异戊烯基化吲哚类生物碱的化学酶合成2009

ISS N 100727626C N 1123870ΠQ中国生物化学与分子生物学报Chinese Journal of Biochemistry and M olecular Biology2009年6月25(6):580～584收稿日期:2008211205;接受日期:2009203210国家自然科学基金资助(N o 130670019)3联系人　T el :010*********;E 2mail :liuxq @Received :N ovember 5,2008;Accepted :M arch 10,2009Supported by National Natural Science F oundation of China (N o.30670019)3C orresponding author T el :010*********;E 2mail :liuxq @异戊烯基化吲哚类生物碱的化学酶合成王　璐1),　尹文兵2),　李书明2),　刘晓晴1)3(1)首都师范大学生命科学学院,北京　100048;2)马尔堡大学药物生物研究所,马尔堡　35037,德国)摘要　异戊烯基化吲哚类生物碱广泛存在于麦角菌、青霉菌和曲霉菌中,具有一定的药理学活性,与未异戊烯基化的前体在生物活性方面具有明显的差异.曲霉菌中的某些异戊烯基化吲哚类生物碱具有抗癌活性,如烟曲霉毒素C (fumitrem orgin C )、tryprostatin B ,但其天然产量低且不易分离,利用化学酶合成法可很容易地将前体转化为异戊烯基化吲哚类生物碱.异戊烯基转移酶FtmPT 1对二甲丙烯基二磷酸(dimethylallyl diphosphate ,DMAPP )具有专一性,但可以接受不同的芳香族底物.早期研究发现,FtmPT 1能接受含色氨酸的不同环二肽为底物,但以cyclo 2L 2T rp 2L 2T yr 和cyclo 2L 2T rp 2L 2Phe 为底物时,酶的相对活性很低,其产物量少,无法用于合成产物.本实验通过优化酶反应条件来提高其产量.将已构建的含ftmPT 1的质粒在大肠杆菌中诱导表达,经Ni 2NT A 亲和柱纯化后用于酶反应.实验结果表明,通过增加酶量(终浓度218μm ol ΠL )、延长培养时间(37℃,24h ),以cyclo 2L 2T rp 2L 2T yr 和cyclo 2L 2T rp 2L 2Phe 为底物的酶反应产率分别达到4913%和2113%,产物经1H 2NMR 、1H 21H 2C OSY 和ESI 2MS 鉴定,其结果与预期吻合.据检索,这2个化合物均为新化合物,分别命名为cyclo 2C221′2DMA 2L 2T rp 2L 2T yr 和cyclo 2C221′2DMA 2L 2T rp 2L 2Phe.关键词　异戊烯基转移酶FtmPT 1;异戊烯基化吲哚类生物碱;化学酶合成中图分类号　Q81Chemoenzymatic Synthesis of Prenylated I ndole AlkaloidsW ANGLu 1),YI N Wen 2Bing 2),LI Shu 2Ming 2),LI U X iao 2Qing1)3(1)College o f Life Science ,Capital Normal Univer sity ,Beijing 100048,China ;2)Institut f ür Pharmazeutische Biologie ,Univer sity o f Marburg ,Marburg 35037,G ermany )Abstract Different from the non 2prenylated precurs ors ,prenylated indole alkaloids produced by Claviceps ,Penicillium and Aspergillus strains exert diverse biological and pharmacological activities ,for exam ples ,fumitrem orgin C and tryprostatin B inhibit the growth of a number of tum or cell lines.H owever ,these com pounds are usually produced at low concentrations in the natural hosts ,thus limits their use in research and development.Chem oenzymatic synthesis has recently been applied to s olve this problem.FtmPT 1,a prenyltrans ferase from Aspergillus f umigatus ,has a high specificity to dimethylallyl diphosphate (DMAPP ),and is als o flexible to tryptophan 2containing cyclic dipeptides as the substrates.H owever ,the yields of FtmPT 1react with cyclo 2L 2T rp 2L 2T yr or cyclo 2L 2T rp 2L 2Phe was not su fficient for success ful production of prenylatedcom pounds in previous studies.In this paper ,by increasing the enzyme dose to 218μm ol ΠL and extending the incubation time to 24hours ,the conversion rates of cyclo 2L 2T rp 2L 2T yr or cyclo 2L 2T rp 2L 2Phe to theirprenylated derivatives was increased to 4913%and 2113%,respectively.1H 2NMR ,1H 21H 2C OSY and ESI 2MS analyses con firmed that the products of the enzymatic reactions were cyclo 2C221′2DMA 2L 2T rp 2L 2T yr and cyclo 2C221′2DMA 2L 2T rp 2L 2Phe ,as expected.K ey w ords prenyltrans ferase FtmPT 1;prenylated indole alkaloids ;chem oenzymatic synthesis 生物碱一般指存在于生物体内的碱性含氮化合物,多数具有复杂的含氮杂环,有光学活性和显著的生理效应,广泛存在于植物、真菌、细菌中.异戊烯基化吲哚类生物碱含有芳香环和异戊烯基,主要存在于真菌的麦角菌、青霉菌和曲霉菌中[1,2],它与未异戊烯化的前体在生物活性方面具有明显的差异[3,4].异戊烯基化吲哚类生物碱具有广泛的药理学活性,例如,fumitrem orgin C 是乳腺肿瘤抗药蛋白的抑制剂,fumitrem orgin B 是一种真菌毒素[5],其生物合成前体tryprostatin B 是细胞周期的抑制剂,干扰细胞分裂G 2ΠM 期[6],tryprostatin B 和其甲氧基衍生物tryprostatin A[4,7]还能抑制微管的聚合[8,9],这类衍生物可成为新的先导化合物,用于研发抗有丝分裂及抗癌的药物.这些异戊烯基化吲哚类生物碱在曲霉菌中含量低且不易分离,不利于药理的研究.因此,对真菌代谢途径的基因工程研究是获取具有潜在药用价值药物的一条重要途径,也是提高活性成分含量的有效手段.异戊烯基转移酶是一类催化C5单位转移的蛋白质.目前,烟曲霉中的7个吲哚异戊烯基转移酶基因已被鉴定出来,其中6个异戊烯基转移酶被过量表达,表达的蛋白质具有生物化学功能,可以将异戊烯基转移到吲哚环不同的位置上[5,10～16].例如,在烟曲霉麦角碱fumigaclavine C 生物合成的第1步中,FgaPT 2催化C 24异戊烯基化反应,将L 2色氨酸转变为42异戊烯基化色氨酸[10,11].最后一步中,FgaPT 1将异戊烯基加到吲哚环的C 22上,使fumigaclavine A 转变为fumigaclavine C [12];72DMATS 催化C 27异戊烯基化反应,将L 2色氨酸转变为72异戊烯基化色氨酸[13];CdpNPT 能在环二肽色氨酸吲哚环的N 21上引入反式异戊烯基[14];FtmPT 1可使brevianamide F (cyclo 2L 2T rp 2L 2Pro )吲哚环的C 22位置异戊烯化,从而合成tryprostatin B [15];FtmPT 2将异戊烯基转移到12,132二羟基2fumitrem orgin C 的N1位生物合成fumitrem orgin B [16].这些酶对DMAPP 具有专一性,但可以接受不同的芳香族底物.在早期的研究中,FtmPT 1在接受含色氨酸的不同环二肽为底物时,其特异性有明显不同,如cyclo 2D 2T rp 2L 2Pro 和cyclo 2L 2T rp 2L 2Leu 的相对活性是其天然底物brevianamide F 的33%和22%,而cyclo 2L 2T rp 2L 2T yr 和cyclo 2L 2T rp 2L 2Phe 的相对活性仅为5%和314%[15].最近有研究报道,加入大量的酶和延长培养时间可使CdpNPT 和FtmPT 1在以L 2T rp 等为底物时的产物量提高[17].因此,本室通过优化酶反应条件对FtmPT 1催化的反应进行再研究,以cyclo 2L 2T rp 2L 2T yr 和cyclo 2L 2T rp 2L 2Phe 为底物(1mm ol ΠL ),218μm ol ΠL FtmPT 1,50mm ol ΠL T ris 2HCl (pH 715),5mm ol ΠL MgCl 2和1mm ol ΠL DMAPP (Fig 11),37℃培养24h 后,使其产物转化率达到4913%和2113%.Fig.1　The enzym atic synthesis of prenylated indole alk aloids with FtmPT 1,cyclo 2L 2T rp 2L 2Tyr (A)and cyclo 2L 2T rp 2L 2Phe (B)1　材料和方法111　材料含有ftmPT 1基因的质粒pAG 012,大肠杆菌X L12Blue ,环二肽(cyclo 2L 2T rp 2L 2T yr ,cyclo 2L 2T rp 2L 2Phe )和DMAPP 见前文[15];亲和层析填料Ni 2NT A 购自Qiagen 公司.185第6期王　璐等:异戊烯基化吲哚类生物碱的化学酶合成112　蛋白质的表达、纯化和鉴定将pAG012重组质粒转化到大肠杆菌X L12Blue中,挑取单菌落接种于含羧苄青霉素(终浓度50μgΠml)LB培养基中,37℃摇培过夜,次日以2%量接入含同样抗生素浓度的LB培养液中,37℃培养至A600为016～017,加入IPTG(终浓度为011mm olΠL) 30℃诱导表达过夜,离心收集菌体.菌体重悬于裂解缓冲液(50mm olΠL NaH2PO4,300mm olΠL NaCl,10 mm olΠL咪唑,pH810),加溶菌酶(终浓度1mgΠml)冰上放置30min,超声波破碎,将离心结合后的上清液加入Ni2NT A亲和柱中,分别用冲洗液(50mm olΠL NaH2PO4,300mm olΠL NaCl,20mm olΠL咪唑)和洗脱液(50mm olΠL NaH2PO4,300mm olΠL NaCl,250mm olΠL 咪唑)冲洗杂蛋白和收集目的蛋白,将洗脱液经PD10柱交换缓冲液(100mm olΠL T ris2HCl,pH715, 15%甘油),S DS2PAGE鉴定收集的目的蛋白.113　FtmPT1的酶活性分析小量酶反应体系(100μl)包含218μm olΠL FtmPT1,50mm olΠL T ris2HCl,pH715,5mm olΠL MgCl2, 1mm olΠL环二肽和1mm olΠL DMAPP,37℃培养24 h,100μl甲醇终止反应,离心除去变性蛋白质,取上清液进行HP LC分析(Agilent HP LC series1200, LiChrospher RP1825column,125mm×4mm,5μm). HP LC溶剂为甲醇和水1∶1混合液(溶剂A)、甲醇(溶剂B),为了分离酶反应混合物,用10%～50%溶剂B梯度洗脱15min,之后用100%溶剂B冲洗5min,最后用10%溶剂B平衡柱5min(流速1mlΠmin). 114　大量合成酶促反应产物大量酶反应体系(10ml)包含218μm olΠL FtmPT1,50mm olΠL T ris2HCl,pH715,5mm olΠL MgCl2, 1mm olΠL环二肽和2mm olΠL DMAPP,37℃培养36 h,向混合物中加10ml水,用乙酸乙酯萃取3次(每次20ml),旋转蒸发萃取液,将萃取物溶于200μl甲醇中.之后用HP LC分离并收集酶反应产物(Agilent HP LC series1200,RP182column,10mm×250mm,5μm).在分离cyclo2C221′2DMA2L2T rp2L2T yr酶反应产物时,用与分析时相同的溶剂和方法,只是流速不同,215mlΠmin;分离酶反应产物cyclo2C221′2DMA2L2 T rp2L2Phe时,溶剂相同,用30%～70%溶剂B梯度洗脱12min,用100%溶剂B冲洗8min,最后用30%溶剂B平衡柱5min.115　酶反应产物结构的鉴定将分离收集的纯酶反应产物,用旋转蒸发仪蒸干,然后用干燥器或冷冻干燥过夜,大约012～015 mg样品送样,用核磁共振法(1H2NMR,1H21H2C OSY,仪器J E O L EC A2500)鉴定其结构.另外,取纯样品,电喷雾离子化质谱(ESI2MS,仪器Qtrap Applied Biosysytems)鉴定其分子量.2　结果211　FtmPT1蛋白的表达和纯化含有ftmPT1的质粒经转化后,在大肠杆菌X L12 Blue中诱导表达,表达的蛋白质为融合蛋白质His62 FtmPT1,经纯化后通过S DS2PAGE得到单一条带(Fig12),其分子量与His62FtmPT1分子量的计算值5314kD基本一致.Fig.2　Analysis of overproduction and purification of H is62 FtmPT1 The proteins were separated on a12%S DS2 PAGE and stained with C oomassie brilliant blue R2250.1, m olecular mass standards;2,s oluble protein before induction; 3,s oluble protein after IPTG induction(011mm olΠL IPTG at 30℃for16hours);4,wash fraction of Ni2NT A agarose;5, Purified His62FtmPT1212　FtmPT1的酶活性分析小量酶反应终止后,将反应混合物通过HP LC 反相层析对其进行分析,以环二肽cyclo2L2T rp2L2T yr 为底物的酶反应,在312min和519min出现化合物峰(Fig13A),分别为底物峰和产物峰.以环二肽cyclo2L2T rp2L2Phe为底物的酶反应,在511min出现底物峰,1013min出现产物峰(Fig13B).213　酶反应产物结构的鉴定为了鉴定酶反应产物的结构,本室进行大规模制备培养,纯化的产物进行1H2NMR、1H21H2C OSY和ESI2MS分析,核磁共振光谱数据见T able1.对比产物和其底物的核磁共振光谱数据,产物中异戊烯基的信号分别出现在3144～3148(d)(H21′),5133～5136(t)(H22′),1178～1179(s)(H24′)和1179～285中国生物化学与分子生物学报25卷Fig.3　HP LC chrom atograms of incub ation mixtures of recombinant FtmPT 1with cyclo 2L 2T rp 2L 2Tyr (A),and cyclo 2L 2T rp 2L 2Phe(B) The reaction mixtures contained Mg2+(5mm ol ΠL ),substrate (1mm ol ΠL )and DM APP (1mm ol ΠL )were incubated at 37℃for 24hours.Detection was carried out using a Photo Diode Array detector T able 1　1H 2N MR d ata of the enzym atic products of FtmPT 1cyclo 2C221′2DM A 2L 2T rp 2L 2T yrcyclo 2C221′2DM A 2L 2T rp 2L 2Phe Proton in C D 3OD in C DCl 3NH 21H 24H 25H 26H 27H 210H 211H 212H 214H 215H 217H 219H 220H 221H 222H 223H 21′H 22′H 24′H 25′-7152,d ,7137100,td ,810,1107107,td ,810,1107126,d ,7193106,dd ,1519,4143110,dd ,1519,6114118,t ,511-3179,dd ,1013,312-1121,m2155,dd ,1016,3166131,dd ,815,2136155,d ,81526155,d ,8156131,dd ,815,2133148,d ,7115136,t ,7101178,s 1179,s7194,s7129,dd ,710,1177125,t ,7137117,td ,712,1157156,dd ,617,1183101,dd ,1417,7173127,dd ,1417,3174127,m 5175,s 4105,d ,14105153,s1194,dd ,1716,13163113,dd ,1217,3127123,t ,6106179,dd ,717,1127115,td ,711,1146179,dd ,717,1127123,t ,6103144,d ,8105133,t ,7191179,s 1182,sDM A :3′,3′2dimethylallyl ;chemical shifts are given in ppm and coupling constants in H z1182ppm (s )(H 25′).相对于底物吲哚环上的2位碳上的氢消失了,说明异戊烯基加在吲哚环上的2位碳上,这些数据与G rundmann 等[15]报道的2位碳异戊烯基化的吲哚类生物碱数据完全吻合,这2个化385第6期王　璐等:异戊烯基化吲哚类生物碱的化学酶合成　合物分别命名为cyclo2C221′2DMA2L2T rp2L2T yr和cyclo2C221′2DMA2L2T rp2L2Phe.ESI2MS鉴定结果如下:对于cyclo2C221′2DMA2L2T rp2L2T yr,[M+H]+是41811,[M+NH4]+是43512,[M+Na]+是44011;对于cyclo2C221′2DMA2L2T rp2L2Phe,[M+H]+是40211, [M+NH4]+是41912,[M+Na]+是42411.这些产物的分子量比底物大68,也证明了异戊烯基的存在.3　讨论在前一个研究中,FtmPT1虽能接受含色氨酸的不同环二肽,但对某些化合物的转化率较低,不能有效用于合成化合物,如cyclo2L2T rp2L2T yr、cyclo2L2 T rp2L2Phe相对于brevianamide F的反应活性仅为5%和314%[15],产率分别为015%和0134% (G rundmann and Li,未发表结果).在本实验中通过改变酶反应条件,如增加酶量,延长培养时间,达到了有效合成化合物的目的,如:以cyclo2L2T rp2L2T yr 和cyclo2L2T rp2L2Phe为底物的酶反应产率分别为4913%和2113%,产物量提高较大.近年来的研究证明,利用芳香族异戊烯基转移酶的化学酶合成法,可获得一系列异戊烯基化的吲哚类生物碱[5,11,17,18],此途径可做为化合物修饰的新策略.与化学合成相比较,化学酶合成是在温和的条件下进行的,因此可以避免不必要的反应和重排.酶反应产物cyclo2C221′2DMA2L2T rp2L2T yr和cyclo2C22 1′2DMA2L2T rp2L2Phe是2个新化合物,其结构与有抗癌作用的tryprostatin A、tryprostatin B、cyclo2 tryprostatin A,B,C,D、fumitrem orgin C相似[5,9],因此,新合成的这两种化合物也可能具有抗癌的潜力.本室将进一步研究其药理作用,并在细胞水平上研究其对微管的影响.参考文献(R eferences)[1]　W illiams R M,S tocking E M,Sanz2Cervera J F.Biosynthesis ofprenylated alkaloids derived from tryptophan[J].T op Curr Chem,2000,209:972173[2]　S tocking E M,W illiams R M,Sanz2Cervera J F.Reverse prenyltrans ferases exhibit poor facial discrim ination in the biosynthesis ofparaherquam ide A,brevianam ide A,and austam ide[J].J Am ChemS oc,2000,122(38):908929098[3]　Botta B,Vitali A,M enendez P,et al.Prenylated flav onoids:pharmacology and biotechnology[J].Curr M ed Chem,2005,12(6):7172739[4]　Usui T,K ondoh M,Cui C B,et al.T ryprostatin A,a specific andnovel inhibitor of m icrotubule assembly[J].Biochem J,1998,333(Pt3):5432548[5]　S teffan N,G rundmann A,Y in W2B,et al.Indole prenyltrans ferasesfrom fungi:a new enzyme group with high potential for the productionof prenylated indole derivatives[J].Curr M ed Chem,2009,16(2):2182231[6]　Sanz2Cervera J F,S tocking E M,Usui T,et al.Synthesis andevaluation of m icrotubule assembly inhibition and cytotoxicity ofprenylated derivatives of cyclo2L2T rp2L2Pro[J].Bioorg M ed Chem,2000,8(10):240722415[7]　Jain H D,Zhang C,Zhou S,et al.Synthesis and structure2activityrelationship studies on tryprostatin A,a potent inhibitor of breastcancer resistance protein[J].Bioorg M ed Chem2008,16(8):462624651[8]　Cui C B,K akeya H,Okada G,et al.T ryprostatins A and B,novelmammalian cell inhibitors produced by Aspergillus fumigatus[J].J Antibiot,1995,48(11):138221384[9]　K ondoh M,Usui T,M ayum i T,et al.E ffects of tryprostatin derivativeson m icrotubule assembly in vitro and in situ[J].J Antibiot,1998,51(8):8012804[10]　Uns ld I A,Li S M.Overproduction,purification and characterizationof FgaPT2,a dimethylallyl tryptophan synthase from Aspergillusfumigatus[J].M icrobiology,2005,151(Pt5):149921505[11]　S teffan N,Uns ld I A,Li S M.Chem oenzymatic synthesis of prenylatedindole derivatives by using a42dimethylallyltryptophan synthase fromAspergillus fumigatu[J]s.Chembiochem,2007,8(11):129821307 [12]　Uns ld I A,Li S M.Reverse prenyltrans ferase in the biosynthesis offum igaclavine C in Aspergillus fumigatus:gene expression,purificationand characterization of fum igaclavine C synthase FgaPT1[J].Chembiochem,2006,7(1):1582164[13]　K remer A,W estrich L,Li S M.A72dimethylallyltryptophan synthasefrom Aspergillus fumigatus:overproduction,purification andbiochem ical characterization[J].M icrobiology,2007,153(Pt10):340923416[14]　Y in W B,Ruan H L,W estrich L,et al.CdpNPT,an N2prenyltrans ferase from Aspergillus fumigatus:overproduction,purification and biochem ical characterisation[J].Chembiochem,2007,8(10):115421161[15]　G rundmann A,Li S M.Overproduction,purification andcharacterization of FtmPT1,a brevianam ide F prenyltrans ferase fromAspergillus fumigatus[J].M icrobiology,2005,151(Pt7):219922207 [16]　G rundmann A,K uznets ova T,A fiyatullov S S,et al.FtmPT2,an N2prenyltrans ferase from Aspergillus fumigatus,catalyses the last step inthe biosynthesis of fum itrem orgin B[J].Chembiochem,2008,9(13):205922063[17]　Z ou H,Zheng X,Li S M.Substrate prom iscuity of the cyclic dipeptideprenyltrans ferases from Aspergillus fumigatus[J].J Nat Prod,2009,72(1):44252[18]　K remer A,Li S M.P otential of a72dimethylallyltryptophan synthase asa tool for production of prenylated indole derivatives[J].ApplM icrobiol Biotechnol,2008,79(6):9512961485中国生物化学与分子生物学报25卷。