000Daasindicatedbysodiumdodecylsulfate-gelelectrophoresisandgelfiltrationmethods.
Oxidizedflavinspectraofpropionate-3-nitronateoxidaserapidlyconvertsintotheanionicsemiquinoneflavinspectrauponanaerobicadditionofpropionate-3-nitronate,followedbytheslowdecompositionofsemiquinonetothefullyreducedflavin,asindicatedbytheanaerobicspectrophotometricstudy.
Substratespecificitystudyontheenzymerevealedthattheenzymeiscapableofcatalyzingnumerousbranchednitronatessuchasbutyrate-4-nitronate,2-hydroxypropionate-3-nitronate,23propyl-amine-3-nitronateand2-aminopropionate-3-nitronate.
Inthesamestudy,aping-pongmechanismhasbeenassignedtotheenzymebasedonthesteadystatekineticsoftheenzymewithpropionate-3-nitronate.
4.
Goals2-NitropropanedioxygenasefromHansenulamrakiiisaflavindependentenzymethatcatalyzesthedenitrificationofanionicnitroalkanesintocorrespondingcarbonylcompoundsandnitrite,withoxygenasanelectronacceptor.
Thestudyofthisenzymeisofimportanceforappliedreasons,sincenitroalkaneshavebeenusedwidelyinchemicalindustrybuthavebeenfoundtobetoxicandcarcinogenic.
Thestudyof2-nitropropanedioxygenasehasthepotentialforthedevelopmentofbioremediationagentsthattargetthedenitrificationoftoxicandcarcinogenicnitrocompounds.
Fromafundamentalstandpoint,2-nitropropanedioxygenaseisanadditiontotwowell-characterizedflavinenzymesthatcatalyzeneutralandbothneutralandanionicnitroalkanesasintrinsicsubstrates,nitroalkaneoxidasefromFusariumoxysporumandNeurosporacrassaenzyme,respectively.
Forthisreason,thegoaloftheresearchpresentedhereinistoinvestigatethebiophysical,biochemical,spectrophotometrical,kinetic,andmechanisticpropertiesofthe2-nitropropanedioxygenasepurifiedfromrecombinantsource.
Thebiophysical,biochemical,structural,andmechanisticcharacterizationofanenzymeusuallyrequireslargequantitiesofthepure,activeandstableformofsuchenzyme.
Consequently,thefirststepinthisprojectwillbeaimedatthepurificationoftherecombinantenzyme.
Efficientapproachesforproteinpurification,suchasfractionationwithammoniumsulfateandtheionicexchangerdiethylamminoethylcelloulosecolumn,willberequiredinordertoobtainlargequantitiesofthepureenzyme.
24Foranewlypurifiedenzyme,understandingthebiochemicalandkineticpropertiesisoftheimportanceforpossibleuseoftheenzymeforappliedreasons.
Thisrequirestheestablishmentofthecofactor,steadystatekinetics,pH-dependence,substratespecificityaswellasidentifyingthecompoundsthatcaninhibittheenzyme.
Inaddition,examiningthestoichiometryofthereactioncatalyzedby2-nitropropanedioxygenasecanshedfurtherlightintothemechanismofthisenzyme.
Anin-depthcharacterizationofthefunctionandmechanismof2-nitropropanedioxygenasealsocallsforthedeterminationofthethree-dimensionalstructurethroughx-raycrystallography.
Structuraldatacangiveinsightintowhytheenzymeefficientlyutilizesanionicbutnotneutralnitroalkanes.
Therefore,obtainingthecrystalsofthe2-nitropropanedioxygenaseaspartofthestructuredeterminationwillbeperformed.
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Abe,andT.
Yamano,OnthereactionofD-aminoacidoxidasewith-chloro-D-alanine.
JBiochem73(1973)1-11.
39ChapterIIOXIDATIONOFALKYLNITRONATESCATALYZEDBY2-NITROPROPANEDIOXYGENASE(ThischapterhasbeensubmittedverbatiminMijatovic,S.
andGaddaG.
,(2008),Arch.
Biochem.
Biophys.
2008ElsevierInc.
)Abstract2-NitropropanedioxygenasefromHansenulamrakiiwasexpressedinEscherichiacolicellsandpurifiedinactiveandstableformusing60%saturationofammoniumsulfateandasinglechromatographicstepontoaDEAEcolumn.
MALDI-TOFmassspectrometricandspectrophotometricanalysesoftheflavinextractedbyheatandaciddenaturationoftheenzymeindicatedthatFMN,andnotFADaserroneouslyreportedpreviously,ispresentina1:1stoichiometrywiththeprotein.
InductivelycoupledplasmamassspectrometricanalysisoftheenzymeestablishedthatH.
mrakii2-nitropropanedioxygenasecontainsnegligibleamountsofiron,manganese,zinc,andcopperions,whicharenotcatalyticallyrelevant.
AnaerobicsubstratereductionandkineticdatausingaClarkoxygenelectrodetomeasureratesofoxygenconsumptionindicatedthattheenzymeisactiveonabroadrangeofalkylnitronates,withamarkedpreferenceforunbranchedsubstratesoverpropyl-2-nitronate.
Interestingly,theenzymereactspoorly,ifatall,withnitroalkanes,assuggestedbylackofbothanaerobicreductionoftheenzyme-boundflavinandconsumptionofoxygenwithnitroethane,nitrobutane,and2-nitropropane.
Finally,boththetightbindingofsulfite(Kd=90M,atpH8and15oC)totheenzymeandtheformationoftheanionicflavosemiquinoneuponanaerobicincubationwithalkyl40nitronatesareconsistentwiththepresenceofapositivelychargedgroupinproximityoftheN(1)-C(2)=OatomsoftheFMNcofactor.
41IntroductionNitroalkanesarewidelyusedinchemicalindustrybecausetheyprovideaquickandeffectivemethodofsynthesizingcommonreagents[1;2].
ThisstemsprimarilyfromthelowpKavaluesintherangefrom7to10andtheslowratesforprotonationanddeprotonationoftheα-carbon[3;4],whichallowforthepresenceofeither(anionic)alkylnitronatesor(neutral)nitroalkanesundermildconditions(Figure1).
However,manynitroalkanes,andtheircorrespondentalkylnitronates,areanticipatedtobetoxic[5;6;7;8;9;10],mutagensinbacteria[11;12],andhepatocarcinogensinrats[5;6].
Consequently,thestudyofthebiochemicalandkineticpropertiesofenzymeswiththeabilityofoxidizingnitroalkanesandalkylnitronateshaspotentialforbioremediationapplications.
CHH3CNOOpKa=8.
5H+CHH3CNOOHCHH3CNOOScheme2.
1.
Theionizationofnitroethane(left)toyieldethylnitronate(right)insolution.
FollowingtheinitialreportsofLittlein1951ontheoxidativedenitrificationofnitrocompoundscarriedoutbyusingextractsofNeurosporacrassa[13]andpeaseedlings[14],twonitroalkane-oxidizingenzymeshavebeenextensivelycharacterizedinthepastdecadeintheirbiochemicalandmechanisticproperties,i.
e.
,nitroalkaneoxidasefromFusariumoxysporum[15],and2-nitropropanedioxygenasefromNeurosporacrassa[16;17;18].
Nitroalkaneoxidasehasbeenshowntooxidizenitroalkanes,butnotalkylnitronates[19],viaacarbanionmechanismofcatalysisinvolvingtheformationofatransient,flavinN(5)-substrateadduct[20;21].
N.
crassa2-nitropropanedioxygenasehasbeenshowntoeffectivelyutilizeboththeneutralandanionicnitronateformsofthesubstrate[17],withcatalysisoccurringthroughasingle-electron42transferreactioninvolvingthetransientformationofananionicflavosemiquinone[17].
Otherenzymeswiththeabilitytooxidizealkylnitronates,suchashorseradishperoxidase[22;23],andtheflavoenzymesglucoseoxidase[24]andD-aminoacidoxidase[25;26],havebeenshowntobesignificantlymoreefficientwiththeirinherentsubstrates,resultingintheirnitronate-oxidizingactivitiesbeingregardedasnon-physiologicalreactions.
Theflavin-dependentenzyme2-nitropropanedioxygenase1fromHansenulamrakiihasbeenreportedtocatalyzetheoxidativedenitrificationofbothalkylnitronatesandnitroalkanesintotheircorrespondingcarbonylcompoundsandnitrite,withoxygenaselectronacceptor[27;28].
InitialreportsshowingthepresenceofironatomsinenzymaticpreparationswerefollowedbystudiesfromthesameauthorsindicatingthatH.
mrakii2-nitropropanedioxygenaseisaflavin-dependentenzymewithnorequirementforiron[29].
Inthepresentstudy,wehaveexpressedandpurifiedtohomogeneityrecombinant2-nitropropanedioxygenasefromH.
mrakii,andcharacterizedthepurifiedenzymeforitssubstratespecificityandcoenzymecontent.
Ourdataindicatethattheenzymecontainstightly,butnon-covalently,boundFMNascofactor,doesnotrequiremetalatomsforactivity,andutilizesalkylnitronatesforcatalysis,butnotnitroalkanes.
12-Nitropropanedioxygenasemaybeamisnomerforthealkylnitronate-oxidizingenzymefromH.
mrakii,assuggestedbythebroadsubstratespecificityoftheenzymewithalkylnitronatesandthepoorreactivitywithnitroalkanesreportedinthisstudy.
However,sincemechanisticstudiesaimedattheelucidationofthemechanismofcatalysisandthestoichiometryoftheenzymaticreactionhavenotyetbeencarriedout,weprefertousetheofficialIUBMBname,2-nitropropanedioxygenase,fortheenzymeinvestigatedheretoensurecontinuitywithpreviousstudyreportedbyotherauthors.
43MaterialsandmethodsMaterials.
TheplasmidpUC25-I3containingthegeneencodingfor2-nitropropanedioxygenasefromH.
mrakiiwasakindgiftfromDr.
NobuyoshiEsakiandDr.
TatsuoKurihara,KyotoUniversityinJapan.
Isopropyl-1-thio-β-D-galactopyranoside(IPTG),calfintestinalalkalinephosphatase,T4DNAligaseandrestrictionendonucleasesNdeIandBamHIwereobtainedfromPromega(Madison,WI).
TheplasmidvectorpET20b(+)wasfromNovagen(LaJolla,CA).
PrimersandprimerextensionreactionproductswerepurifiedusingminikitsfromQiagen(Valencia,CA).
EscherichiacolistrainsBL21(DE3)fromNovagenandXL1-BluefromStratagene(LaJolla,CA)wereusedforproteinexpressionandcloningprocedures,respectively.
Bothstrainswerestoredat-80oCas7%dimethylsulfoxidesuspensions.
Luria-Bertaniagarandbroth,phenylmethylsulfonylfluoride(PMSF),lysozyme,nitroalkanes,superoxidedismutase,catalaseandchloromphenicolwerefromSigma-Aldrich.
DNAse,RNAseandtheRapidDNAligationkitwerefromRocheAppliedScience(Indianapolis,IN).
TheDEAE-SepharoseresinusedforpackagingtheDEAEcolumnwasobtainedfromGEHealthcare(Piscataway,NJ).
Allreagentswereofthehighestpuritycommerciallyavailable.
Instruments.
AnAppliedBiosystemmodelABI377DNAsequencerattheDNACoreFacilityoftheBiologyDepartmentofGeorgiaStateUniversitywasusedforDNAsequencingutilizinganAppliedBiosystemsBigDyekit.
AnABIVoyagerDE-promassspectrophotometerwasusedforrecordingthematrix-assistedlaserdesorption/ionization-timeofflight(MALDI-TOF)spectra.
AnAgilentTechnologiesdiode-arrayspectrophotometerModelHP8453PC,withthermostatedwaterbathwasusedforacquiringUV-visibleabsorbancespectra.
AHansatechInstrumentscomputer-interfacedOxy-32oxygen-monitoringsystemwasusedformeasuringenzymeactivity.
44Cloningof2-NpdintopET20b(+).
E.
colistrainXL1-BluecompetentcellsweretransformeddirectlyusingplasmidpUC25-I3carryingthegeneencodingfor2-nitropropanedioxygenase(GenBankaccessioncodeU13900),utilizingtheheatshockmethodofInoueetal.
[30].
AfterisolationoftheamplifiedpUC25-I3plasmidusingaQIAquickSpinminiprepkit,plasmidDNAwasusedastemplateforprimerextensionamplificationofthegeneencodingfor2-nitropropanedioxygenasebyusingoligonucleotidesenseandantisenseprimerscontainingNdeIandBamHIrestrictionendonucleasesitesdesignedtoannealtothe5'-and3'-endsofthegene,respectively.
Theengineeredrestrictionsitesalloweddirectionalcloningofgeneencodingfor2-nitropropanedioxygenaseintocorrespondingsitesofpET20b(+)usingaRapidDNALigationkit(RocheAppliedScience).
E.
colistrainXL-1Bluecompetentcellsweredirectlytransformedusingtheligationmixture.
TheresultingplasmidpET/2NPDhmwassequencedinbothdirectionsbyusingoligonucleotideprimersdesignedtobindtoDNAregionsofpETflankingtheinsertedgene.
CompetentE.
colistrainBL21(DE3)cellsweretransformedforproteinexpression.
Expressionandpurificationof2-nitropropanedioxygenase.
PermanentfrozenstocksofE.
colistrainBL21(DE3)cellsharboringthepET/2NPDhmplasmidwereusedtoinoculate4x1LofLuria-Bertanibrothmediumcontaining50g/mLampicillinand34g/mLchloramphenicolat37oCundershakingconditions.
OncetheO.
D.
600nmreached0.
8,thebacterialcultureswereinducedwith0.
2mMIPTGfor22hoursat20oC.
Afterharvestingthecellsbycentrifugation,thecellwetpastewassuspendedin4volumesof1mMEDTA,0.
2mg/mLlysozyme,0.
1mMPMSF,10mMofMgCl2,5g/mLofDNAse,20g/mLofRNAseand50mMpotassiumphosphate,pH7.
4,beforesonication.
Theresultingcellfreeextractwascollectedbycentrifugationat12,500xgfor30min,andwastreatedwith60%saturationwith45ammoniumsulfatebystirringonicefor30minutes.
Aftercentrifugationat12,500xgfor30mintheresultingsupernatant(~150mL)wasdialyzedagainst5x2Lchangesof5mMpotassiumphosphate,pH7.
4,over16hours,andappliedtoaDEAEFastFlowcolumn(2.
5x30cm)previouslyequilibratedwith5mMpotassiumphosphate,pH7.
4.
Theenzymewaselutedfromthecolumnusingalineargradientfrom0to0.
5Msodiumchloridein5mMpotassiumphosphate,pH7.
4,ataflowrateof5mL/min.
Fractionsofthehighestpuritywerepooled,dialyzedagainst50mMpotassiumphosphate,pH7.
4,andstoredat-20oC.
Biochemicalmethods.
Theconcentrationofpurified2-nitropropanedioxygenasewasdeterminedwiththeBradfordassay[31],usingtheBio-Radproteinassaykitwithbovineserumalbuminasastandard.
SDS-PAGEwasperformedforallthepurificationstepswithmolecularweightmarkers(Sigma)containingproteinsrangingfrom6,500to200,000Da.
Themolarratioofflavintoproteinwasdeterminedinduplicatefromtheconcentrationofflavinremovedfromtheenzymeuponheatdenaturation,usinganε450nmvalueof12,200M-1s-1forFMNfreeinsolution[32],andtheconcentrationofproteindeterminedbyBradfordassay.
Identificationoftheflavincofactorof2-nitropropanedioxygenasewascarriedoutusingmatrix-assistedlaserabsorption/ionization-timeofflight(MALDI-TOF)massspectroscopicanalysisinthenegativeionmodeusinga50:50methanol/acetonitrilematrix.
Thesamplewaspreparedbydenaturingtheenzymewithheattreatmentat100oCforeither10or30min,followedbycentrifugationandcollectionofthesupernatant.
Alternatively,denaturationoftheenzymewasachievedbyincubationwith10%coldTCA(v/v)for20minonice.
Determinationofthemetalcontentof2-nitropropanedioxygenasewascarriedoutbyinductivelycoupledplasma(ICP)massspectroscopicanalysisattheChemicalAnalysisFacilityoftheUniversityofGeorgiaonasampledialyzedagainst5x2LchangesofMilliQwateroveraperiodof24hat4oC.
For46denaturingexperimentsaimedatthedeterminationofthemolarextinctioncoefficient,2-nitropropanedioxygenasewasincubatedat100oCfor15or30min,followedbytheremovalofthedenaturedproteinbycentrifugation.
Themolarextinctioncoefficientoftheflavincofactorboundtotheenzymewasdeterminedbyfollowingthechangeinabsorbanceat446nmin50mMpotassiumphosphate,pH7.
4,beforeandafterheattreatmenttoextracttheflavinfromtheenzyme.
Anaerobicsubstratereductionstudieswereperformedusingananaerobiccuvettewithtwosidearms.
Onesidearmwasloadedwiththenitroalkanebufferedsolution,whereastheothersidearmwasloadedwiththecorrespondingalkylnitronate.
Thecuvettewasmadeanaerobicbyrepeatedcyclesofvacuumingandflushingwithultra-pureargonforatleast15times,beforemixingtheenzymewiththenitroalkanesolutions.
Afterapproximately10to15minofincubation,wherenospectralchangeswereobserved,theenzymesolutionwasfurthermixedwiththealkylnitronatetoensurethattheenzymewasfunctional.
FormationofanN(5)flavin-sulfiteadductwasdeterminedbyacquiringtheUV-visiblespectraevery5secondsovera15-minuteperiodaftertheadditionof55Mto130Mofsodiumsulfitetotheenzymesolutionin50mMTris-Cl,pH8,at15oC.
Reversibilityoftheflavin-sulfitecomplexwasdeterminedbyfollowingtheincreaseinabsorbanceat455nmuponremovaloftheunboundsulfiteusinggelfiltrationofSephadexG-25column(PD-10column,GEHealthcare)equilibratedwith50mMTris-Cl,pH8.
Enzymekineticassays.
Theenzymaticactivityofthepurifiedenzymewasmeasuredinair-saturated50mMpotassiumphosphate,pH8,usingthemethodoftheinitialrates[33]bymonitoringtherateofoxygenconsumptionat30oC.
EnzymeconcentrationwasexpressedperenzymeboundFMNcontent,usingtheexperimentallydetermined446nmvalueof13,100M-1cm-1(thisstudy).
Stocksolutionsofnitroalkaneswerepreparedin100%ethanol;thoseofalkylnitronatewerepreparedbyallowingthenitroalkanestoreactwitha1.
2molarexcessofKOHfor4724hatroomtemperaturein100%ethanol.
Thefinalconcentrationofethanolineachassaymixturewaskeptconstantat1%tominimizepossibleeffectsonenzymaticactivity.
Thereactionswerestartedbytheadditionoftheorganicsubstratetopre-equilibratedreactionmixtures,inordertominimizechangesintheionizationstateofthenitroalkanesoralkylnitronatesubstrates.
Thesecond-orderrateconstantsfordeprotonationofnitroalkanes(5to6M-1s-1[4])andprotonationofalkylnitronates(15to75M-1s-1[3])weretakenintoaccount,ensuringthatthedeterminationofinitialrates(typically~30s)wasperformedwithfullyprotonatedorunprotonatedsubstrates,respectively.
Formationofsuperoxideduringcatalysiswasdeterminedbymeasuringtherateofoxygenconsumptionwith10mMorganicsubstrateineitherabsenceorpresenceof150unitsofsuperoxidedismutase.
Productionofhydrogenperoxidewasmonitoredusingasimilarapproachasforthesuperoxidedismutaseexperiments,butbyusing170unitsofcatalaseinsteadofsuperoxidedismutase.
Dataanalysis.
KineticdatawerefitwithKaleidaGraph(SynergySoftware,Reading,PA)andEnzfitter(Biosoft,Cambridge,UK)software.
Sincetheenzymecouldnotbesaturatedwiththeorganicsubstrate,theapparentsecondorderrateconstants(kcat/Km)appinatmosphericoxygenweredeterminedbyfittingtheinitialreactionratesdeterminedatvaryingconcentrationoforganicsubstratetoequation1.
AKkevappacat=(1)48ResultsCloning,expressionandpurificationof2-nitropropanedioxygenase.
Asafirststeptowardscharacterizingthepropertiesof2-nitropropanedioxygenasefromH.
mrakii,thegeneencodingfortheenzymewassubclonedinapET20b(+)plasmidexpressionvector,andtheresultingenzymewasexpressedtohighlevels.
Afractionationstepwith60%saturationofammoniumsulfateandasinglechromatographicstepusingaDEAEcolumnwererequiredtoobtainstable,active,andhomogeneouspreparationsoftheenzyme,asdeterminedbySDS-PAGE(Figure2.
1).
Typically,33gofcellwetpasteyielded85mgofenzyme,withaspecificactivityof380molO2min-1mg-1using20mMethylnitronateassubstrateat30oCandpH8.
Figure2.
1.
Purificationofrecombinant2-nitropropanedioxygenasefromHansenulamrakii.
Lane1,markerproteins;lane2,purifiedenzyme.
Cofactorcontent.
TheUV-visibleabsorbancespectrumofpurified2-nitropropanedioxygenaseatpH7.
4showedthetypicalfeaturesofaflavin-containingenzymeintheoxidizedstate[34],withtwomaximacenteredat372nmand446nm(Figure2.
2).
AMALDI-TOFspectrometricanalysisoftheflavinextractedfromtheenzymeupondenaturationwithheat,acid,49oranionicdetergent,yieldedapeakwithanm/z–ratioof455.
1(Figure2.
2),consistentwiththeflavincofactorbeingFMN.
Theextinctioncoefficientfornon-covalentlyboundflavinat446nmwasdeterminedtobe13,100M-1cm-1atpH7.
4,basedontheabsorbanceratioofboundtofreeFMN[32].
Furthermore,astoichiometryof0.
80±0.
01molofflavinpermolofproteinwasestablishedfromtheratiooftheconcentrationofextractedflavintotheproteinconcentrationobtainedbytheBradfordassay.
051015300400500600700800ε,mM-1cm-1Wavelength,nm04080400600800Count%m/z-455.
1Figure2.
2.
UV-visibleabsorbancespectrumof2-nitropropanedioxygenaseaspurified.
Theabsorbancespectrumwasrecordedin50mMpotassiumphosphate,pH7.
4.
Inset,MALDI-TOFmassspectrometricanalysisoftheflavincofactorextractedfrom2-nitropropanedioxygenase.
Themassspectrometricspectrumwasrecordedinnegativeionmodewitha50:50methanol/acetonitrilematrixusingasamplepreparedbytreatingtheenzymewithheat,centrifugationandcollectionofthesupernatant.
Inadditiontoexaminingthepropertiesoftheflavin,aninductivelycoupledplasma(ICP)massspectrometricanalysisoftheenzymetreatedwithextensivedialysisagainstMilliQwaterwasperformedtodeterminewhethermetalcofactorswerepresentintheenzyme.
Asshownin50Table2.
1,negligibleamountsofseveralmetalswereidentified(e.
g.
,≤8%),withtheironcontentbeingonly6%withrespecttotheprotein.
Alltakentogether,thedataindicatethat2-nitropropanedioxygenaseexpressedintheheterologousbacterialsystemcontainstightly,butnon-covalently,boundFMNandisdevoidofmetalcofactors.
Metalconcentration(M)%inproteinFe+25.
86Mg+26.
27Mn+22.
43Co+20.
20.
02Ni+20.
30.
3Zn+27.
68Cu+22.
33aTheanalysiswasperformedona94M2-nitropropanedioxygenasetreatedwith5x2LdialysisagainstmilliQwater.
ThelastchangeofmilliQwaterwasusedasablankforanalysis.
Table2.
1.
InductivelycoupledplasmamassspectroscopicanalysisofH.
mrakii2-nitropropanedioxygenaseaAlkylnitronatesassubstratesfor2-nitropropanedioxygenase.
2-Nitropropanedioxygenasewasmixedanaerobicallywitheithernitroalkanesoralkylnitronatestodeterminewhetherinthepresenceoftheorganicsubstratetheenzyme-boundflavinisreducedtothesemiquinoneorhydroquinonestate.
AsshowninFigure4,uponanaerobicmixingoftheenzymewith1mMethylnitronateatpH7.
4twopeaksat372nmand481nmimmediatelyappearedintheUV-visibleabsorbancespectrum,consistentwiththestabilizationofananionicsemiquinoneformoftheflavin.
SimilarresultswereobtainedatpH8withethylnitronateorbutyl-1-nitronate(Figure2.
3B),oratpH6withethylnitronate,propyl-1-nitronate,propyl-2-nitronate(Figure2.
3C),butyl-1-nitronate,pentyl-1-nitronate,orhexyl-1-nitronate,suggestingthattheanionic51semiquinonewasstabilizedintheenzyme-substratecomplexirrespectiveoftheidentityofthealkylnitronateusedandthepH.
Surprisingly,anaerobicmixingoftheenzymewith1mMnitroethanedidnotresultinanysignificantspectralchangeswithinthefirst10minofincubationatpH7.
4(Figure2.
3A)2.
Inasimilarfashion,noreductionoftheenzyme-boundflavinwasobservedwhennitroethanewasusedatpH6or8,orwhenthenitroalkanewasreplacedwithnitrobutaneor2-nitropropane(Figures2.
3Band2.
3C),suggestingthattheenzymereactspoorly,ifatall,withneutralnitroalkanes.
0100200024[O2],nmol/mLTime,min1200.
10.
2300400500600700800AbsorbanceWavelength,nm12A0.
00.
10.
2300400500600AbsorbanceWavelength,nm12B2Theslowformationofthesemiquinonespeciesoftheenzyme-boundflavinwasobserveduponanaerobicincubationoftheenzymewithnitroethaneovertimessignificantlylongerthan10min.
ThisisduetothefactthatinaqueoussolutionatpH7.
4,nitroethaneisdeprotonatedslowlyyieldingovertimeincreasingamountsofethylnitronate,whichisasubstratefortheenzyme.
520.
00.
10.
2300400500600AbsorbanceWavelength,nm12CFigure2.
3.
Anaerobicsubstratereductionof2-nitropropanedioxygenasewithalkylnitronates.
PanelA,enzymeuponanaerobicincubationwith1mMethylnitronate(line1)or1mMnitroethane(line2)assubstratefortheenzymeatafinalconcentrationof56nM,in50mMpotassiumphosphatepH7.
4and15oC.
Inset,oxygenconsumptionduringtheenzymaticturnoverwith1mMethylnitronate(line1)or1mMnitroethane(line2)in50mMpotassiumphosphatepH7.
4and30oC.
PanelB,2-nitropropanedioxygenaseuponanaerobicincubationwith1mMbutyl-1-nitronate(line1)or1mMnitrobutane(line2)in50mMpotassiumphosphatepH8and15oC.
PanelC,2-nitropropanedioxygenaseuponanaerobicincubationwith1mMpropyl-2-nitronate(line1)or1mM2-nitropropane(line2)in50mMpotassiumphosphatepH6and15oC.
AllUV-visibleabsorbancespectrawithnitroalkaneswererecordedafter10minofanaerobicincubation;thosewithalkylnitronateswereacquiredimmediatelyafteranaerobicmixingoftheenzymewiththesubstrate,i.
e.
,ca.
15s.
Asanindependentapproachtoexaminingwhether2-nitropropanedioxygenasefromH.
mrakiicanusealkylnitronatesassubstrates,butnotnitroalkanes,theratesofoxygenconsumptionweremeasuredwith1mMethylnitronateor1mMnitroethaneassubstratefortheenzymeinair-saturatedbufferatpH7.
4and30oC.
AsillustratedintheinsetofFigure2.
3A,theoxygenelectrodetracesshowedthatundertheseconditionsoxygenwascompletelydepleted53fromtheassayreactionmixturecontainingethylnitronatewithin4minofincubation,consistentwithethylnitronatebeingoxidizedbytheenzyme.
Incontrast,nooxygenconsumptionwasobservedwhennitroethanewasusedassubstratefortheenzyme,suggestingthatnitroethaneisnotasubstratefortheenzyme.
Inthisregard,theapparentrateofoxygenconsumptionwith1mMethylnitronateassubstratefortheenzymeinair-saturatedbufferatpH7.
4and30oCwasnotaffectedwhennitroethanewaspresentintheassayreactionmixtureataconcentrationashighas20mM(Table2.
2),consistentwiththeenzymenotbeingabletobindnitroethaneaseitherinhibitororpoorsubstrate.
Table2.
2.
EnzymaticactivityofH.
mrakii2-nitropropanedioxygenaseawith1mMethylnitronateasasubstrateintheabsenceandpresenceofnitroethanein50mMpotassiumphosphatepH7.
4and30oCEffectofsuperoxidedismutaseandcatalaseontheenzymaticactivity.
Theeffectofsuperoxidedismutaseorcatalaseontheenzymaticactivityof2-nitropropanedioxygenasewithunbranchedalkylnitronatesorpropyl-2-nitronatewasdeterminedatpH8and30oCtoestablishwhethersuperoxideorhydrogenperoxidewereproducedandreleasedfromtheactivesiteoftheenzymeinturnover.
AssummarizedinTable2.
3,theapparentratesofoxygenconsumptionwithunbranchedalkylnitronatesofvariouschainlengthsweresimilarirrespectiveofthepresenceorabsenceofsuperoxidedismutaseorcatalase.
Withpropyl-2-nitronate,asignificantlylowernitroethane,mMrate,bs-1050.
4±0.
7150.
5±0.
41050.
3±0.
12050.
7±1.
3aFinalconcentrationoftheenzymeintheassaywas56nM.
bKineticdataaretheaverageoftwoindependentmeasurements.
54apparentrateofoxygenconsumptionwasobservedinthepresenceofsuperoxidedismutase,butnotinthepresenceofcatalase.
Thekineticdataindicatethatwiththeexceptionofpropyl-2-nitronate,forwhichasignificantamountofsuperoxideisreleased,thereisnoreleaseofsuperoxideorhydrogenperoxidefromtheactivesiteoftheenzymeduringturnoverwithunbranchedalkylnitronates.
Substratecontrols-1+superoxidedismutasebs-1+catalasebs-1Ethylnitronate55±156±155±2propyl-1-nitronate127±1126±1125±2propyl-2-nitronate55±119±156±1butyl-1-nitronate96±195±196±1pentyl-1-nitronate95±195±295±1hexyl-1-nitronate157±2158±2155±1aEnzymeactivitywasmeasuredwith10mMorganicsubstrateintheabsenceorpresenceof151unitsofsuperoxidedismutaseor168unitsofcatalaseinairsaturated50mMTris-Cl,atpH8.
0and30oC.
bKineticdataaretheaverageoftwoindependentmeasurements.
Table2.
3.
EffectofsuperoxidedismutaseandcatalaseontheenzymaticactivityofH.
mrakii2-nitropropanedioxygenaseaSubstratespecificity.
Apparentratesofoxygenconsumptionweremeasuredasafunctionoftheconcentrationofanumberofalkylnitronatesinatmosphericoxygentodeterminethesubstratespecificityoftheenzyme.
AsillustratedintheexamplesofFigure2.
4forethylnitronateandpropyl-1-nitronate,theinitialratesofreactionwerelinearlydependentontheconcentrationofsubstrateupto20mMatpH6and30oC.
Thereforetheapparentsecondorderrateconstantskcat/Kmcouldbeestimatedforthedifferentsubstrates,butnottheturnovernumbers(kcat)andtheMichaelisconstants(Km).
AssummarizedinTable4,theenzymeshowedsimilar(kcat/Km)appvaluesinthelower105M-1s-1rangewithunbranchedalkylnitronatesof55variouschainlengthfrom2to6carbon.
Incontrast,the(kcat/Km)appvaluewithpropyl-2-nitronatewastwoordersofmagnitudelower,suggestingthattheenzymehasamarkedpreferenceforunbranchedalkylnitronates.
Substrate(kcat/Km)app,M-1s-1Ethylnitronate129,000±1,600propyl-1-nitronate170,000±2,300propyl-2-nitronatea1,600±5butyl-1-nitronate112,000±1,600Pentyl-1-nitronate121,000±2,000Hexyl-1-nitronate130,000±2,000aMeasuredinpresenceof170Uofsuperoxidedismutase.
Table2.
4.
Apparentsteadystatesecondorderrateconstants,kcat/KM,foralkylnitronatesassubstratesforH.
mrakii2-nitropropanedioxygenasein50mMpotassiumphosphatepH6and30oC.
010002000300040000510152025vo/e,s-1[alkylnitronate],mMFigure2.
4.
Apparentsteadystatekineticsof2-nitropropanedioxygenasewithethylnitronate()orpropyl-1-nitronate(),asdeterminedin50mMpotassiumphosphate,pH6and30oC.
Datawerefitwithequation1.
FormationofanN(5)-flavinadductwithsulfite.
AsshowninFigure2.
5,incubationof2-nitropropanedioxygenasewithsodiumsulfiteresultedinthebleachingofabsorbancepeakat56446nmwithconcomitantformationofanovelpeakat320nm.
SuchchangesintheUV-visibleabsorbancespectrumoftheenzyme-boundflavinareconsistentwiththeformationofanN(5)-flavinadductwithsulfite[35].
TheprocesswassufficientlyslowatpH8and15oCtoallowforthedeterminationoftheobservedrateofbleachingoftheflavinat446nmasafunctionoftheconcentrationofsulfiteusingaspectrophotometer.
Thevaluesfortherateofformation(kon)anddissociation(koff)oftheflavin-sulfiteadductcouldthenbeestimatedtobe64±6M-1s-1and0.
0056±0.
0006s-1fromtheplotofkobsversus[sulfite].
Thesevalues,inturn,wereusedtocalculateaKdvalueforsulfitebindingof88±10M,consistentwithtightbindingofsulfitetotheflavin.
Formationoftheflavin-sulfiteadductwasfullyreversibleasestablishedbytheincreaseinabsorbanceat446nmslowlyensuingaftertheremovalofexcesssulfitebygelfiltrationusingaPD-10column.
0.
000.
050.
100.
150.
20300400500600700800AbsorbanceWavelength,nm1200.
00.
51.
01.
500.
050.
1kobsx10-2,s-1[Sulfite],mMFigure2.
5.
Reactionof2-nitropropanedioxygenasewithsodiumsulfite.
Enzymewasincubatedwithvaryingconcentrationsofsodiumsulfiteintherangefrom55Mto130Minair-saturated50mMTris-Cl,pH8,at15oC.
UV-visibleabsorbancespectrawererecordedevery15secondsfor15minaftereach57additionofsodiumsulfite.
Forclarityonlyselectedspectraareshown:curve1,absorbancespectrumof2-nitropropanedioxygenaserecorded10saftertheadditionof130Msulfite;curve20,samesampleafter15minofincubation.
Theinsetshowstheobservedratesofbleachingoftheabsorbanceat452nmasafunctionofsulfiteconcentration.
58DiscussionInthisstudy,recombinant2-nitropropanedioxygenasefromtheyeastH.
mrakiiwasexpressedandpurifiedtohighlevelsinE.
colicells,andcharacterizedinregardtoitssubstratespecificity,coenzymecontent,andreactivitywithsodiumsulfite.
H.
mrakii2-nitropropanedioxygenaseoxidativelydenitrifiesalkylnitronatesofvariouschainlengths,assuggestedbyresultsofbothanaerobicsubstratereductionandkineticdatawithanumberofdifferentsubstrates.
Underatmosphericoxygenconditionstheenzymehasamarkedpreferenceforunbranchedprimaryalkylnitronatesascomparedtopropyl-2-nitronate,asindicatedbytheapparentkcat/KmvaluesdeterminedatpH6inthe105M-1s-1rangeascomparedto103M-1s-1.
Interestingly,theenzymeshowsminimaldiscriminationamongunbranchedsubstrateswithchainlengthsspanningfrom2to6carbonatoms,forwhichtheapparentkcat/Kmvalueswerecomprisedbetween1.
1x105M-1s-1and1.
7x105M-1s-1.
Inthisregard,H.
mrakii2-nitropropanedioxygenaseissimilartothewell-characterized2-nitropropanedioxygenasefromNeurosporacrassa,whichshowedsimilarsubstratespecificity[17].
Withallthesubstratestested,H.
mrakii2-nitropropanedioxygenaseisimmediatelyreducedtotheoneelectronanionicflavosemiquinonespeciesuponanaerobicmixingoftheenzymewiththesubstrate.
Moreover,noproductionandreleaseofhydrogenperoxideorsuperoxidewasobservedduringturnoveroftheenzymewithunbranchedprimaryalkylnitronates.
ThelattertwofeatureswerealsorecentlyreportedfortheN.
crassa2-nitropropanedioxygenase[17],suggestingthatthetwoenzymesmaysharesimilarcatalyticmechanismsfortheoxidationofnitronatesubstratesinwhichatransientflavosemiquinoneisobservedinturnover.
H.
mrakii2-nitropropanedioxygenasereactspoorly,ifatall,withunbranchedandsecondarynitroalkanes,assuggestedbyseveralindependentobservations.
First,uponincubating59anaerobicallytheenzymewithnitroethane,nitrobutane,or2-nitropropane,therewerenosignificantchangesintheUV-visibleabsorbancespectrumoftheoxidizedenzyme-boundflavin,irrespectiveofthepHused.
Incontrast,thesameenzymewasimmediatelyreducedtotheflavosemiquinonestateuponadditionofthecorrespondingalkylnitronatesincontrolexperiments,consistentwiththeenzymebeingfunctionalinthepresenceofpropersubstrates.
Second,nooxygenwasconsumedoveratleast180secondswhentheenzymewasincubatedwith1mMnitroethaneinareactionchamberofaClarkoxygenelectrodesystem.
Incontrast,oxygenwascompletelydepletedfromareactionmixturecontaining1mMethylnitronateinsteadofnitroethaneunderthesameconditions,suggestingthatthelackofreactivitywithnitroethaneisnotduetotheenzymebeinginactive.
Finally,theapparentratesofoxygenconsumptionwith1mMethylnitronateassubstratefortheenzymewerenotchangedwhennitroethaneuptoaconcentrationof20mMwaspresentintheenzymereactionmixture,consistentwithnitroethanenotbindingintheactivesiteoftheenzyme,eitherasaslowsubstrateoraninhibitor.
Theapparentincongruenceoftheresultsreportedinthisstudywithresultsshowingthattheenzymecanutilizenitroalkanesassubstratepreviouslyreportedbyotherauthorsisreadilyexplaineduponconsideringthatanend-pointassayforthemeasurementofthenitriteproduceduponturnoveroftheenzymewith2-nitropropanefor20minutesatpH8.
0wasused[27;28].
Indeed,oversuchanextendedtimeatpH8.
0significantamountsofnitroalkanearebeingconvertedinanon-enzymaticreactioncatalyzedbythebufferintothecorrespondingalkylnitronate[4],whichisthenoxidizedbytheenzymeasitbecomesavailable.
ThelackofreactivityofH.
mrakii2-nitropropanedioxygenasewithnitroalkanesdifferentiatesthisenzymefromtheothertwowell-characterizedflavin-dependentenzymeswiththeabilitytooxidizenitrocompounds:2-nitropropanedioxygenasefromN.
crassa,whichoxidizesbothalkylnitronatesandnitroalkanes60[17],andnitroalkaneoxidasefromFusariumoxysporum,whichoxidizesnitroalkanes,butnotalkylnitronates.
H.
mrakii2-nitropropanedioxygenasecontainstightly,butnon-covalently,boundFMNina1:1stoichiometrywiththeprotein,asindicatedbytheresultsoftheMALDI-TOFmassspectrometricandthespectrophotometricanalysesoftheflavinextractedbydenaturationoftheenzymewithheatoracid.
ConsistentwithFMNbeingthecofactorforH.
mrakii2-nitropropanedioxygenasetheaminoacidsequenceoftheenzyme(GenBankaccessionno.
AAA64484)doesnotcontainanyconsensussequenceofthetypeGXGXXG/A,whichistypicallyfoundinFAD-bindingproteins[36].
Moreover,nometalionsarepresentinamountsthataresufficientlylargetobeconsideredcatalyticallyrelevant,assuggestedbytheICPmassspectrometricanalysisoftheenzyme.
ThedeterminationoftheflavinasFMNreportedhereapparentlycontrastswithdatathatareavailableintheliterature,showingthatFADmaybethecofactoroftheenzyme[27;28;29].
Inthosestudies,however,theidentityoftheflavinwasproposedbasedontheallegedobservationthattheflavinextractedfromtheH.
mrakiienzymewasabletoconferenzymaticactivitytoanapoproteinformofD-aminoacidoxidase[28].
Indeed,theexperimentaldatawerenotpresentedandthepropercontrolexperimentsshowingeitherthepresenceorlackofenzymaticactivityinD-aminoacidoxidasereconstitutedwithFMNwerenotcarriedout[29].
LackofironintherecombinantenzymefromH.
mrakiiexpressedinE.
coliconfirmsresultspreviouslyreportedbyotherauthors,showingthattheinitialreportsdescribingH.
mrakii2-nitropropanedioxygenaseasaniron-dependentflavoproteinwereflawedbythepresenceofcontaminantiron-containingproteins[27;28].
Thus,tothisdatetheonlythree2-nitropropanedioxygenasesforwhichbiochemicalorcrystallographicdataareavailable,i.
e.
,theenzymes61fromH.
mrakii,N.
crassaandP.
aeruginosa,allrequireFMNforcatalysisanddonotcontainironatomsintheiractivesites[17;37].
Theoxidationofpropyl-2-nitronatecatalyzedbyH.
mrakii2-nitropropanedioxygenasehasasignificantnon-enzymaticcomponentthatoccursoutsidetheactivesiteoftheenzyme,assuggestedbytheeffectofsuperoxidedismutaseontheratesofoxygenconsumptionwiththissubstrate.
Suchaneffectofthedismutasecanbereadilyrationalizedwithsuperoxidebeingnormallyproducedduringturnoveroftheenzymewithorganicsubstrates,butbeingreleasedfromtheenzymesurfaceonlywhentheactivesiteisoccupiedwithpropyl-2-nitronate.
Asignificantamplificationoftherateofoxygenconsumptionisconsequentlyobservedintheabsenceofthescavengingeffectofsuperoxidedismutasebecausethesuperoxidereleasedinsolutionwouldinitiateandpropagateawell-characterizedradicalreactionofoxidationofalkylnitronates[38].
Incontrast,thelackofeffectofsuperoxidedismutasewithunbranchedalkylnitronatesisconsistentwiththeoxidationreactionoccurringexclusivelyattheactivesiteoftheenzymewithnoadventitiousreleaseofthesuperoxideintermediate.
Asimilarmechanisticbehaviorwasrecentlyreportedfortheoxidationofpropyl-2-nitronatecatalyzedbyanother2-nitropropanedioxygenase,i.
e.
,theenzymefromN.
crassa,forwhichasignificantnon-enzymaticradicalcomponentwasestablished[17].
ApositivechargeislocatedclosetotheN(1)-C(2)=OlocusoftheisoalloxazinemoietyoftheFMNcofactor,assuggestedbythestabilizationoftheanionicformoftheflavinsemiquinoneandthereactivityoftheenzymewithsulfite[35].
IntheabsenceofstructuraldataontheenzymefromH.
mrakii,onecanspeculatethatsuchapositivechargemaybeprovidedbyeitherapositivelychargedaminoacidresidueortheelectricaldipoleofanα-helix.
Inthisregard,thecrystallographicstructureof2-nitropropanedioxygenasefromP.
aeruginosaata62resolutionof2showsthepresenceofalysineresidueinproximityoftheN(1)-C(2)=OlocusoftheFMNcofactor[37].
Inconclusion,thebiochemicalcharacterizationof2-nitropropanedioxygenasefromH.
mrakiipresentedinthisstudyhasestablishedthattheenzymerequiresFMNascofactorforcatalysis,doesnotcontainironatomsinitsactivesite,utilizesalkylnitronatesassubstrates,andreactspoorly,ifatall,withnitroalkanes.
Moreover,ananionicflavosemiquinoneisformeduponanaerobicreductionoftheenzymewithnitronatesubstrates.
Theseresultsallowtocompare,andtocontrast,thisenzymewithtwootherwell-characterizedflavindependentenzymeswiththeabilitytooxidizenitroalkanes/alkylnitronates,i.
e.
,2-nitropropanedioxygenasefromN.
crassaandnitroalkaneoxidasefromF.
oxysporum.
Theformerisknowntooxidativelydenitrifybothnitroalkanesandalkylnitronatesandtoutilizemechanismfortheoxidationoftheorganicsubstrateinvolvingaone-electrontransfertotheflavin[17;18].
ThelatterisactiveonlyonnitroalkanesandhasbeenshowntoutilizeacarbanionmechanismofcatalysisproceedingthroughtheformationofaflavinN(5)-substrateadduct[15].
TheavailabilityoflargeamountsofH.
mrakii2-nitropropanedioxygenasewillbeinstrumentalforfuturebiochemicalandmechanisticstudiesaimedattheelucidationofthechemicalmechanismofcatalysisandthereasonsforthepoorreactivityoftheenzymewithnitroalkanes.
Acknowledgment:TheauthorsthankDr.
NobuyoshiEsakiandDr.
TatsuoKurihara,KyotoUniversity,Japan,forthekindgiftofplasmidpUC25-I3;Dr.
SimingWangformassspectroscopicanalysisofflavincofactor;Mr.
DawitSeyfefortheinitialcharacterizationoftheenzyme;andMs.
Baotran'Nguyenforcarryingoutthesodiumsulfiteexperiment.
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68ChapterIIISTOICHIOMETRYOFTHEREACTIONCATALYZEDBY2-NITROPROPANEDIOXYGENASEAbstractInpreviousstudies,nitroalkane-oxidizingenzymefromHansenulamrakiiwasnamed2-nitropropanedioxygenasebasedontheobservationthattheenzymeutilizes2-nitropropaneasasubstratemostefficiently.
Dioxygenasepartofthenamestemsfromtheobservationthathydrogenperoxideisnotformedduringcatalysisandconclusionthatonemoleculeofoxygenisincorporatedintotwomoleculesofcarbonylproductwhen2-nitropropaneisusedasasubstrate.
Sameresultswerenotobtainedwhen2-nitropropanewasreplacedby1-nitropropaneandnitroethane.
Therefore,thefocusofthisstudyistodeterminestoichiometryoftheenzymaticreactionoftherecombinantenzymeusingseveralalkylnitronates.
Theratiooforganicsubstratetooxygenandnitriteof2to1and3to1wasdeterminedwhenhexyl-1-nitronate,pentyl-1-nitronateandbutyl-1-nitronatewereusedassubstrates.
ThesedatasuggestthatHansenulamrakiiisadioxygenaseaspreviouslyreported.
69Introduction2-NitropropanedioxygenasefromHansenulamrakiicatalyzestheconversionofanionicnitroalkanesintocorrespondingcarbonylcompoundsbyremovingthenitriteandutilizingoxygenastheelectronacceptor(Scheme3.
1).
HCRN+–OOHCRONO2-+O2+222Scheme3.
1.
Reactioncatalyzedby2-nitropropanedioxygenase.
ThestoichiometryofthereactionwaspreviouslystudiedontheenzymeobtainedfromtheyeastH.
mrakii.
Formationofhydrogenperoxidewasmonitoredtodeterminewhether2-nitropropanedioxygenasecatalyzesthedenitrificationofsubstratesviaoxidaseoroxygenasemechanism[1].
Thelackofhydrogenperoxideformationduringcatalysisindicatedthattheenzymeactsasanoxygenaseratherthananoxidase.
Basedon18O2massfragmentationstudybothatomsofmolecularoxygenwerefoundtobeincorporatedintotheorganicproductduringtheoxidationof2-nitropropaneby2-nitropropanedioxygenase[2].
Consequently,theH.
mrakiienzymewasclassifiedasadioxygenase.
Incontrast,replacingthe2-nitropropanewith1-nitropropaneandnitroethaneassubstrates,noincorporationof18O2wasobservedinfinalproductspropionaldehydeandacetaldehyde,respectively.
Furthermore,oxygenconsumption,aswellasnitriteandacetoneformationstudies,suggestthat2-nitropropanedioxygenaseisanintermoleculardioxygenasecapableofdenitrifying2moleculesof2-nitropropanebyforming70twomoleculesofnitritewhileincorporatingtwoatomsofoxygenintotwomoleculesoftheacetone[2].
Theenzymewasclassifieddioxygenasebasedonobservationsmadewith2-nitropropaneasasubstratewhiletheobservationsthatnoincorporationof18O2wasobservedinfinalproductswhen1-nitropropaneandnitroethaneareusedassubstrateswereignored.
Also,nodatawasshownin18O2massfragmentationstudiestosupporttheseconclusions.
Therefore,inthisstudystoichiometryoftheenzymaticreactionwasthefocus.
Thereasonsforthisstudyaretoexaminethestoichiometryoftheenzymaticreactionbytherecombinantenzymeandtogainabetterunderstandingofweatherthestoichiometryoftheenzymaticreactionisconsistentforallofthesubstratesused.
Thestudywascarriedoutinthreestepsbyidentifyingtheratiooforganicsubstratetooxygen,carbonylcompoundandnitrite.
Furthermore,futurestudieswillhavetoinvolveMALDI-TOFanalysisontheenzymaticassayobtainedinmillQwaterinordertoidentifyalltheproductsofthe2-nitropropanedioxygenasereaction.
UsingabufferedassayforMALDI-TOFanalysisyieldsuslessresultsduetotheinteractionsofthebufferwiththematrixusedfortheanalysis.
Also,usingHPLCprotocoldescribedinthischapterfordeterminationofcarbonylcompoundswouldallowfordeterminationofnanomolarconcentrationofthecarbonylproduct.
71ExperimentalProceduresMaterials,enzymeandorganicsubstratepreparations.
NitroalkaneswerefromSigma-Aldrich(St.
Louis,MO).
GreissreagentsystemwasfromPromega.
Allotherreagentswereofthehighestpuritycommerciallyavailable.
2-NitropropanedioxygenasewaspreparedasdescribedinChapter2andstoredin50mMpotassiumphosphatepH7.
4at-20oC.
EnzymeconcentrationwasexpressedperenzymeboundFMNcontent,usingtheexperimentallydeterminedε445nmvalueof13,130M–1cm–1.
Stocksolutionsofalkylnitronateswerepreparedbyallowingthenitroalkanestoreactwitha1.
2molarexcessofKOHfor24hatroomtemperaturein100%ethanol.
Thefinalconcentrationofethanolineachassaymixturewaskeptconstantat1%tominimizepossibleeffectsonenzymaticactivity.
Thereactionswerestartedbytheadditionoftheorganicsubstratetopre-equilibratedreactionmixturesinordertominimizechangesintheionizationstateofthenitroalkanesoralkylnitronatesubstrates.
Thesecond-orderrateconstantsfordeprotonationofnitroalkanes(5to6M-1s-1[3])andprotonationofalkylnitronates(15to75M-1s-1[4])weretakenintoaccount,ensuringthatthedeterminationofinitialrates(typically~30s)wasperformedwithfullyprotonatedorunprotonatedsubstrates,respectively.
Themassspectroscopyanalysiswasperformedonthesample,containingtheproductsandtheenzyme,preparedbyheatingat100oCfor30minandcentrifugedtoremovetheprotein.
Instruments.
OxygenconsumptionduringturnoverwasmeasuredpolarographicallywithaHansatechoxygenelectrode(HansaTechOxy-32)thermostatedat30oC.
UV-visibleabsorbancespectrawererecordedonaHewlett-Packard8453Adiodearrayspectrophotometer.
Determinationoforganicsubstratetooxygenratio.
Consumptionofoxygenduringtheturnoveroftheenzymewasmeasuredbycomparingtheoxygenconcentrationinsolutionbeforeandaftertheenzymaticreaction.
Thebuffersolutionof50mMpotassiumphosphatepH7.
4or6and50mMsodiumpyrophosphatepH9wereequilibratedat30oCforaperiodof1572minutes,inordertoachieveastablereadingoftheoxygenconcentration.
Enzymaticreactionwenttocompletionduetothelimitedconcentrationoftheorganicsubstrate(100M,80Mor60M),whichwassmallerthantheconcentrationoftheoxygen(~250Mat30oC).
Theratiooforganicsubstratetooxygenwascalculatedbycomparingthealreadyknownconcentrationoftheorganicsubstratetotheconcentrationoftheoxygenconsumed.
Determinationoftheorganicsubstratetonitriteratio.
TheamountofnitriteproducedduringthecatalyticturnoveroftheenzymewithalkylnitronateswasdeterminedspectrophotometricallyusingaGreissmethodmodifications[5].
Briefly,calibrationcurvewasconstructedfor0-20Mnitritein2MincrementswiththenitritesolutionprovidedintheGreissreagentsystem.
Theincubationwithboth1%sulfilamideand1%ofN-1-napthylethyleneamine(NED)wereperformedonthe23oCbyprotectingthesamplesfromlightwithaluminumfoilforaperiodof5-10minutes.
The900Lofstandardnitritesolutionsaswellastotheunknownwereincubatedfirstwitha50Lofsulfilamidesolution,providedinGreissreagentsystem.
Uponincubationofthesampleswitha50LofNEDsolution,alsoprovidedinGreissreagentsystem,UV-visibleabsorbancewasmeasured.
Thestandardnitritecurvewasconstructedcomparingtheconcentrationofnitritetotheabsorbanceat450nm.
Determinationoforganicsubstratetocarbonylcompoundratio.
Theamountofcarbonylcompoundsproducedduringthecatalyticturnoveroftheenzymewithalkylnitronatesassubstrateswasdeterminedspectrophotometricallyusing2,4-dinitrophenylhydrazinebyJellinecket.
al.
[6]procedurewithmodifications.
Briefly,0.
5M2-nitropropanedioxygenasewasincubatedwith60,80or100Mhexyl-1-nitronatein1mlof50mMpotassiumphosphate(pH7.
4or6)or50mMsodiumpyrophosphate(pH9)and30°C.
Ice-coldtrichloroaceticacidatafinalconcentrationof12%(v/v)wasaddedtothemixtureandincubatedonicefor20minbeforecentrifugationat14,000*gfor15mintoremovetheprotein.
Afteradditiontothe73resultingsupernatantsof250Lof0.
4%2,4-dinitrophenylhydrazinepreparedin2MHCl,thesampleswereheatedat100°Cfor5min.
Aftercoolingthesampleswithtapwater,0.
25mlofsupernatantwasmixedwith1mlof1.
2MNaOH,andallowedtostandfor10minat25°Cbeforemeasuringtheabsorbanceat440nm.
QuantificationoftheproductsbyMALDI-TOF.
Fortheidentificationofproductsofthecatalyticturnoveroftheenzymewithhexyl-1-nitronate,MALDI-TOFmassspectrometrywasusedinboththepositiveandnegativeionmodewith50:50methanol/acetonitrilematrix.
Thesamplewaspreparedbyrunningtheenzymaticreactionandboilingitat100oCfor10minfollowedbycentrifugationtoremovetheprotein.
DataAnalysis.
DatawerefitwithKaleidaGraphsoftware(SynergySoftware,Reading,PA).
Thenitritestandardcurvewasfittedwithlineequation,whereyistheabsorbanceat450nmcorrespondingtoconcentrationofnitriteandmistheslopeoftheline.
74ResultsTheratioofbutyl-1-nitornatetooxygenconsumedduringtheenzymaticturnoverwithsubstratesis2to1(Table3.
1).
ThedeterminedratioisnotdependentonthepHoftheexperiment,assupportedbytheindependentstudiesperformedatpH6,7.
4and9.
Similarresultswereobtainedwhentheconcentrationofthebutyl-1-nitronatewasvaried(60M,80Mto100M).
Theratiooforganicsubstrateconsumedtonitriteformedwasdeterminedtobe3to1in50mMpotassiumphosphatepH6.
Theexpectationwasthatforeverymoleculeofalkylnitronatedenitrifiedduringtheenzymaticreaction,onemoleculeofnitritewouldbeformed.
ThepHofthereactionortheconcentrationofthebutyl-1-nitronatedidnotplayarole,assimilarresultswereobtainedatvaryingconcentrationsofbutyl-1nitronateinthreedifferentbuffers.
BufferOrganicsubstrate:O2Organicsubstrate:Nitrite50mMKPipH60.
52±0.
010.
34±0.
0150mMKPipH7.
40.
51±0.
020.
27±0.
0350mMNaPPipH90.
66±0.
10.
24±0.
01aDataistheaverageofthreeindependentmeasurementsperformedfor60,80and100Mofbutyl-1-nitronate.
Table3.
1.
Oxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwithbutyl-1-nitronateassubstrates.
aTheratioof2:1fororganicsubstratetooxygenwasalsodeterminedwhenpentyl-1-nitronatewasusedasasubstrate,asseeninthetable3.
2.
ThepHofthereactiondidnotplayarole,assimilarresultsweredeterminedin50mMpotassiumphosphatepH6and7.
4and50mMsodiumpyrophosphatepH9.
Foreverythreepentyl-1-nitronatemoleculesconsumedonemoleculeofnitritewasdetermined,asopposedtodeterminingtheonemoleculeofnitriteforeverymoleculeofthealkylnitronatedenitrified.
Thedeterminedratioofpentyl-1nitornateto75nitriteisnotdependentonthepHoftheexperiment.
Similarresultsfororganicsubstratetooxygenornitriteratiowereobtainedwhenhexyl-1-nitronatewasusedasasubstrate(datanotshown).
Varyingtheconcentrationofthehexyl-1-nitronateandpHofthereactionyieldedsimilarresults.
BufferOrganicsubstrate:O2Organicsubstrate:Nitrite50mMKPipH60.
50±0.
010.
32±0.
0150mMKPipH7.
40.
51±0.
020.
31±0.
0250mMNaPPipH90.
51±0.
030.
27±0.
01aDataistheaverageofthreeindependentmeasurements.
Table3.
2.
Oxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwith100Mpentyl-1-nitronateassubstrates.
aDuetothepublishedresultsonreactionofnitritewithanionicspecies,suchassuperoxideandperoxide[7;8],andreportsoftheflavindependentenzymesproducingsuperoxidetransiently[9;10],determinationoforganicsubstratetonitriteratiowasperformedinabsenceandpresenceofsuperoxidedismutase.
Ifsuperoxide,orhydrogenperoxide,isformedduringcatalysisandisreleasedinsolution,thenitritecouldreadilyreactwithsuperoxideanionsandtheconcentrationofthenitritewoulddecrease.
Inthepresenceortheabsenceofsuperoxidedismutase,theratioofhexyl-1-nitronatetooxygenandnitritewasdeterminedtobe2to1and3to1,respectively(Table3.
3).
Uponsubstitutionofhexyl-1-nitronatewithpentyl-1-nitronateandbutyl-1-nitronate,similarresultswereobtained,indicatingthattheproductionandthereleaseofthesuperoxideandthesubsequentreactionwiththenitritedonotoccur.
Inadditiontoperformingtheexperimentwithsuperoxidedismutase,theeffectofcatalasewasexaminedduetoproductionofthehydrogenperoxidebymanyknownoxidases[9].
Noeffectwasobservedwhencatalasewasaddedintotheassaywithhexyl-1-nitronateasasubstratein50mM76potassiumphosphatepH7.
4.
Similarresultswereobtaineduponsubstitutinghexyl-1-nitronatewithpentyl-1-nitronateorbutyl-1-nitronate,asseenintheTable3.
3.
substrate+/-SOD(L)Organicsubstrate:O2Organicsubstrate:Nitritehexyl-1-nitronate00.
520.
3250.
500.
32200.
510.
32pentyl-1-nitronate00.
660.
3950.
640.
41200.
650.
35butyl-1-nitronate00.
590.
3750.
630.
39200.
630.
33substrate+/-Catalase(L)Organicsubstrate:O2Organicsubstrate:Nitritehexyl-1-nitronate00.
490.
3550.
480.
35200.
490.
33pentyl-1-nitronate00.
680.
3950.
720.
40200.
710.
39butyl-1-nitronate00.
610.
3750.
580.
40200.
600.
37aMeasuredin50mMpotassiumphosphate,pH7.
4and30oC.
Table3.
3.
Effectofsuperoxidedismutaseandcatalaseonoxygenconsumptionandnitriteproductionduring2-nitropropanedioxygenaseturnoverwith10mMalkylnitronatesassubstrates.
aDeterminingthehexanalasaproductoftheenzymaticturnoverwithhexyl-1-nitronatebyJellinecketalmethod[6]wasunsuccessfulduetotheinsolubilityofthehexanalintheaqueoussolution(datanotshown).
Severalindependentattemptsmadewithcommerciallyavailablehexanaltoconstructthestandardcurvewereunsuccessful.
Inordertoconfirmifthemethodisusable,twostandardcurves,oneforbetainealdehydeandoneforhexanal,wereconstructedbyfollowingthesameprotocol.
ThestandardcurveforbetainealdehydeyieldedahighR2of0.
99953whilethehexanalstandardcurvedidnotevenproducethelinewiththepositiveslope.
77-0.
2500.
250.
50.
75100.
511.
522.
5Absorbanceat440nm[Substrate],mMFigure3.
1.
Standardcurveconstructedbycomparingtheconcentrationofthecompoundwiththeabsorbanceof440nm.
()Standardcurveforbetaine-aldehydeand()hexanal.
78DiscussionMeasuringtheoxygenconcentrationbeforeandaftercatalysisandcomparingittotheconcentrationoftheorganicsubstratehaveyieldedtheratiooforganicsubstratetooxygenof2to1.
Thisratioisindependentoftheconcentrationoforganicsubstrate,aswellasthelengthofthecarbonchainonthesubstrate,assuggestedbystudiesperformedonthehexyl-1-nitronate,pentyl-1-nitronateandbutyl-1-nitronateatdifferentconcentrations.
ThepHdidnotplayaroleontheratiooforganicsubstratetooxygen,asseenfromtheratiodeterminedatpH6,7.
4and9.
Thedeterminedratiooforganicsubstratetonitriteof3to1doesnotmatchthetheoreticallyexpectedratioof1to1.
ThirdofthenitritecanbedeterminedregardlessoflengthorconcentrationoftheorganicsubstrateorthepHusedduringcatalysis.
Itisexpectedthatdenitrificationofonemoleculeofalkylnitronatewouldyieldonemoleculeofnitrite.
SinceGreissreagentisspecificfornitrite,quantificationofnitrate,nitrousornitricacidmoleculesasaresultofnitriteconversionwouldhavetobedeterminedwithothermethods.
Therefore,atthisstage,weareunabletoaccountfor2/3ofthenitrite.
AnionicoxygenspeciessuchassuperoxideO2-canbeproducedbyflavin-dependentenzymesinreducedstateuponreactionwithmolecularoxygen[9;11;12].
Inaddition,hydrogenperoxidehasbeenfoundtobeaproductofflavin-dependentenzymes[13;14].
Bothanionicspecies,superoxideO2-andperoxideO22-,havebeenidentifiedtobestableinmanyionicsolvents[7].
Nitriteiscapableofreactingwithsuperoxidetoproducenitrateandperoxide,inadditionofbeingcapableofreactingwithperoxide[8].
Theformationandreleaseofsuperoxideorhydrogenperoxidewasnotobservedduringenzymaticreaction.
Theconversionofnitritetonitrateortootherspecieswillhavetobetestedwiththemethodsspecificforthementionedcompounds.
79Inabilitytoconstructthestandardcurvewithcommerciallyavailablehexanalisduetoprecipitationofthecompoundinaqueoussolutionuponreactionwith2,4-dinitrophenylhydrazine.
Futurestudiesfocusingonthedeterminationofcarbonylproductswillhavetobecarriedoutbyamethodthatallowsdeterminationofthealdehydeandketonecompoundsinnanomolaramounts.
Studyondeterminationofpropionaldehyde,formaldehydeandacetaldehydeinnanomolaramountswascarriedoutonthesamplesobtainedbymixingthecompoundswith2,4-dinitrophenylhydrazineandusingtheHPLC[15].
Inthatstudy,C18(5m4.
6mmi.
D.
x150mmlength)reverse-phasecolumnwasusedataflowrateof1mLmin-1byelutingthesampleswithtwomobilephases,phaseA(70%milliQwater,20%tetrahydrofuran(THF)and10%methanol)andphaseBconsistingof90%acetonitrileinmilliQwater.
Thechromatographywascarriedoutwithanisocraticelutionat90%Bover6minutes,followedbya90%Ato60%Agradientover24minutes,followedby60%Ato0%Agradientover10minutes.
Theprocesswasfinishedwithanisocraticgradientof90%Bfor10minutes.
TheUV-visibleabsorptiondetectorwasoperatedat365nm.
Inconclusion,MALDI-TOFanalysisofthereactionmixtureinmillQwaterisrequiredinordertoidentifytheproductsofthe2-NPDenzymaticreaction.
Furthermore,utilizingtheHPLCmethoddescribedinpreviousparagraphwillallowfordeterminationoftheconcentrationofthecarbonylproduct.
80References[1]T.
Kido,T.
Yamamoto,andK.
Soda,Purificationandpropertiesofnitroalkane-oxidizingenzymefromHansenulamrakii.
JBacteriol126(1976)1261-5.
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Kido,K.
Soda,T.
Suzuki,andK.
Asada,Anewoxygenase,2-nitropropanedioxygenaseofHansenulamrakii.
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Gadda,D.
Y.
Choe,andP.
F.
Fitzpatrick,UseofpHandkineticisotopeeffectstodissecttheeffectsofsubstratesizeonbindingandcatalysisbynitroalkaneoxidase.
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T.
Nielsen,TheChemistryoftheNitroandNitrosoGroupsIntersciencePublishers,NewYork,1969.
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S.
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H.
Snyder,Nitricoxide:aphysiologicmessengermolecule.
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R.
Strength,andS.
A.
Thayer,Isolationandidentificationoftheproductsoftheoxidationofcholine.
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G.
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Cavaggioni,Redoxmechanisminanionicmatrix.
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Paniccia,andP.
G.
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Messner,andJ.
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Imlay,TheidentificationofprimarysitesofsuperoxideandhydrogenperoxideformationintheaerobicrespiratorychainandsulfitereductasecomplexofEscherichiacoli.
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Massey,Thechemicalandbiologicalversatilityofriboflavin.
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Massey,Activationofmolecularoxygenbyflavinsandflavoproteins.
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Fridovich,Quantitativeaspectsoftheproductionofsuperoxideanionradicalbymilkxanthineoxidase.
JBiolChem245(1970)4053-7.
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Cona,G.
Rea,R.
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Federico,andP.
Tavladoraki,Functionsofamineoxidasesinplantdevelopmentanddefence.
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Ozimek,M.
Veenhuis,andI.
J.
vanderKlei,Alcoholoxidase:acomplexperoxisomal,oligomericflavoprotein.
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Takeda,S.
Katoh,N.
Nakatani,andH.
Sakugawa,Rapidandhighlysensitivedeterminationoflow-molecular-weightcarbonylcompoundsindrinkingwaterandnaturalwaterbypreconcentrationHPLCwith2,4-dinitrophenylhydrazine.
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83ChapterIVINHIBITIONOF2-NITROPROPANEDIOXYGENASEWITHCHLORIDEIONSAbstractTheflavin-dependentenzyme2-nitropropanedioxygenasefromHansenulamrakiicatalyzestheoxidativedenitrificationofalkylnitronatesintotheircorrespondingcarbonylcompoundsandnitrite,withoxygenaselectronacceptor.
Flavin-dependentenzymes,whichdenitrifynitroalkanes,suchasglucoseoxidaseandD-aminoacidoxidase,areknowntobeinhibitedbythechlorideionsorcompoundscontainingchloride.
Inthischapter,theeffectofchlorideionsontheenzymaticactivityof2-nitropropanedioxygenaseisexamined.
Theinactivationofthe2-nitropropanedioxygenasebychlorideionswasdependentontheconcentrationofthehalide.
Theapparentsecondorderrateconstants(kcat/Km)appfollowedsaturationcurve,wheresaturationregionwasobservedoncetheratioofchlorideionstoflavinwas2to1,suggestingthatchlorideionsdonotactascompetitiveinhibitorswhenethylnitronateisusedasasubstrate.
Inabilitytosaturateenzymewithorganicsubstratepreventeddeterminationofthechlorideionsinhibitionpattern.
84Introduction2-Nitropropanedioxygenase(2NPD)fromHansenulamrakiicatalyzestheconversionofanionicnitroalkanesintocorrespondingcarbonylcompoundsbyremovingnitriteandutilizingoxygenastheelectronacceptor(Scheme4.
1).
HCRN+–OOHCRONO2-+O2+222Scheme4.
1.
Reactioncatalyzedby2-nitropropanedioxygenase.
Withtheinitialcharacterizationof2-nitropropanedioxygenase,inhibitoryeffectsofseveralcompoundsaswellasfewchelatingagentshavebeentested,duetothepresenceofiron-dependentproteinboundedto2-nitropropanedioxygenase[1;2;3].
Chelatingagentsfordivalentandtrivalentmetalssuchα,α'-dipyridylando-phenanthrolineactedasactivatorsfortheenzyme.
However,EDTA,p-chloromercuribenzoate,N-ethylmaleimideandiodoacetatehavebeenfoundtoinhibitenzymaticactivity.
Tiron,cysteineandglutathionewerefoundtoinhibit2-nitropropanedioxygenasecompletely;whileHgCl2andoxinewerefoundtobepowerfulinhibitors.
AsimilarstudywasalsoperformedonnitroalkaneoxidasefromFusariumoxysporum,whereHgCl2aswellasp-chloromercuribenzoatewerefoundtocompletelyinhibittheenzyme[4].
EvenwiththeinitialcharacterizationoftheNeurosporacrassa2-nitropropanedioxygenase,LittleidentifiedNaCl,KCl,andNH4Clasinhibitorsoftheenzyme[5].
Halideshavebeenfound85tobecompetitiveinhibitorsofglucoseoxidasewiththerespectofD-glucoseor2-deoxy-D-glucoseassubstrate,sothepurificationstepsaswellasthesolutionsusedforstoringorstudyingtheenzymehavetobedevoidofchloride[6;7].
ThebindingofthechlorideionstotheoxidizedformofglucoseoxidaseresultsinaperturbationoftheUV-visibleabsorbtionspectrumoftheenzyme-boundflavin[6].
ChlorideionsalsoperturbthepKaoftheionizablegroupontheproteinthatisresponsibleforthebindingofthesubstrate,asindicatedbysteady-statekineticstudies[6].
TheflavindependentD-aminoacidoxidase(DAAO)iscompletelyinactivatedby1-chloro-1-nitroethane,byratioof1:1.
5of1-chloro-1-nitroethanetoflavin[8].
Furthermore,theinhibitionoftheDAAObyN-chloro-D-leucinewasstudiedwithrespecttothesiteandmechanismofchlorination[9].
Inthatstudyitwasshownthatflavinreductionslowsdownbyafactorof2x103duetochlorinationofthetyrosinethatisconvertedto3,5-dichlorotyrosine,assupportedbytheaminoacidanalysisandspectraltitrationstudy.
FurtherstudieswithN-chloro-D-leucineconfirmedthatthechlorinationofDAAOintheactivesiteregionisconsistentwithconsecutivechlorinationofanaminoacidresiduebythetwomoleculesofN-chloro-D-leucine[10].
AnaerobicandaerobicspectralstudiesofDAAOwithD-alanineor-chloro-D-alanineassubstratefurthersupportedthenotionthatchlorinatedderivativesinhibittheenzymecompetitively.
Furthermore,kineticstudiesrevealedthattheenzymereactswith-chloro-D-alaninefourtimeslessefficientlythatwithD-alanine[11;12].
Therefore,theobjectiveofthisstudywastoidentifythepatternofinhibitionbythechlorideionsontheenzymaticactivityof2-nitropropanedioxygenase.
Chlorideionsinhibit60%oftheenzymaticactivityuponadditionof2:1ratioofchloridetoflavin.
Chlorideionsarenotcompetitiveinhibitorsof2-nitropropanedioxygenasewiththerespecttoethylnitronate,sincetheinhibitioneffectofchlorideionsontheapparentsecondorderrateconstants(kcat/Km)appfollowed86saturationcurve.
However,duetoinabilitytosaturatetheenzymewiththeorganicsubstrate,distinguishingbetweenthenoncompetitiveanduncompetitivepatternoftheinhibitioncouldnotbedetermined.
87ExperimentalProcedureMaterials.
NitroalkaneswerefromSigma-Aldrich.
PotassiumphosphateandKClwerefromFisher.
TrisbasewasfromJ.
TBarker.
Allreagentswereofthehighestpuritycommerciallyavailable.
Methods.
Enzyme,2-nitropropanedioxygenasewaspreparedasdescribedinChapter2andstoredin50mMpotassiumphosphatepH7.
4at-20oC.
EnzymeconcentrationwasexpressedperenzymeboundFMNcontent,usingtheexperimentallydetermined445nmvalueof13,130M–1cm–1.
Enzymeactivitywasmeasuredwiththemethodoftheinitialrates[13]inairsaturated50mMpotassiumphosphateorTris-ClatpH6bymonitoringtherateofoxygenconsumptionwithacomputer-interfacedOxy-32oxygen-monitoringsystem(HansatechInstrumentLtd.
)thermostatedat30oC.
Stocksolutionsofalkylnitronateswerepreparedbyallowingthenitroalkanestoreactwitha1.
2molarexcessofKOHfor24hatroomtemperaturein100%ethanol.
Thefinalconcentrationofethanolineachassaymixturewaskeptconstantat1%tominimizepossibleeffectsonenzymaticactivity.
Thereactionswerestartedbytheadditionoftheorganicsubstratetopre-equilibratedreactionmixtures,inordertominimizechangesintheionizationstateofthenitroalkanesoralkylnitronatesubstrates.
Thesecond-orderrateconstantsfordeprotonationofnitroalkanes(5to6M-1s-1[14])andprotonationofalkylnitronates(15to75M-1s-1[15])weretakenintoaccount,ensuringthatthedeterminationofinitialrates(typically~30s)wasperformedwithfullyprotonatedorunprotonatedsubstrates,respectively.
Dataanalysis.
KineticdatawerefitwithKaleidaGraph(SynergySoftware,Reading,PA)andEnzfitter(Biosoft,Cambridge,UK)software.
Sincetheenzymecouldnotbesaturatedwiththeorganicsubstrate,theapparentsecondorderrateconstants(kcat/Km)appinatmosphericoxygenweredeterminedbyfittingtheinitialreactionratesdeterminedatvaryingconcentrationof88organicsubstratetoequation1.
Here,KarepresentstheMichaelisconstantsfororganicsubstrate(A)andkcatistheturnovernumberoftheenzyme(e).
AKkevappacat=(1)89ResultsandDiscussionTheenzymaticactivityof2-nitropropanedioxygenasewith20mMethylnitronatein50mMpotassiumphosphatepH6and50mMTris-ClpH6(Fig4.
1)wasmeasuredtobe770s-1and200s-1,respectively.
Theexperimentwasperformedwiththesamesampleoftheenzymeandthesubstrate.
01002003000246810[O2],nmol/mLTime,min21Figure4.
1.
Oxygenconsumptionof2-nitropropanedioxygenasewith20mMethylnitronateduringturnover.
Line1-in50mMpotassiumphosphatepH6and30oC;line2-in50mMTris-ClpH6and30oC.
Furthermore,theapparentsecondorderrateconstants(kcat/Km)appinabsenceandpresenceofdifferentconcentrationsofpotassiumchloridewereobtained.
AsseeninTable4.
1,theenzymaticactivityof2-nitropropanedioxygenasewithethylnitronateisinhibitedinthepresenceofdifferentconcentrationsofpotassiumchloride.
Intherangeof0.
11-1nMofpotassiumchlorideinhibitionoftheenzymaticactivityincreaseslinearlyastheconcentrationofpotassiumchlorideincreases.
Thefurtherincreaseofthechlorideconcentrationupto11nMdidnotleadto90furtherinhibitionoftheenzyme.
Furthermore,evenwhen0.
9mMpotassiumchloridewasusedintheassaysimilarresultswereobtained(datanotshown).
Therefore,chlorideionscannotbecompetitiveinhibitorsof2-nitropropanedioxygenasesincethedegreeofinhibitiondoesnotincreasewiththeincreaseoftheinhibitorconcentration.
[KCl]nM(kcat/KM)0amM-1s-1(kcat/KM)KClbmM-1s-10.
11129±297±3.
30.
33152±3109±4.
21133±488±3.
11.
9131±577±3.
83.
3152±387±2.
89133±477±1.
911127±375±1.
1a(kcat/KM)0representsapparentsteadystatesecondorderrateconstantobtainedintheabsenceofthepotassiumchloride.
b(kcat/KM)0representsapparentsteadystatesecondorderrateconstantobtainedinthepresenceofthepotassiumchloride.
Table4.
1.
Apparentsteadystatesecondorderrateconstants,kcat/KM,inabsenceandpresenceofpotassiumchloridein50mMpotassiumphosphatepH6and30oC.
Thefinalconcentrationof2-nitropropanedioxygenaseintheenzymaticassaywas1.
1nM.
AsitiseasilyseenintheFigure4.
2,maximuminhibitionof40%bychlorideionson2-nitropropanedioxygenaseactivityisobservedevenwhenonlytwochlorideionspereveryflavinmoleculearepresentinthesolution.
Itcouldbehypothesized,thattherearetwositesontheproteinwherechlorideionscouldbindandinduceconformationalchangesthatinhibitcatalyticpoweroftheenzyme.
Beingthatenzymaticassayswereperformedin50mMpotassiumphosphate,theadditionofonly0.
11to11nMofpotassiumchloridedoesnotaffecttheionic91strength.
Therefore,theobservedreducedrateofthe2-nitropropanedioxygenasecannotbeassignedtotheionicstrengthofthesolution.
11.
21.
41.
61.
82024681012(kcat/KM)0/(kcat/KM)KCl[KCl]/[2NPD]Figure4.
2.
EffectofKClonkcat/KMof2-nitropropanedioxygenasein50mMpotassiumphosphatepH7.
4and30oC.
The[KCl]/[2NPD]representstheratioofpotassiumchloridetoenzymeconcentrationusedintheenzymaticassays.
(kcat/KM)0representsapparentsteadystatesecondorderrateconstantobtainedintheabsenceofthepotassiumchloride.
(kcat/KM)0representsapparentsteadystatesecondorderrateconstantobtainedinthepresenceofthepotassiumchloride.
(kcat/KM)0/(kcat/KM)KClrepresentstheratioofsecondorderrateconstantinabsenceandpresenceofpotassiumchloride.
Theconcentrationof1.
1nMof2-nitropropanedioxyganasewasusedthroughoutallexperiments.
Ifthechlorideionswerecompetitiveinhibitorsoftheenzymewiththerespecttotheethylnitronateasasubstrate,theinhibitionoftheenzymewouldbefurtherincreasedwiththeincreaseofthechlorideionconcentrationlargerthan1.
9nM.
Therefore,chlorideionsarenotcompetitiveinhibitorsfor2-nitropropanedioxygenasewiththerespecttoethylnitronate.
Inordertofurtherdistinguishbetweennoncompetitiveanduncompetitiveinhibitionpatternofchlorideions,saturationoftheenzymewiththeorganicsubstrateisneeded.
Becauseorganicsubstrateis92madeinethanolandthepercentageofethanolintheenzymaticassayiskeptunder1%,theconcentrationoftheorganicsubstratehigherthan20mMinenzymaticassaycannotbeachived.
Steady-statekineticsmeasuredwithethylnitronateasasubstratein50mMpotassiumphosphatepH6and8and50mMsodiumpyrophosphatepH10.
5at30oChaveshownthatthehighestsaturationoftheenzymewiththeorganicsubstrateisachievedatpH8.
Inordertodetermineinhibitionpatternofchlorideionsinfutureexperiments50mMpotassiumphosphatepH8shouldbeused.
Additionally,alcoholscontainingthenitrogrouparemoresolubleinwaterthannitroalkanes,e.
g.
2-nitro-1-propanolismoresolubleinwaterthan2-nitropropane[16].
If2-nitropropanedioxygenasecaneffectivelydenitrifythesealcohols,usingthemassubstratesmightprovideawaytosaturatetheenzymewithorganicsubstrateanddeterminetheinhibitionpatternofthechlorideions.
01000200030000510152025vo/e,s-1[Ethylnitronate],mMFigure4.
3.
Apparentsteadystatekineticsof2-nitropropanedioxygenasewithethylnitronate(),inpresenceof()1mMpotassiumiodide,()potassiumbromideorpotassiumfluoride()asdeterminedin50mMpotassiumphosphate,pH6and30oC.
Datawerefitwithequation1.
93Inaddition,theapparentsecondorderrateconstants(kcat/Km)appinabsenceandpresenceof1mMpotassiumiodide,potassiumbromideorpotassiumfluorideweredetermined(Figure4.
1).
Theapparentsecondorderrateconstants(kcat/Km)appof2-nitropropanedioxygenasewithethylnitronatein50mMpotassiumphosphatepH6wasdeterminedtobe120,000M-1s-1,whileinpresenceof1mMpotassiumiodide,potassiumbromideorpotassiumfluoridewasdeterminedtobe99,000M-1s-1,51,000M-1s-1or97,500M-1s-1,respectively.
Theenzymaticactivityof2-nitropropanedioxygenasewithethylnitronateisinhibited18%,57.
5%or19%inpresenceofpotassiumiodide,potassiumbromideorpotassiumfluoride.
Halideionscanpossiblyinduceconformationalchangesin2-nitropropnaedioxygenaseandthereforeinfluencetheactivityoftheenzyme.
InthecaseofcytochromeP450itwasestablishedthatconformationoftheproteinishighlydependentontheconcentrationofNaCl,asα-helixcontentincreasedandβ-sheetdecreasedwiththeincreaseinthesaltconcentration,coincidingwithhigheractivityoftheenzyme[17].
Inaddition,conformationalchangesbychlorideionsareresponsibleforinhibitionofshikimatekinasefromMycobacteriumtuberculosis[18].
Inthiscontext,futurestudiesfocusingontheeffectofhalideionsontheenzymaticactivityof2-nitropropanedioxygenasewillhavetoincludeexaminingpossibleconformationalchangesoftheenzymeinthepresenceofhalideions.
Inconclusion,usingnitrocompounds,whicharereadilysolubleinwater,assubstratescouldleadtosaturationoftheenzymewiththeorganicsubstrateandthereforeallowforthedeterminationoftheinhibitionpatternofhalideions.
Inaddition,futurestudiesfocusedonreasonswhy2NPDisimhibitedbyhalideionswillhavetoincludestudiesonconformationchanges.
94References[1]T.
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Soda,T.
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Yamamoto,andK.
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96ChapterVPHSTUDIESON2-NITROPROPANEDIOXYGENASEAbstractThestudiesonpHdependenceofthekineticparameterscouldprovidethevaluableinsightintotheenzymaticreaction.
Thesestudieshaveprovidedtheevidencesupportingtheneedforprotonatedandunprotonatedgroupforcatalysisforbothnitroalkaneoxidaseand2-nitropropanedioxygenasefromNeurosporacrassa.
Eventhoughcrystallographicandmutagenesisstudieshelpedwithidentifyingtheionizablegroups,thepHprofilesofthesetwonitroalkane-oxidizingenzymeswerefirstevidenceinestablishingtheneedforacidorabaseincatalysis.
Inthisstudy,thepHdependenceofapparentsecondorderrateconstants(kcat/Km)appwithethylnitronateassubstrateshowedtwoplateauregions,consistentwiththerequirementoftwoionizablegroupsthathavetobeprotonatedforcatalysis.
Withmixtureofnitroethane/ethylnitronateassubstratesthe(kcat/Km)appvaluesyieldedbell-shapedpHprofile.
SimilarresultswereobtainedforpHdependenceofapparentsecondorderrateconstants(kcat/Km)appforbutyl-1-nitronateandthemixtureofbutyl-1-nitronate/nitrobutane.
97IntroductionThestudiesonpHdependenceofthekineticparameterscanrevealhowmanyionizablegroupsareneededincatalysisandifthosegroupsneedtobeprotonatedordeprotonatedforcatalysistooccur.
ThepHdependencestudieshavebeencarriedoutforbothwell-characterizednitroalkaneoxidizingenzymes,nitroalkaneoxidase[1]and2-nitropropnaedioxygenasefromNeurosporacrassa[2].
ThepHdependencestudiesonthekineticparametersofnitroalkaneoxidasewithnitroethaneassubstrateshowedtherequirementfortwoionizablegroups,onethatmustbeunprotonatedandthesecondprotonatedforcatalysis[1].
Theaminoacidthatneedstobeprotonatedforcatalysiswaslateridentifiedastyrosineresiduethatparticipatesinsubstratebindingbasedontheinactivationstudieswithtyrosinedirectedreagent,tetranitromethane[3].
Basedoncrystallographicandmutagenesisstudies,thegroupthatneedstobeunprotonatedforcatalysiswasidentifiedtobeAsp402,whichabstractstheα-carbonofnitroalkanesubstrates[4-6].
Inthecaseof2-nitropropanedioxygenasefromNeurosporacrassa,oxidationoftheneutralnitroalkanesubstratesrequiresacatalyticbase,assupportedbythepHprofilesofthekineticparametersobtainedwithnitroethaneandnitrobutaneassubstrates(figure5.
1).
Inaddition,thepHprofilesofthekineticparameterswithbothalkylnitronatesandnitroalkanesshowedtherequirementofionizablegroupthatneedstobeprotonatedforcatalysis,asshowninFigure5.
2foralkylnitronates[2].
98Figure5.
1.
pHdependenceofthekcat/Kmvalueof2-nitropropanedioxygenasefromNeurosporacrassawithnitroalkanes.
Takenwithoutpermission[2].
Figure5.
2.
pHdependenceofthekcatvalueof2-nitropropanedioxygenasefromNeurosporacrassawithalkylnitronates.
Takenwithoutpermission[2].
ThefocusofthisstudyisonobtainingthepHdependenceofsecondorderrateconstants(kcat/Km)appwithtwoalkylnitronatesandthemixtureofnitroalkane/alkylnitronate.
99ExperimentalprocedureMaterials.
NitroalkaneswerefromSigma-Aldrich.
PotassiumphosphateandpotassiumpyrophosphatewerefromFisher.
Allreagentswereofthehighestpuritycommerciallyavailable.
Methods.
Enzyme,2-nitropropanedioxygenasewaspreparedasdescribedinchapter2andstoredin50mMpotassiumphosphatepH7.
4at-20oC.
EnzymeconcentrationwasexpressedperenzymeboundFMNcontent,usingtheexperimentallydeterminedε445nmvalueof13,130M–1cm–1.
Enzymeactivitywasmeasuredwiththemethodoftheinitialrates[7]in100mMsodiumpyrophosphate(intherange5.
5-6and8.
5-10.
5)andpotassiumphosphate(intherangeof6.
5-8and11)bymonitoringtherateofoxygenconsumptionwithacomputer-interfacedOxy-32oxygen-monitoringsystem(HansatechInstrumentLtd.
)thermostatedat30oC.
Preparationofsubstrates.
Stocksolutionsofalkylnitronateswerepreparedbyallowingthenitroalkanestoreactwitha1.
2molarexcessofKOHfor24hatroomtemperaturein100%ethanol.
Thefinalconcentrationofethanolineachassaymixturewaskeptconstantat1%tominimizepossibleeffectsonenzymaticactivity.
Thereactionswerestartedbytheadditionoftheorganicsubstratetopre-equilibratedreactionmixtures,inordertominimizechangesintheionizationstateofthenitroalkanesoralkylnitronatesubstrates.
Thesecond-orderrateconstantsfordeprotonationofnitroalkanes(5to6M-1s-1[8])andprotonationofalkylnitronates(15to75M-1s-1[9])weretakenintoaccount,ensuringthatthedeterminationofinitialrates(typically~30s)wasperformedwithfullyprotonatedorunprotonatedsubstrates,respectively.
Mixturesofnitroalkanes/alkylnitronateswerepreparedbydilutingthenitroalkanesinbufferandadjustingthepHpriortoperformingtheassay.
100Dataanalysis.
KaleidaGraph(SynergySoftware,Reading,PA)orEnzfitter(Biosoft,Cambrage,UK)softwarewasusedtoanalyzethedata.
Sincetheenzymecouldnotbesaturatedwiththeorganicsubstrate,theapparentsecondorderrateconstants(kcat/Km)appinatmosphericoxygenweredeterminedbyfittingtheinitialreactionratestoMichaelis-Mentenequationforonesubstrate.
Here,KarepresentstheMichaelisconstantsfororganicsubstrate(A)andkcatistheturnovernumberoftheenzyme(e).
AKkevappacat=(1)Whenbothoxygenandtheorganicsubstrateconcentrationwerevariedkineticparametersweredeterminedbyfittingthedatawithequations2and3,describingasequentialandaping-pongsteadystatekineticmechanism.
Here,KaandKbrepresenttheMichaelisconstantsfororganicsubstrate(A)andoxygen(B),respectivelyandkcatistheturnovernumberoftheenzyme(e).
Apparentkineticparametersinatmosphericoxygenweredeterminedbyfittingthedatawithequation3,describingMichaelis-Mentenequation,whereKarepresentstheMichaelisconstantfororganicsubstrate(A)andkcatistheturnovernumberoftheenzyme(e).
biabacatKKABAKBKABkev+++=(2)ABAKBKABkevbacat++=(3)Whenethylnitronatewasusedassubstrate,thepHdependenceoftheapparentsteadystatekineticparameterswasdeterminedbyfittinginitialratedataobtainedatvaryingconcentrationsoforganicsubstratetoequations2,whichdescribeacurvewithaslopeof–1andaplateauregionatlowpH.
101+=pHpK10101CloglogY2a(4)Whenmixtureethylnitronate/nitroethanewasused,thepHdependenceoftheapparentsteadystatekineticparameterswasdeterminedbyfittinginitialratedataobtainedatvaryingconcentrationsoforganicsubstratetoequations3whichdescribeabell-shapedcurvewithaslopeof+1atlowpHandaslopeof–1athighpH.
CisthepHindependentvalueofthekineticparameterofinterest.
++=pHpKpKpH101010101CloglogY2aa1(5)102ResultsanddiscussionThesteadystatekineticparametersfor2-nitropropanedioxygenaseweredeterminedwithethylnitronatebyvaryingbothconcentrationofoxygenandorganicsubstrateatpH6,8and10.
5and30oC.
AsshowninFigure5.
3,5.
4and5.
5,theinitialratesofoxygenconsumptionasafunctionofsubstrateconcentrationoverlapwithvaryingoxygen.
Plotsforvaryingorganicsubstrateandoxygenwerefittedwithbothequation2and3describingasequentialandpingpongmechanism.
Usingthisapproachsteadystatemechanismwasnotdistinguishablebetweensequentialandpingpong.
TheKO2wasdeterminedtobe5MorbelowforexperimentsdeterminedatpH6,8and10.
5,thereforeobtainingthekineticparameterswithethylnitronateasasubstrateatathmosphericoxygenwouldbeagoodestimateoftruekineticparametersmeasuredatsaturatingoxygen.
Subsequently,determiningthesubstratespecificityfor2-nitropropanedioxygenasewithanionicsubstratesofdifferentalkylchainlengthswasperformedbymeasuringtheinitialratesofenzymaticactivityinair-saturatedoxygen.
05001000150020000510152025vo/e,s-1[ethylnitronate],mMFigure5.
3.
Steadystatekineticof2-nitropropanedioxygenasecatalyzedreactionwithethylnitronateandoxygenassubstrates.
Enzymaticactivitywasmeasuredbymethodofinitialratesbyvarying103concentrationsofbothethylnitronateandoxygenin50mMpotassiumphosphatepH6and30oC.
Concentrationofoxygenwas:()82M,()102Mand()130M.
Datafitwithequation1.
01503004506000510152025vo/e,s-1[ethylnitronate],mMFigure5.
4.
Steadystatekineticof2-nitropropanedioxygenasecatalysedreactionwithethylnitronateandoxygenassubstrates.
Enzymaticactivitywasmeasuredbymethodofinitialratesbyvaryingconcentrationsofbothethylnitronateandoxygenin50mMpotassiumphosphatepH8and30oC.
Concentrationofoxygenwas:()58M,()41Mand()20M.
Datafitwithequation1.
03060901200510152025vo/e,s-1[ethylnitronate],mMFigure5.
5.
Steadystatekineticof2-nitropropanedioxygenasecatalysedreactionwithethylnitronateandoxygenassubstrates.
Enzymaticactivitywasmeasuredbymethodofinitialratesbyvarying104concentrationsofbothethylnitronateandoxygenin50mMsodiumpyrophosphatepH10.
5and30oC.
Concentrationofoxygenwas:()69M,()82Mand()98M.
Datafitwithequation1.
ThepHdependenceoftheapparentsecondorderrateconstants(kcat/Km)appof2-nitropropanedioxygenasewithethylnitronateandbutyl-1-nitronateassubstratesweremeasuredinair-saturatedbufferintheaccessiblepHrange.
Withethylnitronateasasubstrate(kcat/Km)appvaluesincreasewithdecreasingpH(Figure5.
6).
Twoplateauregionswereobserved,consistentwiththerequirementofatwoionizablegroupsthathavetobeprotonatedforcatalysis.
TheparticipationofanacidwithpKaof6.
5and9.
9issuggestedbythepH-dependenceofthe(kcat/Km)appvalueswithethylnitronate,showingtheinvolvementoftwoprotonatedgroupsincatalysis.
-2-1012356789101112logkcat/KMmM-1s-1pHFigure5.
6.
pHdependenceonkcat/Kmwithethylnitronateandmixtureofethylnitronateandnitroethane.
Initialratesweremeasuredinairsaturated100mMsodiumpyrophosphateintherange5.
5-6and8.
5-10.
5andpotassiumphosphateintherangeof6.
5-8and11at30oC.
()pHdependencewhenethylnitronatewasusedasasubstrate,()pHdependencewhenmixtureofethylnitronateandnitroethanewasusedforasubstrate.
Datawerefitwithequation4.
105Withmixtureofnitroethane/ethylnitronateassubstratethe(kcat/Km)appvaluesyieldedbell-shapedpHprofile(figure5.
6).
Thisresultcanbeinterpretedasbeingconsistentwithinvolvementoftwoionizablegroupsthatmustbeprotonatedandunprotonatedforthecatalysis.
ThispHprofileisconsistentwiththerequirementofasingleionizablegroupontheenzymethatmustbeprotonatedforcatalysis.
Aspointedoutinchapter2,2-nitropropanedioxygenaseutilizesalkylnitronatesassubstratesbutisincapableofoxidizingtheneutralnitroalkanes.
Therefore,thepKaof8.
4determinedforthepHprofilewithmixtureofnitroethane/ethylnitronatecorrespondstothepKaofthesubstrate,assupportedbythepKaof8.
5fornitroethanedeterminedinsolution[9].
ThesecondpKaof9.
6issimilartothepKaof9.
9determinedfromthepHprofilewithethylnitronate.
ThegroupontheenzymethatcancorrespondtothepKawithvalueof9.
9couldbetheN3ontheisoaloxazineringoftheflavin.
Similarresultswereobtainedwhenethylnitronatewassubstitutedwithbutyl-1-nitronateorwhenthemixtureofethylnitronate/nitroethanewasreplacedwithbutyl-1-nitronate/nitrobutane,asseeninfigure5.
4.
TwopKavaluesforthepHprofileofbutyl-1-nitronatearenotclearlydefinedasforethylnitronatepHprofileduetothequalityofdata.
-2-10123456789101112logkcat/KMmM-1s-1pHFigure5.
7.
pHdependenceonkcat/Kmwithbutyl-1-nitronateandmixtureofbutyl-1-nitronateandnitrobutane.
Initialratesweremeasuredinairsaturated100mMsodiumpyrophosphateintherange5.
5-6106and8.
5-10.
5andpotassiumphosphateintherangeof6.
5-8and11at30oC.
()pHdependencewhenbutyl-1-nitronatewasusedasasubstrate,()pHdependencewhenmixtureofbutyl-1-nitronateandnitrobutanewasusedforasubstrate.
Datawerefitwithequation4.
TwopKavaluesforthepHprofileofthemixtureofbutyl-1-nitronate/nitrobutaneare8.
7and9.
9.
InthiscasethepKaof8.
7doesnotcorrespondtothepKaofthesubstrateduetothemaskingofthesubstrate'sintrinsicpKabythegroupontheenzymethatneedstobeprotonatedforcatalysis.
Inordertoidentifythegroupsinvolvedincatalysisfuturemutagenisisstudieswillhavetobeperformed.
Inaddition,groupwiththepKa~9.
9inpHpresentinallpHprofilescouldpossiblybeassignedtotheN3ontheisoaloxazineringoftheflavin.
TofurthersupportthisclaimpHprofileoftheenzymehastobeperformedspectrophotometrically.
107References[1]C.
J.
Heasley,P.
F.
Fitzpatrick,Kineticmechanismandsubstratespecificityofnitroalkaneoxidase,BiochemBiophysResCommun225(1996)6-10.
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Francis,B.
Russell,G.
Gadda,Involvementofaflavosemiquinoneintheenzymaticoxidationofnitroalkanescatalyzedby2-nitropropanedioxygenase,JBiolChem280(2005)5195-5204.
[3]G.
Gadda,A.
Banerjee,P.
F.
Fitzpatrick,Identificationofanessentialtyrosineresidueinnitroalkaneoxidasebymodificationwithtetranitromethane,Biochemistry39(2000)1162-1168.
[4]A.
Nagpal,M.
P.
Valley,P.
F.
Fitzpatrick,A.
M.
Orville,Crystalstructuresofnitroalkaneoxidase:insightsintothereactionmechanismfromacovalentcomplexoftheflavoenzymetrappedduringturnover,Biochemistry45(2006)1138-1150.
[5]M.
P.
Valley,P.
F.
Fitzpatrick,Inactivationofnitroalkaneoxidaseuponmutationoftheactivesitebaseandrescuewithadeprotonatedsubstrate,JAmChemSoc125(2003)8738-8739.
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F.
Fitzpatrick,Reductivehalf-reactionofnitroalkaneoxidase:effectofmutationoftheactivesiteaspartatetoglutamate,Biochemistry42(2003)5850-5856.
[7]R.
D.
Allison,D.
L.
Purich,Practicalconsiderationsinthedesignofinitialvelocityenzymerateassays,MethodsEnzymol63(1979)3-22.
[8]G.
Gadda,D.
Y.
Choe,P.
F.
Fitzpatrick,UseofpHandkineticisotopeeffectstodissecttheeffectsofsubstratesizeonbindingandcatalysisbynitroalkaneoxidase,ArchBiochemBiophys382(2000)138-144.
108[9]A.
T.
Nielsen,TheChemistryoftheNitroandNitrosoGroupsIntersciencePublishers,NewYork,1969.
109ChapterVIGROWINGTHECRYSTALSOFRECOMBINANT2-NITROPROPANEDIOXYGENASEAbstractCrystallographicanalysiscanbeapowerfultoolusedtogainthemechanisticinsightsintotheenzymes.
Thefocusofthischapterisonobtainingthecrystalsforrecombinant2-nitropropanedioxygenasefromHansenulamrakii.
Needle-shapedyellowcrystalsgrewin15%(v/v)(+/-)-2-methyl-2,4-pentanediol(MPD),0.
02Mcalciumchloridedehydrateand0.
1MsodiumacetatetrihydratepH4.
6.
Byrepeatingthesameconditionsandre-seedingthecrystals,biggersinglecrystalswereobtained.
Heavyprecipitationandsubsequentformationoftheskininthevapordropwereaddressedbyaddingtheglyceroltocrystallizationconditions.
110IntroductionThecrystalstructureofnitroalkaneoxidase(NAO)fromFusariumoxysporumat2.
2resolutionwassolved[1;2],providingthesupportingevidenceofacovalentN5–FADadduct.
Thetetramericstructure(Figure6.
1)withasymmetricunitswasrevealedtohavethreesolvent-accessiblechannels.
Thebiggestchannel,whichextendsfromtheexterioroftheprotein,terminatesatAsp402andtheN5positiononoftheflavinatthesidechainofPhe273.
Figure6.
1.
CrystalstructureofnitroalkaneoxidasefromFusariumoxysporum.
EachsubunitofthehomotetramerisdepictedasadifferentlycoloredCαribbontrace[2].
Eachsubunitconsistsofaβ-sheetdomaininthecentralregionandtwoα-helicaldomains,oneattheC-terminalregionandoneattheN-terminalregion.
TheFADbindsnon-covalentlyineachsubunitthroughseveralhydrogenbonds.
Theadeninemoietyextendstothedimer–dimerinterfacewhereitinteractswithHis313viaπ-stackingandhydrogenbondswiththesidechainsofGln314andAsn141.
WiththehelpofcrystalstructureimportantresiduesintheactivesitewereidentifiedtobeAsp402,Arg409andSer276(Figure6.
2).
111Figure6.
2.
ActivesiteofnitroalkaneoxidaseillustratinganalternativeorientationofAsp402andSer276.
Takenwithoutpermission[2].
Asp402wasidentifiedtobeincorrectpositiontoserveastheactive-sitebasetoabstracttheα-protonfromneutralnitroalkanes.
TheoxygenatomsonAsp402formhydrogenbondswiththeOHmoietyofSer276andwiththeguanidinogroupofArg409.
CrystalstructuredataalsoprovidedcluesonhowthecovalentflavinN(5)-substrateadductisstabilized.
Beingexcludedfromthebulksolvent,theflavinadductissequesteredfromadditionalmolecularinfluences.
HydrogenbondingoftheisoalloxazineN1andO2atomswithamideprotonsandthehydroxylgroupofSer134reducethechargedensityontheN1atomandconsequentlystabilizetheadduct.
ThenitrogrouponthesubstrateaspartofN(5)-substrateadductinteractswiththesidechainofAsp402topotentiallyreducethecapacityofNO2toserveasafavorableleavinggroup.
Thecrystalstructureof2-nitropropanedioxygenasefromP.
aeruginosaat2.
0resolutionwassolvedasthemodelofthebinarycomplexwithFMNandtheternarycomplexwithFMNand2-nitropropane.
Themonomericenzymeconsistsoftwodomainsconnectedbytwoloopsandhasapproximatedimensionsof40x40x60(Figure6.
3).
112Figure6.
3.
Overallstructureof2-nitropropanedioxygenasefromPseudomonasaeruginosa.
Borrowedwithoutpermission[3].
AmoleculeofFMNisnon-covalentlyboundinthedeepactivesitepocketandislocatedneartheinterfacebetweenthetwodomains.
ThephosphatemoietyofFMNisnotsolvent-accessiblesinceitisburiedcompletelyinsidethepocket.
TheFMN-bindingpocketconsistsoftenresiduesGly22,Gln24,Thr75,Lys124,Asp145,Ala150,Ser178,Gly180,Gly201,andThr202(Figure6.
4).
ThehydroxylgroupofThr75ishydrogen-bondedtotheN3andO2atomsofFMN.
ThesidechainofLys124ishydrogen-bondedtotheO2atomontheisoalloxazinemoiety,resultsconsistentwithpositivelychargedgroupclosetotheN1-C2=O2locusofflavin.
Thesubstratemolecule,onthesifaceoftheisoalloxazineringofFMN,isprotectedfromthebulksolvent.
His152andSer288arelocatedinproximityof2-nitropropaneintheactivesite.
His152isorientedinsuchawaythatitsunprotonatedN1atominteractswiththeα-carbonof2-nitropropaneandissuitablypositionedtoabstracttheα-protonoftheneutralsubstrate.
ThesidechainofSer288recognizesthenitrogroupof2-nitropropaneviaahydrogenbond.
Thishydrogenbondpotentiallyincreasestheacidityoftheadjacentcarbon,facilitatingitsdeprotonationbyHis152.
113Figure6.
4.
Activesiteof2-nitropropanedioxygenasefromPseudomonasaeruginosa.
Borrowedwithoutpermission[3].
Thefocusofthischapterisoninitialtrialstoobtainthecrystalsfor2-nitropropanedioxygenasefromHansenulamrakiiandtheattempttooptimizetheconditionsinwhichthecrystalswerefound.
114ExperimentalProceduresMaterialsandMethods.
AllreagentswereofthehighestpuritycommerciallyavailableandwereobtainedfromHamptonResearch(HamptonResearch,AlisoViejo,CA,USA).
2-NitropropanedioxygenasewaspreparedasdescribedinChapter2andstoredin50mMpotassiumphosphatepH7.
4at-20oC.
EnzymeconcentrationwasexpressedperenzymeboundFMNcontent,usingtheexperimentallydeterminedε445nmvalueof13,130M–1cm–1.
Theconcentrationofpurified2-nitropropanedioxygenasewasdeterminedwiththeBradfordassay[4],usingtheBio-Radproteinassaykitwithbovineserumalbuminasastandard.
Crystallization.
Crystalsofrecombinant2-nitropropanedioxygenasefromHansenulamrakiiweregrownbythehangingdropvapordiffusionmethod,andinitialcrystallizationconditionswereobtainedusingcommerciallyavailablesparse-matrixscreeningkits(CrystalScreenHR2-110andCrystalScreenLiteHR2-128).
Thecrystalsweregrownbymixing2Lof2-nitropropanedioxygenasewith2Lofreservoirsolution.
Theenzymedropswereequilibratedover1mLofthereservoirsolutionat23oCor4oC.
Priortobeingflash-frozenbyquicksubmersionintoliquidnitrogen,singlecrystalswerecryoprotectedbybeingtransferredintoglycerolandallowedtoincubatefor2minat23oC.
115ResultsanddiscussionNomicrocrystalswereinitiallyobtainedfrom2Lof2-nitropropanedioxygenase(2.
3mg/mL)mixedwith2LofreservoirsolutioncontainingreagentsfromCrystalScreenHR2-110(Table1).
Growingcrystalsinsecondkit,CrystalScreenLiteHR2-128(Table2),yieldedbetterresultswith5.
6mg/mLof2-nitropropanedioxygenase.
Colorlessrodlikecrystalsgrewin10%(w/v)polyethyleneglycol(PEG)8000,0.
1MsodiumcacodylatetrihydratepH6.
5and0.
2Mmagnesiumacetate(CrystalScreenLitesolution18).
Tooptimizethecrystallizationconditions,theconcentrationofPEGwasvariedfrom12to17%with1%increments.
Inaddition,thesizeofPEGwasalsovariedbyusing3000,5000,6000and10,000DaPEG.
Crystals,obtainedinpolyethyleneglycol,0.
1MsodiumcacodylatetrihydratepH6.
5and0.
2Mmagnesiumacetate,didnotappeartohaveyellowcolorsothefurtheroptimizationwasnotperformed.
Colorlessfusedcrystalsgrewin0.
75Mlithiumsulfateand0.
1MsodiumHepespH7.
5(CrystalScreenLitesolution16).
Tooptimizethecrystallizationconditions,theconcentrationoflithiumsulfatewasvariedfrom0.
5to1Mwith0.
1Mincrements.
Inaddition,theconcentrationofHepeswasalsovariedfrom0.
05to0.
20Mwithincrementsof0.
05M.
Crystals,obtainedinlithiumsulfateand0.
1MsodiumHepespH7.
5,didnotappeartohaveyellowcolorsothefurtheroptimizationwasnotperformed.
Needle-shapedyellowcrystalsinitiallygrewin15%(v/v)(+/-)-2-methyl-2,4-pentanediol(MPD),0.
02Mcalciumchloridedehydrateand0.
1MsodiumacetatetrihydratepH4.
6(CrystalScreenLitesolution1).
Whenneedle-shapedcrystalswerere-seededusingsameconditions,singlebiggercrystalsgrew.
Whenmotherliquorwasmadefromcommerciallyavailablecompounds,asopposedtousingtheCrystalScreenLitesolution1,nocrystalswereobserved116evenwhenconcentrationofenzymewasinrangefrom3.
1to12.
3mg/mLorwhen0.
5or4Mnitroethanewasusedasligand.
Consequently,subsequentstudieswerecarriedoutwiththeCrystalScreenLitesolution1.
Heavyprecipitationandsubsequentformationoftheskinonthesurfaceofthevapordropwasobservedwhenenzymeconcentrationwasatleast4.
5mg/mLinthewell.
Theformationoftheskinwasaddressedbyaddingtheglyceroltothemotherliquor(5,10and15%)ortotheenzyme(10and20%).
Alighterskinformedonthesurfaceofthevapordropwhenglycerolwasaddedtocrystallizationconditions.
Futurestudiesshouldfocusonre-seedingtheexistingcrystalsintothewellscontainingthe15%(v/v)(+/-)-2-methyl-2,4-pentanediol(MPD),0.
02Mcalciumchloridedehydrateand0.
1MsodiumacetatetrihydratepH4.
6(CrystalScreenLitesolution1).
Inaddition,theheavyprecipitationandformationoftheskinonthesurfaceofthevapordropshouldbeaddressedbyadditionofanotherviscouscompound.
Alternativelygrowingthecrystalsincompletelydifferentconditionscouldprovidelargeenoughcrystalsfordeterminationofthestructure.
Forexample,conditionstestedwithCrystalScreenHR2-110couldbere-testedwith4.
5mg/mLorlargerfinalproteinconcentrationinthewell.
117Table6.
1.
CrystalScreenHR2-110ReagentFormulationTubeSaltTubeBufferTubePrecipitant###1.
0.
02MCalciumchloridedihydrate1.
0.
1MSodiumacetatetrihydratepH4.
61.
30%v/v(+/-)-2-Methyl-2,4-pentanediol2.
None2.
None2.
0.
4MPotassiumsodiumtartratetetrahydrate3.
None3.
None3.
0.
4MAmmoniumphosphatemonobasic4.
None4.
0.
1MTrishydrochloridepH8.
54.
2.
0MAmmoniumsulfate5.
0.
2MSodiumcitratetribasicdihydrate5.
0.
1MHEPESsodiumpH7.
55.
30%v/v(+/-)-2-Methyl-2,4-pentanediol6.
0.
2MMagnesiumchloridehexahydrate6.
0.
1MTrishydrochloridepH8.
56.
30%w/vPolyethyleneglycol4,0007.
None7.
0.
1MSodiumcacodylatetrihydratepH6.
57.
1.
4MSodiumacetatetrihydrate8.
0.
2MSodiumcitratetribasicdihydrate8.
0.
1MSodiumcacodylatetrihydratepH6.
58.
30%v/v2-Propanol9.
0.
2MAmmoniumacetate9.
0.
1MSodiumcitratetribasicdihydratepH5.
69.
30%w/vPolyethyleneglycol4,00010.
0.
2MAmmoniumacetate10.
0.
1MSodiumacetatetrihydratepH4.
610.
30%w/vPolyethyleneglycol4,00011.
None11.
0.
1MSodiumcitratetribasicdihydratepH5.
611.
1.
0MAmmoniumphosphatemonobasic12.
0.
2MMagnesiumchloridehexahydrate12.
0.
1MHEPESsodiumpH7.
512.
30%v/v2-Propanol13.
0.
2MSodiumcitratetribasicdihydrate13.
0.
1MTrishydrochloridepH8.
513.
30%v/vPolyethyleneglycol40014.
0.
2MCalciumchloridedihydrate14.
0.
1MHEPESsodiumpH7.
514.
28%v/vPolyethyleneglycol40015.
0.
2MAmmoniumsulfate15.
0.
1MSodiumcacodylatetrihydratepH6.
515.
30%w/vPolyethyleneglycol8,00016.
None16.
0.
1MHEPESsodiumpH7.
516.
1.
5MLithiumsulfatemonohydrate17.
0.
2MLithiumsulfatemonohydrate17.
0.
1MTrishydrochloridepH8.
517.
30%w/vPolyethyleneglycol4,00018.
0.
2MMagnesiumacetatetetrahydrate18.
0.
1MSodiumcacodylatetrihydratepH6.
518.
20%w/vPolyethyleneglycol8,00019.
0.
2MAmmoniumacetate19.
0.
1MTrishydrochloridepH8.
519.
30%v/v2-Propanol20.
0.
2MAmmoniumsulfate20.
0.
1MSodiumacetatetrihydratepH4.
620.
25%w/vPolyethyleneglycol4,00021.
0.
2MMagnesiumacetatetetrahydrate21.
0.
1MSodiumcacodylatetrihydratepH6.
521.
30%v/v(+/-)-2-Methyl-2,4-pentanediol22.
0.
2MSodiumacetatetrihydrate22.
0.
1MTrishydrochloridepH8.
522.
30%w/vPolyethyleneglycol4,00023.
0.
2MMagnesiumchloridehexahydrate23.
0.
1MHEPESsodiumpH7.
523.
30%v/vPolyethyleneglycol40024.
0.
2MCalciumchloridedihydrate24.
0.
1MSodiumacetatetrihydratepH4.
624.
20%v/v2-Propanol25.
None25.
0.
1MImidazolepH6.
525.
1.
0MSodiumacetatetrihydrate26.
0.
2MAmmoniumacetate26.
0.
1MSodiumcitratetribasicdihydratepH5.
626.
30%v/v(+/-)-2-Methyl-2,4-pentanediol27.
0.
2MSodiumcitratetribasicdihydrate27.
0.
1MHEPESsodiumpH7.
527.
20%v/v2-Propanol28.
0.
2MSodiumacetatetrihydrate28.
0.
1MSodiumcacodylatetrihydratepH6.
528.
30%w/vPolyethyleneglycol8,00029.
None29.
0.
1MHEPESsodiumpH7.
529.
0.
8MPotassiumsodiumtartratetetrahydrate30.
0.
2MAmmoniumsulfate30.
None30.
30%w/vPolyethyleneglycol8,00031.
0.
2MAmmoniumsulfate31.
None31.
30%w/vPolyethyleneglycol4,00032.
None32.
None32.
2.
0MAmmoniumsulfate33.
None33.
None33.
4.
0MSodiumformate34.
None34.
0.
1MSodiumacetatetrihydratepH4.
634.
2.
0MSodiumformate35.
None35.
0.
1MHEPESsodiumpH7.
535.
0.
8MSodiumphosphatemonobasicmonohydrate0.
8MPotassiumphosphatemonobasic36.
None36.
0.
1MTrishydrochloridepH8.
536.
8%w/vPolyethyleneglycol8,00037.
None37.
0.
1MSodiumacetatetrihydratepH4.
637.
8%w/vPolyethyleneglycol4,00038.
None38.
0.
1MHEPESsodiumpH7.
538.
1.
4MSodiumcitratetribasicdihydrate39.
None39.
0.
1MHEPESsodiumpH7.
539.
2%v/vPolyethyleneglycol4002.
0MAmmoniumsulfate40.
None40.
0.
1MSodiumcitratetribasicdihydratepH5.
640.
20%v/v2-Propanol20%w/vPolyethyleneglycol4,00041.
None41.
0.
1MHEPESsodiumpH7.
541.
10%v/v2-Propanol20%w/vPolyethyleneglycol4,00042.
0.
05MPotassiumphosphatemonobasic42.
None42.
20%w/vPolyethyleneglycol8,00043.
None43.
None43.
30%w/vPolyethyleneglycol1,50044.
None44.
None44.
0.
2MMagnesiumformatedihydrate45.
0.
2MZincacetatedihydrate45.
0.
1MSodiumcacodylatetrihydratepH6.
545.
18%w/vPolyethyleneglycol8,00046.
0.
2MCalciumacetatehydrate46.
0.
1MSodiumcacodylatetrihydratepH6.
546.
18%w/vPolyethyleneglycol8,00047.
None47.
0.
1MSodiumacetatetrihydratepH4.
647.
2.
0MAmmoniumsulfate48.
None48.
0.
1MTrishydrochloridepH8.
548.
2.
0MAmmoniumphosphatemonobasic49.
1.
0MLithiumsulfatemonohydrate49.
None49.
2%w/vPolyethyleneglycol8,00050.
0.
5MLithiumsulfatemonohydrate50.
None50.
15%w/vPolyethyleneglycol8,000118Table6.
2.
CrystalScreenLiteHR2-128ReagentFormulationTubeSaltTubeBufferTubePrecipitant###1.
0.
02MCalciumchloridedihydrate1.
0.
1MSodiumacetatetrihydratepH4.
61.
15%v/v(+/-)-2-Methyl-2,4-pentanediol2.
None2.
None2.
0.
2MPotassiumsodiumtartratetetrahydrate3.
None3.
None3.
0.
2MAmmoniumphosphatemonobasic4.
None4.
0.
1MTrishydrochloridepH8.
54.
1.
0MAmmoniumsulfate5.
0.
2MSodiumcitratetribasicdihydrate5.
0.
1MHEPESsodiumpH7.
55.
15%v/v(+/-)-2-Methyl-2,4-pentanediol6.
0.
2MMagnesiumchloridehexahydrate6.
0.
1MTrishydrochloridepH8.
56.
15%w/vPolyethyleneglycol4,0007.
None7.
0.
1MSodiumcacodylatetrihydratepH6.
57.
0.
7MSodiumacetatetrihydrate8.
0.
2MSodiumcitratetribasicdihydrate8.
0.
1MSodiumcacodylatetrihydratepH6.
58.
15%v/v2-Propanol9.
0.
2MAmmoniumacetate9.
0.
1MSodiumcitratetribasicdihydratepH5.
69.
15%w/vPolyethyleneglycol4,00010.
0.
2MAmmoniumacetate10.
0.
1MSodiumacetatetrihydratepH4.
610.
15%w/vPolyethyleneglycol4,00011.
None11.
0.
1MSodiumcitratetribasicdihydratepH5.
611.
0.
5MAmmoniumphosphatemonobasic12.
0.
2MMagnesiumchloridehexahydrate12.
0.
1MHEPESsodiumpH7.
512.
15%v/v2-Propanol13.
0.
2MSodiumcitratetribasicdihydrate13.
0.
1MTrishydrochloridepH8.
513.
15%v/vPolyethyleneglycol40014.
0.
2MCalciumchloridedihydrate14.
0.
1MHEPESsodiumpH7.
514.
14%v/vPolyethyleneglycol40015.
0.
2MAmmoniumsulfate15.
0.
1MSodiumcacodylatetrihydratepH6.
515.
15%w/vPolyethyleneglycol8,00016.
None16.
0.
1MHEPESsodiumpH7.
516.
0.
75MLithiumsulfatemonohydrate17.
0.
2MLithiumsulfatemonohydrate17.
0.
1MTrishydrochloridepH8.
517.
15%w/vPolyethyleneglycol4,00018.
0.
2MMagnesiumacetatetetrahydrate18.
0.
1MSodiumcacodylatetrihydratepH6.
518.
10%w/vPolyethyleneglycol8,00019.
0.
2MAmmoniumacetate19.
0.
1MTrishydrochloridepH8.
519.
15%v/v2-Propanol20.
0.
2MAmmoniumsulfate20.
0.
1MSodiumacetatetrihydratepH4.
620.
12.
5%w/vPolyethyleneglycol4,00021.
0.
2MMagnesiumacetatetetrahydrate21.
0.
1MSodiumcacodylatetrihydratepH6.
521.
15%v/v(+/-)-2-Methyl-2,4-pentanediol22.
0.
2MSodiumacetatetrihydrate22.
0.
1MTrishydrochloridepH8.
522.
15%w/vPolyethyleneglycol4,00023.
0.
2MMagnesiumchloridehexahydrate23.
0.
1MHEPESsodiumpH7.
523.
15%v/vPolyethyleneglycol40024.
0.
2MCalciumchloridedihydrate24.
0.
1MSodiumacetatetrihydratepH4.
624.
10%v/v2-Propanol25.
None25.
0.
1MImidazolepH6.
525.
0.
5MSodiumacetatetrihydrate26.
0.
2MAmmoniumacetate26.
0.
1MSodiumcitratetribasicdihydratepH5.
626.
15%v/v(+/-)-2-Methyl-2,4-pentanediol27.
0.
2MSodiumcitratetribasicdihydrate27.
0.
1MHEPESsodiumpH7.
527.
10%v/v2-Propanol28.
0.
2MSodiumacetatetrihydrate28.
0.
1MSodiumcacodylatetrihydratepH6.
528.
15%w/vPolyethyleneglycol8,00029.
None29.
0.
1MHEPESsodiumpH7.
529.
0.
4MPotassiumsodiumtartratetetrahydrate30.
0.
2MAmmoniumsulfate30.
None30.
15%w/vPolyethyleneglycol8,00031.
0.
2MAmmoniumsulfate31.
None31.
15%w/vPolyethyleneglycol4,00032.
None32.
None32.
1.
0MAmmoniumsulfate33.
None33.
None33.
2.
0MSodiumformate34.
None34.
0.
1MSodiumacetatetrihydratepH4.
634.
1.
0MSodiumformate35.
None35.
0.
1MHEPESsodiumpH7.
535.
0.
4MSodiumphosphatemonobasicmonohydrate0.
4MPotassiumphosphatemonobasic36.
None36.
0.
1MTrishydrochloridepH8.
536.
4%w/vPolyethyleneglycol8,00037.
None37.
0.
1MSodiumacetatetrihydratepH4.
637.
4%w/vPolyethyleneglycol4,00038.
None38.
0.
1MHEPESsodiumpH7.
538.
0.
7MSodiumcitratetribasicdihydrate39.
None39.
0.
1MHEPESsodiumpH7.
539.
2%v/vPolyethyleneglycol4001.
0MAmmoniumsulfate40.
None40.
0.
1MSodiumcitratetribasicdihydratepH5.
640.
10%v/v2-Propanol10%w/vPolyethyleneglycol4,00041.
None41.
0.
1MHEPESsodiumpH7.
541.
5%v/v2-Propanol10%w/vPolyethyleneglycol4,00042.
0.
05MPotassiumphosphatemonobasic42.
None42.
10%w/vPolyethyleneglycol8,00043.
None43.
None43.
15%w/vPolyethyleneglycol1,50044.
None44.
None44.
0.
1MMagnesiumformatedihydrate45.
0.
2MZincacetatedihydrate45.
0.
1MSodiumcacodylatetrihydratepH6.
545.
9%w/vPolyethyleneglycol8,00046.
0.
2MCalciumacetatehydrate46.
0.
1MSodiumcacodylatetrihydratepH6.
546.
9%w/vPolyethyleneglycol8,00047.
None47.
0.
1MSodiumacetatetrihydratepH4.
647.
1.
0MAmmoniumsulfate48.
None48.
0.
1MTrishydrochloridepH8.
548.
1.
0MAmmoniumphosphatemonobasic49.
0.
5MLithiumsulfatemonohydrate49.
None49.
2%w/vPolyethyleneglycol8,00050.
0.
5MLithiumsulfatemonohydrate50.
None50.
7.
5%w/vPolyethyleneglycol8,000119References[1]A.
Nagpal,M.
P.
Valley,P.
F.
Fitzpatrick,andA.
M.
Orville,Crystallizationandpreliminaryanalysisofactivenitroalkaneoxidaseinthreecrystalforms.
ActaCrystallogrDBiolCrystallogr60(2004)1456-60.
[2]A.
Nagpal,M.
P.
Valley,P.
F.
Fitzpatrick,andA.
M.
Orville,Crystalstructuresofnitroalkaneoxidase:insightsintothereactionmechanismfromacovalentcomplexoftheflavoenzymetrappedduringturnover.
Biochemistry45(2006)1138-50.
[3]J.
Y.
Ha,J.
Y.
Min,S.
K.
Lee,H.
S.
Kim,J.
Kimdo,K.
H.
Kim,H.
H.
Lee,H.
K.
Kim,H.
J.
Yoon,andS.
W.
Suh,Crystalstructureof2-nitropropanedioxygenasecomplexedwithFMNandsubstrate.
Identificationofthecatalyticbase.
JBiolChem281(2006)18660-7.
[4]M.
M.
Bradford,Arapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.
AnalBiochem72(1976)248-54.
120ChapterVIIGENERALDISCUSSIONTheFMN-dependentenzyme2-nitropropanedioxygenasefromHansenulamrakiicatalyzestheoxidativedenitrificationofanionicnitroalkanesintocorrespondingcarbonylcompoundsandnitrite,withoxygenaselectronacceptor.
Thebiotechnologicalimportanceofthisenzymeisfoundinthepossiblebioremediationapplicationsfordenitrificationofharmfulnitroalkanesthatareusedinchemicalindustrybecausetheyprovideaquickandeffectivemethodofsynthesizingcommonreagents[1;2].
Fromachemicalstandpoint,2-nitropropanedioxygenasefromHansenulamrakiiisanimportantnewadditiontoanitroalkaneoxidizingfamilyduetotheenzymepreferenceforanionicnitroalkanes.
Inthisstudy,thebiophysical,biochemical,structuralanalysisof2-nitropropanedioxygenasearepresented,providingasolidbackgroundforfuturebiochemicalandmechanisticstudiesaimedattheelucidationofthechemicalmechanism.
Therecombinant2-nitropropanedioxygenasefromtheyeastH.
mrakiiwasexpressedtohighlevelsinE.
colicells.
Afractionationstepwith60%saturationofammoniumsulfateandasinglechromatographicstepusingaDEAEcolumnallowedforpreparationsofactiveandhomogeneousenzyme.
Thebiochemicalcharacterizationof2-nitropropanedioxygenasehasestablishedthattheenzymerequiresFMNbutnotironascofactorforcatalysis.
Preferenceoftheenzymeforunbranchedanionicnitroalkanesoralkylnitronateswasconfirmedbyanaerobicsubstratereductionstudiesaswellasseveralkineticstudieswithanumberofdifferentsubstrates.
Theenzymeshowsminimaldiscriminationamongunbranchedalkylnitronateswithchainlengthsrangingfrom2to6carbonatoms,asshownbytheapparentkcat/Kmvaluesbetween1.
1x105M-1s-1and1.
7x105M-1s-1forthesesubstrates.
Thestabilizationofflavosemiquinone121alongwiththeformationofaflavin-N(5)-sulfiteadductin2-nitropropanedioxygenasewasexplainedasaresultofthestabilizationofthenegativechargethatdevelopsonN(1)locusoftheflavinbyapositivechargeclosetothatregion.
Apositivechargemaybeprovidedbyeithertheelectricaldipoleofα-helixorapositivelychargedaminoacidresidue,whichpossiblyisalysineresidueasindicatedbythecrystallographicstructureof2-nitropropanedioxygenasefromPseudomonasaeruginosa[3].
2-Nitropropanedioxygenaseisinhibitedbyhalideions.
Inthepresenceoftwotooneratioofchlorideionstoflavinmoleculeinhibitionof40%on2-nitropropanedioxygenaseactivityisobserved.
Whileinpresenceof1mMpotassiumiodide,potassiumbromideorpotassiumfluorideasdeterminedin50mMpotassiumphosphatepH6withethylnitronateasasubstrate,enzymeactivityisinhibited18%,58%or19%.
ThepHdependenceoftheapparentsecondorderrateconstants(kcat/Km)appof2-nitropropanedioxygenasewithethylnitronateandbutyl-1-nitronateassubstratesisconsistentwiththerequirementofatwoionizablegroupthatmustbeprotonatedforcatalysiswithpKaof6.
5and9.
9.
However,pH-dependentstudiesoftheapparentsecondorderrateconstants(kcat/Km)appwithamixtureofanionicandneutralnitroalkaneswithethylnitronate/nitroethaneandbutyl-1-nitronate/nitrobutaneassubstrates,yieldedbell-shapedpHprofiles.
AsingleionizablegroupontheenzymethatmustbeprotonatedforcatalysiswasidentifiedinbothpHprofilescorrespondtothesecondpKaof9.
9inthepHprofilewithethylnitronate.
TheionizablegroupthatmustbeunprotonatedforcatalysiscorrespondstothepKavalueforthesubstrate.
Asthefirststepinobtainingthecrystalstructureof2-nitropropanedioxygenasecrystalsweresuccessfullygrowninwellscontainingthe15%(v/v)(+/-)-2-methyl-2,4-pentanediol(MPD),0.
02Mcalciumchloridedehydrateand0.
1MsodiumacetatetrihydratepH4.
6.
122Insummary,theresultsobtainedhereinfromthebiochemicalandkineticcharacterizationofa2-nitropropanedioxygenasesuggestthattheenzyme:1)waspurifiedtohighyieldsasrecombinantenzyme;2)hasapreferenceforunbranchedalkylnitronates;3)requiresFMNascofactorforcatalysis;4)hasanegativechargethatdevelopsontheN(1)-C(2)=Olocusoftheboundflavin;5)isinhibitedbyhalideions;6)requirestwounprotonatedgroupsforcatalysiswithethylnitronateandbutyl-1nitronateassubstrates.
Inaddition,crystalsobtainedinthisstudyprovidefirstimportantstepinobtainingthestructuraldatafor2-nitropropnaedioxygenase.
Thecalculatedmolecularweightofthe374-residueunitof2-nitroproanedioxygenase(2NPD)is41467Da.
TheenzymehasatheoreticalpIof5.
76.
Aminoacidsequencecomparisonsindicatethat2NPDishomologoustoothernitroalkaneoxidizingenzymes,withidentitiesof26.
8%and27.
4%tonitroalkaneoxidaseandNeurosporacrassa2NPD,respectively(Appendix1and2).
Moreover,aminoacidsequencecomparisonofH.
mrakii2NPDtoanother2NPDfromBurkholderiaphymatumyieldsthehighestpercentidentityof34.
7%in340residuesoverlap(Appendix3).
Inthisstudy,biochemicalcharacterizationestablishedthat2-nitropropanedioxygenasefromHansenulamrakiiutilizesalkylnitronatesassubstrates,andreactspoorly,ifatall,withnitroalkanes.
PreferenceoftheH.
mrakiienzymefortheanionicnitroalkanesmakesthisenzymeinterestingadditiontotwonitroalkane-oxidizingflavoenzymes,nitroalkaneoxidase[4]andN.
crassa2-nitropropanedioxygenase[5],whichutilizeneutralandbothneutralandanionicnitroalkanes,respectively.
Bothenzymesinitiatetheoxidationofneutralsubstratesbyabstractionoftheprotonfromtheα-carbon.
His196wasproposedtoplayaroleofthebaseinactivesiteofN.
crassaenzyme[6],whileAsp402hasbeenshowntoserveasthecatalyticbaseincrystallographicandmutagenesisstudiesofnitroalkaneoxidase[7;8;9].
His197inH.
mrakiienzyme,whichcorrespondstotheHis196inN.
crassaenzyme,isaconservedresidue(Scheme1237.
1).
Inthiscontext,H.
mrakiienzymehasaresiduewhichcanactasabase,butitdoesnotshowactivitywiththeneutralnitroalkanes,canindicatethattheHis197isnotintherightpositiontoabstracttheα-protonfromthesubstrateandthereforeinitiatethecatalysiswiththeneutralnitroalkanes.
Scheme7.
1.
Aminoacidsequencecomparisonoftheconservedhistidineresidue.
Theoxidationofnitroalkanesbyflavin-dependentenzymescanoccurviatwocatalyticstrategies.
First,asincaseofnitroalkaneoxidasewithaformationofaflavinN(5)-substrateadduct[4].
Second,viasingleelectrontransferreactioninwhichthetransientlyanionicsemiquinoneisobservedfortheN.
crassaenzyme[6].
ThecatalyticstrategyofH.
mrakiienzymeisprobablyverysimilartotheN.
crassaenzyme,sincebothenzymeshavebeenshowntoformanionicsemiquinoneuponincubationwithorganicsubstratesinabsenceofoxygen.
Nitroalkaneoxidase(NAO)hasahydrophobicbindingsitesufficientlylargeenoughtoaccommodatesubstrateswithalkylchain4carbonslong,asindicatedbysubstratespecificityconstantwithprimaryandsecondarynitroalkanesaswellasthenitrocompoundswhichcontainhydroxylgroup[10].
Asmallerbindingpocketwasalsoidentifiedforthenitroalkaneoxidasewhichcanaccommodatebranchedsubstrates[10].
Incontrasttonitroalkaneoxidase,2-nitropropanedioxygenasefromHansenulamrakiidoesnotfollowanytrendbasedonthelength124ofthealkylchain,asthespecificityconstantofethylnitronateissimilartothespecificityconstantofthehexyl-1-nitronate.
However,bothenzymeshaveapreferenceforunbranchedsubstrates.
Inthiscontext,H.
mrakii2-nitropropanedioxygenasepossiblyhasacavitysimilartonitroalkaneoxidase,whichcanaccommodatethebranchedpartofthesecondarysubstrateinadditiontohavingamoreflexiblesubstratebindingsitewhichcaneasilyanchoranylinearnitroalkanes.
Theinabilityofnitroalkaneoxidasetoreactwithanionicnitroalkanesisduethetyrosine398residue,whichmustbeprotonatedforbindingofthenitrogroupofthesubstrate[4;11].
Therefore,theabilityofthe2NPDtobindandcatalyzeanionicnitroalkanescanindicatethattheenzymehasadifferentbindingstrategythanhydrogenbondingtothenitrogroup,asitiscaseforNAO.
Inconclusion,biochemicalcharacterizationof2-nitropropnaedioxygenasefromHansenulamrakiiinthisstudyprovidesasolidbackgroundforfuturestudiesontheenzyme,whichisimportantforbothappliedandfundamentalreasons.
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Fitzpatrick,Inactivationofnitroalkaneoxidaseuponmutationoftheactivesitebaseandrescuewithadeprotonatedsubstrate.
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Biochemistry45(2006)1138-50.
[10]G.
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Biochemistry39(2000)1162-8.
127Appendix126.
8%identityin41residuesoverlap;Score:34.
0;Gapfrequency:0.
0%2NPDhm,91VNEEWLKKYDKIYGKAGIEFDKKELKLLYPSFRSIVDPQHPNAO,157VGNEWVISGEKLWPSNSGGWDYKGADLACVVCRVSDDPSKP25.
9%identityin27residuesoverlap;Score:32.
0;Gapfrequency:0.
0%2NPDhm,159LQASDIKIFVTVTNLQEFQQAYESKLDNAO,327LETSRLLVWKAVTTLEDEALEWKVKLE31.
8%identityin22residuesoverlap;Score:27.
0;Gapfrequency:0.
0%2NPDhm,332ANDAKQSGKGPQYSAFLAGSNYNAO,140ANWLQKGGPGLQTTARKVGNEW33.
3%identityin15residuesoverlap;Score:27.
0;Gapfrequency:0.
0%2NPDhm,153EAVIESLQASDIKIFNAO,368ECVIDAMKAVGMKSY*****44.
4%identityin9residuesoverlap;Score:26.
0;Gapfrequency:0.
0%2NPDhm,148FGLPHEAVINAO,250FHVPHENLL****29.
8%identityin47residuesoverlap;Score:25.
0;Gapfrequency:0.
0%2NPDhm,21APMAGASTLELAATVTRLGGIGSIPMGSLSEKCDAIETQLENFDELVNAO,288ARAAFEEALVFAKSDTRGGSKHIIEHQSVADKLIDCKIRLETSRLLV29.
6%identityin27residuesoverlap;Score:24.
0;Gapfrequency:0.
0%1282NPDhm,26ASTLELAATVTRLGGIGSIPMGSLSEKNAO,89ATSITIVATALGLMPVILCDSPSLQEK57.
1%identityin7residuesoverlap;Score:23.
0;Gapfrequency:0.
0%2NPDhm,150LPHEAVINAO,73LVHESII****50.
0%identityin8residuesoverlap;Score:23.
0;Gapfrequency:0.
0%2NPDhm,311QSSPLASINAO,124EGEPLASL****33.
3%identityin15residuesoverlap;Score:23.
0;Gapfrequency:0.
0%2NPDhm,79FAHKEPRSGRADVNENAO,298FAKSDTRGGSKHIIE*****41.
7%identityin12residuesoverlap;Score:23.
0;Gapfrequency:0.
0%2NPDhm,158SLQASDIKIFVTNAO,111SLQEKFLKPFIS*****50.
0%identityin10residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,30ELAATVTRLGNAO,233ELAGHITTSG*****57.
1%identityin7residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,6QSFLKTFNAO,114EKFLKPF****12923.
5%identityin17residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,150LPHEAVIESLQASDIKINAO,311IEHQSVADKLIDCKIRL****66.
7%identityin6residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,128PQHPTVNAO,197PQDPNV****30.
8%identityin13residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,257AVQLGTVWLPSSQNAO,154ARKVGNEWVISGE****21.
1%identityin19residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,280FQSPKSDTMMTAAISGRNLNAO,250FHVPHENLLCTPGLKAQGL****22.
2%identityin18residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,267SSQATISPEHLKMFQSPKNAO,29SAEYSTQKDQLSRFQATR****35.
7%identityin14residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,171TNLQEFQQAYESKLNAO,245TRFTEFHVPHENLL*****18.
8%identityin16residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,312SSPLASIPDYPLPYDS130NAO,241SGPHTRFTEFHVPHEN***131Appendix227.
4%identityin117residuesoverlap;Score:81.
0;Gapfrequency:6.
8%2NPDhm,155VIESLQASDIKIFVTVTNLQEFQQAYESKLDGVVLQGWEAGGHRGNFKANDVEDGQLKTL2NPDnc,154IIEALHASGFVVFFQVGTVKDARKAAADGADVIVAQGIDAGGHQLATGSGIVS-------2NPDhm,215DLVSTIVDYIDSASISNPPFIIAAGGIHDDESIKELLQFNIAAVQLGTVWLPSSQAT2NPDnc,207-LVPEVRDMLDREFKEREVVVVAAGGVADGRGVVGALGLGAEGVVLGTRFTVAVEAS48.
4%identityin31residuesoverlap;Score:59.
0;Gapfrequency:0.
0%2NPDhm,18IIQAPMAGASTLELAATVTRLGGIGSIPMGS2NPDnc,33IISAPMYLIANGTLAAEVSKAGGIGFVAGGS55.
6%identityin9residuesoverlap;Score:29.
0;Gapfrequency:0.
0%2NPDhm,300TISTPFLRD2NPDnc,108TISVPYVTD*****50.
0%identityin14residuesoverlap;Score:29.
0;Gapfrequency:0.
0%2NPDhm,26ASTLELAATVTRLG2NPDnc,74ALSTELASARSRLG23.
5%identityin34residuesoverlap;Score:29.
0;Gapfrequency:0.
0%2NPDhm,188VLQGWEAGGHRGNFKANDVEDGQLKTLDLVSTIV2NPDnc,154IIEALHASGFVVFFQVGTVKDARKAAADGADVIV13254.
5%identityin11residuesoverlap;Score:28.
0;Gapfrequency:0.
0%2NPDhm,229ISNPPFIIAAG2NPDnc,34ISAPMYLIANG50.
0%identityin12residuesoverlap;Score:27.
0;Gapfrequency:0.
0%2NPDhm,291AAISGRNLRTIS2NPDnc,329AASSGDNSRAVT24.
0%identityin25residuesoverlap;Score:26.
0;Gapfrequency:0.
0%2NPDhm,225DSASISNPPFIIAAGGIHDDESIKE2NPDnc,301DGRAVRNASYDDHAAGVPFEENHKK40.
0%identityin10residuesoverlap;Score:25.
0;Gapfrequency:0.
0%2NPDhm,354HKSWKDTRST2NPDnc,323HKKFKEAASS****30.
8%identityin26residuesoverlap;Score:25.
0;Gapfrequency:0.
0%2NPDhm,65ELVGDSGRIVNLNFFAHKEPRSGRAD2NPDnc,156EALHASGFVVFFQVGTVKDARKAAAD36.
4%identityin11residuesoverlap;Score:24.
0;Gapfrequency:0.
0%2NPDhm,106AGIEFDKKELK2NPDnc,315AGVPFEENHKK****19.
4%identityin31residuesoverlap;Score:23.
0;Gapfrequency:0.
0%2NPDhm,244DESIKELLQFNIAAVQLGTVWLPSSQATISP1332NPDnc,90DRPLTPLPGIGVGLILTHTISVPYVTDTVLP40.
0%identityin10residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,205DVEDGQLKTL2NPDnc,273ETNDGGLNTV****62.
5%identityin8residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,35VTRLGGIG2NPDnc,93LTPLPGIG*****30.
0%identityin10residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,39GGIGSIPMGS2NPDnc,237GVVGALGLGA***40.
0%identityin10residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,118LYPSFRSIVD2NPDnc,207LVPEVRDMLD****44.
4%identityin9residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,92NEEWLKKYD2NPDnc,293NTIWHNVYD****22.
6%identityin31residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,6QSFLKTFEVRYPIIQAPMAGASTLELAATVT2NPDnc,15QAALTKLNSWFPTTKNPVIISAPMYLIANGT13422.
7%identityin22residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,143IVSFHFGLPHEAVIESLQASDI2NPDnc,164VVFFQVGTVKDARKAAADGADV66.
7%identityin6residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,191GWEAGG2NPDnc,57GFVAGG****135Appendix334.
7%identityin340residuesoverlap;Score:354.
0;Gapfrequency:6.
2%2NPDhm,2RSQIQSFLKTFEVRYPIIQAPMAGASTLELAATVTRLGGIGSIPMGSLSEKCDAIETQLE2NPDbp,4RSEGKPLLSLLGISKPIIQAPMAGVTTPALAAAVTNAGGLGSLGVGAM--KAEAARKTIR2NPDhm,62NFDELVGDSGRIVNLNFFAHKEPRSGRADVNEEWLKKYDKIYGKAGIEFDKKELKLLYPS2NPDbp,62DTRALTSGP---FNINVFCHASA-AANAKVEQEWLSWLAPQFAKYGASAPEK-LSEIYTS2NPDhm,122FRSIVDPQHPTVRLLKNLKPKIVSFHFGLPHEAVIESLQASDIKIFVTVTNLQEFQQAYE2NPDbp,117F----GDDQAMLDVFLEEKPAIVSFHFGMPSKETIKALKDAGIVLLASATNLEEAQQVVD2NPDhm,182SKLDGVVLQGWEAGGHRGNFKANDVEDGQLKTLDLVSTIVDYIDSASISNPPFIIAAGGI2NPDbp,173AGVDALVAQGIEAGGHRGVFDPQVFDEG-LGTLALTRLLVEKFDLP-------VIAAGGI2NPDhm,242HDDESIKELLQFNIAAVQLGTVWLPSSQATISPEHLKMFQSPKS-DTMMTAAISGRNLRT2NPDbp,225MDGAGIAAVLALGAQAAQLGTAFVPCPETSIDEGYRRAILGDAALHTTLTSAISGRPARS2NPDhm,301ISTPFLRDLHQSSPLASIPDYPLPYDSFKSLANDAKQSGK2NPDbp,285MVNRFT-ELGRASDRPATPDYPIAYDAGKALHAAAKAKGE50.
0%identityin10residuesoverlap;Score:26.
0;Gapfrequency:0.
0%2NPDhm,244DESIKELLQF2NPDbp,355DEAVGRLRQF*****13642.
9%identityin14residuesoverlap;Score:26.
0;Gapfrequency:0.
0%2NPDhm,26ASTLELAATVTRLG2NPDbp,231AAVLALGAQAAQLG41.
2%identityin17residuesoverlap;Score:26.
0;Gapfrequency:0.
0%2NPDhm,23MAGASTLELAATVTRLG2NPDbp,158LASATNLEEAQQVVDAG33.
3%identityin21residuesoverlap;Score:25.
0;Gapfrequency:0.
0%2NPDhm,299RTISTPFLRDLHQSSPLASIP2NPDbp,4RSEGKPLLSLLGISKPIIQAP57.
1%identityin7residuesoverlap;Score:25.
0;Gapfrequency:0.
0%2NPDhm,123RSIVDPQ2NPDbp,189RGVFDPQ****30.
4%identityin23residuesoverlap;Score:24.
0;Gapfrequency:0.
0%2NPDhm,240GIHDDESIKELLQFNIAAVQLGT2NPDbp,140GMPSKETIKALKDAGIVLLASAT36.
4%identityin11residuesoverlap;Score:24.
0;Gapfrequency:0.
0%2NPDhm,128PQHPTVRLLKN2NPDbp,142PSKETIKALKD****33.
3%identityin12residuesoverlap;Score:23.
0;Gapfrequency:0.
0%1372NPDhm,225DSASISNPPFII2NPDbp,62DTRALTSGPFNI****55.
6%identityin9residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,63FDELVGDSG2NPDbp,192FDPQVFDEG*****50.
0%identityin8residuesoverlap;Score:22.
0;Gapfrequency:0.
0%2NPDhm,355KSWKDTRS2NPDbp,58KTIRDTRA****37.
5%identityin16residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,60LENFDELVGDSGRIVN2NPDbp,211VEKFDLPVIAAGGIMD29.
4%identityin17residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,106AGIEFDKKELKLLYPSF2NPDbp,83ANAKVEQEWLSWLAPQF23.
5%identityin17residuesoverlap;Score:21.
0;Gapfrequency:0.
0%2NPDhm,351SNYHKSWKDTRSTEEIF2NPDbp,111SEIYTSFGDDQAMLDVF****46.
2%identityin13residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,131PTVRLLKNLKPKI2NPDbp,9PLLSLLGISKPII13823.
5%identityin34residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,34TVTRLGGIGSIPMGSLSEKCDAIETQLENFDELV2NPDbp,146TIKALKDAGIVLLASATNLEEAQQVVDAGVDALV29.
4%identityin17residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,211LKTLDLVSTIVDYIDSA2NPDbp,341LPAAELVARLEAELDEA*****66.
7%identityin6residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,63FDELVG2NPDbp,197FDEGLG****46.
2%identityin13residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,26ASTLELAATVTRL2NPDbp,338ARALPAAELVARL80.
0%identityin5residuesoverlap;Score:20.
0;Gapfrequency:0.
0%2NPDhm,64DELVG2NPDbp,355DEAVG****