糖基转移酶与糖苷酶

第二节糖基转移酶及其应用主要内容糖转移酶简介糖基转移酶在合成中的应用•Glycosyltransferases应用‘activated’sugarphosphates作为糖供体，合成glycosidiclinkage，糖受体通常为nucleophilicgroup,usuallyanalcohol.生成的糖苷可以为O-,N-,S-,orC-glycoside糖转移酶简介功能：催化糖苷键的合成（O-,N-,S-,orC-glycoside）供体：活化的糖磷酸受体：亲核基团（蛋白、脂、核酸、糖、小分子），通常为-OHNon-Leloirdonors：Leloirdonors：糖供体底物糖基转移酶根据糖供体中是否含有核苷酸分为两类。Leloir:阿根廷生物化学家，研究核苷酸代谢，1970，诺贝尔化学奖分为94家族（distinctsequence-basedfamilies）(CAZyserver,)人类拥有约270多种糖基转移酶序列，属于33个家族。Alpha-1,4-葡萄糖转移酶；beta-1,4-半乳糖转移酶；2,3-唾液酸转移酶糖基转移酶分类根据氨基酸序列相似性进行分类：根据蛋白结构相似性进行分类：根据糖供体和糖苷键连接方式进行分类：GT-A，GT-B，其他类型Rossmann-typedomains(fornucleotidebinding)NDP-bindingdomaingenerallycontainsaconservedDXDaminoacidmotifGT-Afold:SpsAfromBacillussubtilusGT-Bfold:beta-glucosyltransferasefrombacteriophageT4LeloirGTsTransglycosylasefromStaphylococcusaureusOligosaccharyltransferaseSTT3fromPyrococcusfuriosiusNon-LeloirGTs催化机理•Glycosyltransferasescatalyzethetransferofglycosylgroupstoanucleophilicacceptorwitheitherretentionorinversionofconfigurationattheanomericcentre.Thisallowstheclassificationofglycosyltransferasesaseitherretainingorinvertingenzymes.鎓催化机理Inverting:SN2nucleophilicattackattheC1atomRetaining:doubledisplacementmechanism糖基转移酶辅因子Many,butnotall,glycosyltransferasesutilizedivalentmetalioncofactorssuchasMn2+andMg2+.…mainlyinglycosyltransferasesthatarediphosphonucleoside-dependent.metalioniscoordinatedtoanoxygenofeachofthetwophosphategroups,aswellastoside-chaincarboxylatesderivedfromtheprotein..糖基转移酶抑制剂直接抑制糖基转移酶活性底物类似物；过渡态类似物alpha-2,6-唾液酸转移酶抑制剂Beta-1,4-半乳糖转移酶抑制剂阻断糖供体的合成N-Glycan合成过程中，首先要合成：dolichol-pp-N-acetylglucosamineUDP-GlcANc+dolichol-p------dolichol-pp-GlcNAcN-acetylglucosaminephosphorotransferase糖基转移酶在合成中的应用寡糖的酶法合成具有生物活性含糖天然产物的酶法合成生物制药----糖蛋白药物生产策略生物制药----糖疫苗生产策略OAcOAcOOAcOAcOOAcOAcOOAcOAcOHOOAcOAcOOAcOAcOOAcN3OOAcOOAcOAcOOAcOAcOOAcN3OBnOBnOBnOOBnOOHOOHOHOOHOHOOHNHAcOHOHOHOOH2679N33steps6stepsglycosylation4stepsChemicalSynthesisofa-Gal规模小，过程复杂，立体选择性难OOHHOHOHOOPOPONNHOOOOHHOHOHOOOHHONHAcOOROOHHOHOHOOOHHOOOHOOHHONHAcOOROOOO-O-HOPOPONNHOOOOOO-O-OHHOOHHOGal1,4GlcNAc-R,acceptor-GalepitopeUDPUDP-GalDonor1,3-galactosyltransferaseMn2+++ReactionCatalyzedby1,3-Galactosyltransferase碳水化合物的合成OrganicSynthesisBiosyntheticprocessDerivativesynthesisVersatileNotveryflexibleProcessExpensiveandmultistepEconomicandshortEnvironmentproblemChemicalwasteEnvironment-friendlyCarbohydratepolymersynthesisNotapplicablePracticalapproach体内糖供体合成途径Glc,GlcNAc&ManSugar-6-pSugar-1-pSugar-NDPkinaseMutasepyrophorylaseGal,GalNAc&FucSugar-1-pSugar-NDPkinasepyrophorylaseNeu5AcNeu5AcCMP-Neu5AcpyrophorylaseCTP糖供体相互转化:•GalE:UDP-Gal↔UDP-Glu•GalNAcE:UDP-GalNAc↔UDP-GlcNAc•UGD(UDP-Glcdehydrogenase)：UDP-Glc↔UDP-GlcA•UDP-GlcAdecarboxylase:UDP-GlcA↔UDP-Xyl糖基转移酶介导的寡糖合成PhosphorylasesGlucosyltransferasesFructosyltransferasesCyclodextringlucanotransferasesMany微生物中的糖供体•天然糖苷化合物中的糖一般为C2,C3,C4and/orC6脱氧糖，而且大部分为6-deoxyhexosesfamily（Rhamnose）•原核生物糖转移酶具有糖供体的广泛性•6-deoxyhexoses一般通过TDP-sugars进行底物活化（TDP-Rhamnose）优点:*区域、立体选择性*大量生产不足:*糖基转移酶在大量表达方面存在困难•糖供体比较昂贵.•副产物抑制(i)消除副产物抑制（碱性磷酸酶）(ii)原位产生糖供体(iii)合成廉价的糖供体类似物.针对以上不足解决策略糖基转移酶在工业应用中优势与弱点可作为LgtC底物•碱性磷酸酶酶法再生糖供体策略UDP-sugar(A)andCMP-sugar(B)糖供体的合成糖供体的合成糖供体的合成糖基转移酶在寡糖合成中的应用（一）具有生物活性寡糖的酶法合成(fordetailsvisit:wang.chem.wayne.edu)OHOOHHOHOOHOOHOHOOOOHHONHACOHOOHOHOOOOHHOHOResearchonCarbohydratesinHumanImmunityinWang'sLaboratoryOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBloodtypeBantigenTypeBbloodTypeAbloodAnti-Bantibody?Barrierinbloodtransfusion?Barrierinorgantransplantation?Causeofhyperacuterejectioninanimaltohumanxenotransplantation?Humannaturalimmunityagainstpathogens,tumors,etc.Non-primatemammals(pig,mouse,dogetc.)NewworldmonkeyHumanApeOldworldmonkeyAnti-Galantibody(themostabundantnaturalAb)1-2%totalIgG,3-8%totalIgM-Galepitopeabundant,1million/cellHuman-HumanHuman-AnimalOHOOHOHOHOOHHOHOOHOOHOHOOOOHHONHACOHOOHOHOOOOHHOHOThespecificityofglycosyltransferasesdeterminethesugarsequenceOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBloodtypeBantigenOHOOHOH-GalantigenOOHOHHOOOOOHHONHACOHOOHOHOOOOHHOHOOHOOHOHOOHOHHOHOOOOHHONHACOHOOHOHOOOOHHOHOhuman1,3GalTpig1,3GalTAnti-Galproductionmillionyearsago5-1520-3030-4060-7070-80120-130mammals-galepitopesynthesishumansapesmonkeysOldWorldNewWorldmonkeysprosimiansmarsupialsnonprimateplacentalmammalsInactivationof1,3GTBiomedicaluseof-Galsoluble-Galantagonisttoanti-Gal-Galconjugatesinimmunotherapyagainstbacteria,virus,andcancercells.人工合成的必要性OOHHOHOHOOPOPONNHOOOOHHOHOHOOOHHOOHOOROOHHOHOHOOOHHOOOHOOHHOOHOOROOOO-O-HOPOPONNHOOOOOO-O-OHHOOHHOReactionCatalyzedby1,3-GalactosyltransferaseGal1,4Glc-R,acceptor-GalepitopeUDPUDP-GalDonor1,3-galactosyltransferaseMn2+++1,3GalT催化合成-GalEpitopes及其衍生物HOOOHHOHOOUDPHOOOHHOHOOUDPOHOOHHOOOOHHOR1R2HOOHOOHHOHOOHOOHOHOOOOHHOR1R2UDP-Gal4-epimeraseUDP1,3GalTR1R2OHOHOHOHNHAcN3SPhOAllylabcdNHAcOHeNH2OHfUDP-Gal100mg/$416UDP-Glc5g/$529.5EC5.1.3.2FangJ,ChenX,WangPG,etal.:J.Org.Chem.1999,64,4089-4094.OOHOHOHOOOHOHHON3OHOOOHNHAcHOOOHOHOHOOOHOHHON3OOOOHNHAcHOOOHOHOHOOOHOHOHHOOOHOHOHOOOHOHHON3OOOOHNHA