31310.氘標記1脫氧d木酮糖5磷酸酯的合成研究

1、西北大學碩士學位論文氘標記1脫氧D木酮糖5磷酸酯的合成研究姓名：周躍飛申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：高文運20090609Thesyntheticstudyof1deoxyDxylulose5phosphateallowingdeuteriumlabellingAbstract1deoxyD—xylulose5phosphate(DXP)hasbeenidentifiedasaprecursorinthreema

2、jormetabolicpathways：thebiosynthesisofthiaminediphosphate(vitaminB1，ThPP)andpyridoxolphosphate(vitaminB6)inbacteriaandtheformationofisopentenyldiphosphate(IPP)anddimethylallyldiphosphate(DMAPP)viathe2methylDerythritol4ph

3、osphate(MEP)pathwayforisoprenoidbiosynthesisinmostbacteriaandinthechlotoplastsofphototrophicorganismsDXPissynthesizedbytheDXPsynthase(DXS)，aThPPdependentenzyme，catalyzingthecondensationofpyruvateandD—glyceraldehyde3phosp

4、hateAlso，DXPisthesubstratefortheenzyme1deoxyD—xylulose5phosphatereductoisomerase(OXR)whichisaratelimitingenzymeinMEPpathwayandapotentialtargetforthescreeningofantibacterialdrugsItconvertsDXPintothebranchedcompoundMEPInor

5、dertostudythemechanismofDXRwerequiredapreparationofactiveDXSbymolecularbiologicaltechniquesandaflexiblesynthesisofthesubstratewhichwouldallowtheincorporationofdeuteriumlabelesInordertoobtainpurifiedDXS，therecombinantplas

6、midpFMH30WastransformedintoEcoliBL21(DE3)competentcells，andtheDXSproteininducedbyIPTGWasexaminedbySDS—PAGEanalysisTheresultofSDS—PAGEshowedthatthemolecularweightoffusionproteinDXSwithaHis6tagwassameastheexpectedvalue，app

7、roximately66kDaTherecombinantDXSproteinWaspurifiedusingthe(NH4)2S04precipitationandNi2affinitychromatographyanditsconcentrationWas118mg／ml，determinedbyBradfordreagentwithpurityover90％TheactivitytestbyReversedphaseionpair

8、IIPLCindicatedthatthisfusionproteinCanbeusedfortheenzymicsynthesisofDXPDXPakeymetaboliteintheMEPpathwayforisoprenoidbiosynthesis，isconvenientlysynthesizedfrompyruvateandDGlyceraldehyde一3phosphate(D—GAP)ordihydroxyacetone