12.18细胞色素P450酶系ppt课件

细胞色素P450酶系 孙婉菊2014年12月17日 1 细胞色素P450酶系概述 细胞色素P450单加氧酶系 cytochromeP450monooxygenases P450s NAD P H 细胞色素P450还原酶 CPR 细胞色素P450单加氧酶 CYP 2个组分均通过疏水的膜结合锚点结合在微粒体膜上 CYP450广泛分布在动物 植物 低等真核生物和细菌体内 在同种生物的不同组织中都存在 是自然界中含量最丰富 底物谱最广的催化酶系 NADPH 细胞色素P450还原酶 NADPH 细胞色素P450还原酶是一种以FAD和FMN作为辅基的细胞微粒体复合黄素蛋白 CYP450是以硫醇盐结合血红素为活性中心催化氧原子转移的氧化还原酶 氧由亚铁血红素 HEME 来活化 CPR是CYP的主要电子供体 电子传递给CYP之后 CYP才能与底物发生氧化还原反应 发挥代谢活性 CPR与CYP的电子传递反应是CYP氧化还原反应的限速步骤 2 HumanandMicrobialCYPs HumanCYPsHumanscontain57CYPsmitochondrialCYPs CYP11A1 CYP11B1 CYP11B2microsomalCYPs CYP17A1 CYP19A1 CYP21A1SixP450enzymesareinvolvedinsteroidogenesis InhumanadrenalP450sbeenintensivelystudiedinsteroidsynthesis Inbacteria theP450sareusedincatabolicpathwaysfortheproductionofsteroids bioactivelipidsandchiralsynthons MicrobialCYPsActinomycetesMycobacteriumsmegmatis 39P450s Mycobacteriumtuberculosis 20P450s Streptomycescoelicolor 18P450s 3 甾体羟化反应 甾体羟化酶大都是细胞色素P450依赖型单加氧酶 属于末端氧化酶 能将一摩尔分子氧引入底物 通过NADPH递氢 完成羟化反应 RH NADPH O2ROH NADP OH Steroidogenesis 细胞色素P450酶系羟化反应主要有 9 羟化 11 羟化 11 羟化 16 羟化 17 羟化和19 羟化等 甾体化合物分子中11 羟基的引入在甾体药物化学中占极为重要的地位 TowardsPreparativeScaleSteroidHydroxylationwithCytochromeP450MonooxygenaseCYP106A2 1 CYP106A2fromBacillusmegateriumATCC13368 enablestheoxidationof3 keto 4 enesteroidsmainlyatposition15 2 Weexpressedthisenzymetogetherwiththeelectron transferpartnersbovineadrenodoxinandadrenodoxinreductaseinEscherichiacoli 3 Anenzyme coupledcofactorregenerationsystemwasimplementedbyexpressingalcoholdehydrogenasefromLactobacillusbrevis eg1 Cloning pBARTwin bovineAdR AdxpACYCFHH28 CYP106A2pBARTripel bovineAdR AdX LbADH SelectivityofCYP106A2 Withgrowingcellsasabiocatalyst alimitedselectivityofbothProgandTeshydroxylationwasobserved Nosideproductformationcouldbeobservedwhenrestingcellswereusedasbiocatalysts ComparisonofgrowingandrestingcellsforA progesteroneandB testosteroneconversion Substratesolubilityandtransportintothecell Highestactivitywasobservedinpresenceofeven2m 15 4vol propan 2 ol Duringthesecondreactioncycleonly21 ofProgcouldbeconvertedinthepresenceofpropan 2 ol Withoutthiscosolvent53 15b Progwasformed Thisindicatesthelimitedapplicabilityofpropan 2 olasacosolventinthissystem Influenceofpropan 2 olontheactivity A andstability B ofthewhole cellbiocatalyst 21 53 Substratesolubilityandtransportintothecell biotransformationwith membrane free crudecellextract CCE andthewhole cell ConversionofprogesteronebyusingcrudecellextractofE coliexpressingCYP106A2 AdR AdxandLbADHasbiocatalyst Theinitialreactionrateincreased 0 4U15 Proggwcw 1to9 6U15 Proggwcw 1glyophilisate 1incasesofCCE Alossinselectivitycomparedtorestingcells 20 sideproductsareformed Batchexperiments 15 Progformation 45minreactionconversion 85 Tesconversion 30minconversion nearlycompletearound20 SPsproductivity 3 7gL 1d