細(xì)胞色素P酶系

細(xì)胞色素P酶系第1頁/共14頁細(xì)胞色素P450酶系第2頁/共14頁1.細(xì)胞色素P450酶系概述細(xì)胞色素P450單加氧酶系(cytochromeP450monooxygenases，P450s)：

NAD(P)H-細(xì)胞色素P450還原酶

(CPR)

細(xì)胞色素P450單加氧酶(CYP)2個組分均通過疏水的膜結(jié)合錨點結(jié)合在微粒體膜上。

CYP450廣泛分布在動物、植物、低等真核生物和細(xì)菌體內(nèi)，在同種生物的不同組織中都存在，是自然界中含量最豐富、底物譜最廣的催化酶系。第3頁/共14頁NADPH-細(xì)胞色素P450還原酶NADPH-細(xì)胞色素P450還原酶是一種以FAD和FMN作為輔基的細(xì)胞微粒體復(fù)合黃素蛋白；CYP450是以硫醇鹽結(jié)合血紅素為活性中心催化氧原子轉(zhuǎn)移的氧化還原酶，氧由亞鐵血紅素（HEME）來活化。CPR是CYP的主要電子供體，電子傳遞給CYP之后，CYP才能與底物發(fā)生氧化還原反應(yīng)，發(fā)揮代謝活性，CPR與CYP的電子傳遞反應(yīng)是CYP氧化還原反應(yīng)的限速步驟

。第4頁/共14頁2.HumanandMicrobialCYPsHumanCYPsHumanscontain57CYPsmitochondrialCYPs:CYP11A1、CYP11B1、CYP11B2microsomalCYPs:CYP17A1、CYP19A1、CYP21A1SixP450enzymesareinvolvedinsteroidogenesisInhumanadrenalP450sbeenintensivelystudiedinsteroidsynthesis.Inbacteria,theP450sareusedincatabolicpathwaysfortheproductionofsteroids,bioactivelipidsandchiralsynthons.

MicrobialCYPs

Actinomycetes

Mycobacteriumsmegmatis(39P450s)Mycobacteriumtuberculosis(20P450s)Streptomycescoelicolor(18P450s)第5頁/共14頁3.甾體羥化反應(yīng)甾體羥化酶大都是細(xì)胞色素P450依賴型單加氧酶，屬于末端氧化酶，能將一摩爾分子氧引入底物，通過NADPH遞氫，完成羥化反應(yīng)。RH+NADPH+O2ROH+NADP++OH-第6頁/共14頁Steroidogenesis細(xì)胞色素P450酶系羥化反應(yīng)主要有：9α羥化、11α羥化、11β羥化、16α羥化、17α羥化和19α羥化等。甾體化合物分子中11-羥基的引入在甾體藥物化學(xué)中占極為重要的地位。第7頁/共14頁TowardsPreparativeScaleSteroidHydroxylationwithCytochromeP450MonooxygenaseCYP106A21.CYP106A2fromBacillusmegateriumATCC13368,enablestheoxidationof3-keto-4-enesteroidsmainlyatposition15.2.Weexpressedthisenzymetogetherwiththeelectron-transferpartnersbovineadrenodoxinandadrenodoxinreductaseinEscherichiacoli.3.Anenzyme-coupledcofactorregenerationsystemwasimplementedbyexpressingalcoholdehydrogenasefromLactobacillusbrevis。eg1第8頁/共14頁CloningpBARTwin:bovineAdR、AdxpACYCFHH28:CYP106A2 pBARTripel:bovineAdR、AdX、LbADH第9頁/共14頁SelectivityofCYP106A2Withgrowingcellsasabiocatalyst,alimitedselectivityofbothProgandTeshydroxylationwasobserved.Nosideproductformationcouldbeobservedwhenrestingcellswereusedasbiocatalysts.ComparisonofgrowingandrestingcellsforA)progesteroneandB)testosteroneconversion第10頁/共14頁SubstratesolubilityandtransportintothecellHighestactivitywasobservedinpresenceofeven2m(15.4vol%)propan-2-ol.Duringthesecondreactioncycleonly21%ofProgcouldbeconvertedinthepresenceofpropan-2-ol.Withoutthiscosolvent53%15b-Progwasformed;Thisindicatesthelimitedapplicabilityofpropan-2-olasacosolventinthissystem.Influenceofpropan-2-olontheactivity(A)andstability(B)ofthewhole-cellbiocatalyst21%53%第11頁/共14頁Substratesolubilityandtransportintothecellbiotransformationwith“membrane-free”crudecellextract(CCE)andthewhole-cellConversionofprogesteronebyusingcrudecellextractofE.coliexpressingCYP106A2,AdR,AdxandLbADHasbiocatalystTheinitialreactionrateincreased:0.4U15?-Proggwcw-1to9.6U15?-Proggwcw-1glyophilisate-1incasesofCCE.

Alossinselectivitycomparedtorestingcells.20%sideproductsareformed.第12頁/共14頁Batchexperiments

15?-Progformation:45minreactionconversion:85%Tesconversion:30minconversion:nearlycompletearound20%SPsproductivity:3.7gL-1d-1for15?-Prog

5.5gL-1d-1