结核分枝杆菌的ALD基因编码的丙氨酸脱氢酶和假定的甘氨酸脱氢酶.

aldofMycobacteriumtuberculosisEncodesboththeAlanineDehydrogenaseandthePutativeGlycineDehydrogenase结核分枝杆菌的ALD基因编码的丙氨酸脱氢酶和假定的甘氨酸脱氢酶Introduction•Mycobacteriumtuberculosis(结核分枝杆菌)isthecausativeagentoftuberculosis,andoneofthemostsuccessfulhumanpathogens.•Itwasresponsibleforapproximately2milliondeathsin2008,whilecurrentlyalmostone-thirdoftheworld’spopulationisinfectedwiththisorganism.ResearchwithM.tuberculosishasdescribedapathogenuniquelyadaptedtothewiderangeofharshenvironmentspresentedbythehost.Introduction•Muchofthisworkhasfocusedonthemicrobe’smetabolism（新陈代谢）,withtheideaofidentificationofnovelenzymes（识别新的酶）orpathwaystotargetfordrugdevelopment（药物靶向途径的开发）.•Oneoftheseenvironmentalfactorsisnitrogenavailability.VerylittleisknownaboutthenitrogensourcesusedbyM.tuberculosisinvivo.M.tuberculosiscanutilizemanyaminoacidsfornitrogen,includingalanineandglycine.Introduction•TheenzymeglycinedehydrogenasewasfirstdescribedinM.tuberculosisin1962.ThisenzymewasdetectedbythereductiveaminationofglyoxylatetoglycineconcurrentwiththeoxidationofNADHtoNAD(Fig.1)。Theactivitycorrespondingtothereversereaction,catalyzedbyglycinedehydrogenase(GDH),wasnotdetected.Introduction•TheexpressionofglyoxylatereductiveaminationbyaputativeglycinedehydrogenaseinM.tuberculosishasbeencharacterizedinnonreplicatingpersistent(NRP)cultures（非复制持久性培养基）.•IntheWaynemodelofdormancy,sealedculturesofM.tuberculosiscreateamicroaerobicenvironment(NRP-1),whichsubsequentlydevelopsintotheanaerobicstage(微氧环境)(NRP-2).GxRAactivitywasinducedduringmicroaerobicNRP-1,withthestrongestactivityinanaerobicNRP-2（无氧阶段）cultures.ItwasproposedthattheroleofthisenzymewastomaintainredoxbalancebyrecyclingNADH/NADduringinterruptionofaerobicrespiration.Introduction•Thenamingoftheglycinedehydrogenasewasbasedonthesimilarityoftheglyoxylatereductiveaminasereaction(乙醛酸还原氨基反应)tothatcatalyzedbyL-alaninedehydrogenase.•InMycobacteriumsmegmatis（耻垢分枝杆菌）pyruvateandglyoxylateaminaseactivitiescomigratedonanativepolyacrylamidegel,suggestingoneenzymeforbothactivities.However,aknockoutmutantofthealaninedehydrogenasegenealdinM.smegmatislostalaninedehydrogenaseactivitybutretainedglycinedehydrogenaseactivity.Introduction•Moreover,M.bovis（牛分支杆菌）doesnotproducealaninedehydrogenase,butglycinedehydrogenaseactivityhasbeenreported.•Inthisstudyaldwasshowntoencodebothalaninedehydrogenaseandglyoxylatereductiveaminase(glycinedehydrogenase)activities.Thiswasdeterminedbybothbiochemicalandgeneticmethods.Thisdualfunctionenzymewaslocalizedtothecellmembraneandcytosol.Itplaysanessentialroleintheutilizationofalanine,butnotglycine,asanitrogensource.Results-Isolationoftheputativeglycinedehydrogenase.•M.tuberculosisH37Rv•two-steppurification.Firsthydrophobicinteraction(疏水作用色谱法)andthenanion-exchangechromatography（离子交换色谱法）wereused.Results-Inactivationofald.•(A)DNAwasisolatedfromeachstrain,cutwithSacI,andanalyzedwithaprobespecificforald.RVW7,ald::hyg;Ald21,RVW7(pAld-Gen21).Theapproximatesizesareindicatedontheleft.•pUC-GM-INT+ald+527bpResults-Inactivationofald.•B.Geneorganizationofald,showingtheinsertionofhyg.Arrowsindicateopenreadingsframesandthedirectionoftranscription.X,XbaIsite;B,BamHIsite;S,SacIsite.ald=isthedisruptedald.Thealdprobebindingsiteisshown.Results-Inactivationofald.•(AandB)NADHbreakdownwithglyoxylateinaerobiccultures(A)ornonreplicatingpersistentstage2cultures(B).Filledsymbolsandsolidlinesshowdataforcompletereactions,whileemptysymbolsanddottedlinesareforreactionswithoutammoniumsulfate.Circles,WT;triangles,RVW7;squares,RVW7(pAld-Gen21).Results-Inactivationofald.•PvRAandGxRAactivitiesofthealdmutantinaerobiccultures(C)ornonreplicatingpersistentstage2cultures(D).PvRAisinvolvedinthereductiveaminationofpyruvatetoalanine.GxRAisinvolvedinthereductiveaminationofglyoxylatetoglycine.RVW7,ald;Ald21,RVW7(pAld-Gen21).SpecificactivityisreportedinmolesofNADHoxidized/min/gofprotein.Results-EnzymaticactivityofAld.•FIG4ActivityofHis-taggedAldatdifferentpHlevels.(A)Pyruvatereductiveaminationactivity(circles)andalaninedehydrogenaseactivity(squares).(B)Glyoxylatereductiveaminationactivity(circles),glycinedehydrogenaseactivity(squares),andglyoxylatereductiveaminationactivitywithglutamineinplaceofammoniumsulfate(triangles).SpecificactivityisshowninmolesofNADHoxidized/min/gofprotein.Results-EnzymaticactivityofAld.•(A)Reductiveaminationreaction（氨化反应）withpyruvate(Pyv),glyoxylate(Glx),α-ketoglutarate(aKT),hydroxypyruvate(Hpv),malate(Mal)（苹果酸）,acetate(Ace)（乙酸）,glycolate(Glc)（乙醇酸）,oxaloacetate(Oaa)（草酰乙酸）,andmethylglyoxal(Mg)（甲基乙二醛）.Results-EnzymaticactivityofAld.•(B)Oxidativedeaminationreaction（氧化脱按反应）withalanine(Ala),glutamate(Glu),proline(Pro),aspartate(Asp),serine(Ser),andglycine(Gly).Results-Enzymekinetics.（酶动力学）Forpyruvate,theVmaxwas5.8mmol/min/mgofprotein,andforglyoxylateitwas2.5mmol/min/mgofprotein.Results-SubcellularlocalizationofAld.ThelocationofAldforM.tuber-culosiswasexaminedinaerobicandNRPculturesbytwometh-ods.Forthefirstmethod,enzymeactivitywasmeasured,andforthesecondanAld-specificantibodywasused.Results-SubcellularlocalizationofAld.Results