安丝菌素生物合成中酰胺N-糖基化和氨甲酰化的后修饰
其他题名Amide N-Glycosylation and Carbamoylation in the Post-PKS Modifications of Ansamitocin
赵沛基
学位类型博士
导师沈月毛
2008-05-26
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词安丝菌素 糖基转移酶(asm25) 氨甲酰基转移酶(asm21) 安丝菌素酰胺n-(氨甲酰基)糖苷 异源表达 二重催化功能
摘要近期我们在分析橙色束丝放线菌固体培养物时,不但分离到大量的安丝菌素,还分离到一系列安丝菌素酰胺N-葡萄糖苷类化合物,其中一类是正常的葡萄糖基,另一类是氨甲酰基修饰的葡萄糖基(4-OH 或6-OH)。本论文通过遗传学、生物化学和化学的分析,对橙色束丝放线菌产生的安丝菌素后修饰糖基转移酶基因asm25异源表达及其体外催化特性进行了深入研究;而在分离到安丝菌素酰胺N-(氨甲酰基)糖苷类化合物后,分析安丝菌素生物合成基因簇,只发现一个编码氨甲酰基转移酶的基因asm21,文献报道基因的功能是催化C-7 位羟基的氨甲酰化,因此我们推测Asm21除催化C-7 位羟基的氨甲酰化外,还可能催化安丝菌素葡萄糖苷糖基的氨甲酰化,具有催化的二重性。对后修饰基因asm21进行敲除、回补和对突变株无细胞提取物进行活性测定,体内验证Asm21的催化功能;通过asm21异源表达,确定了Asm21体外的双重催化功能。 通过asm25的克隆和异源表达及包涵体复性获得可溶性活性蛋白,发现温度、pH、重金属离子对Asm25催化活性的影响较大:根据检测结果确定Asm25的最适温度为37 oC;在两个缓冲系统中(柠檬酸-磷酸二氢钠缓冲液、Tris-HCl)的pH 4.0~9.0范围内,pH 6.0~8.0均适于Asm25的催化反应,其中pH 7.5为最适pH值;在金属离子的影响上,Cu2+和Hg2+对Asm25的活性有抑制作用,在浓度达10 mM时,活性完全丧失;Zn2+在低浓度下(0.1 mM)有促进作用,而在高浓度下,表现为抑制作用;Li+和Mn2+在低浓度下(0.1 mM)有明显的促进作用,而在高浓度下对酶活没有影响,但Mn2+离子促进作用相对较弱。在最适条件下,获得了Asm25的动力学参数,Km(PND-3) = 17.79 M,KB (UDP-glucose) = 87.30 M。kcat= Vmax / [E]=0.0021/0.16=1.3×10-2 s-1。在不同的安丝菌素(合成的中间产物)、安沙类化合物(格尔德霉素、利福霉素及naphthomycin A)以及indolin-2-ones作为受体,以UDP-glucose, ADP-glucose, GDP-glucose, UDP-galatose, UDP-N-acetyl glucosamine 和 UDP-glucuronic acid作为供体,发现Asm25在体外催化过程中,对糖基供体有严格的选择性,而对苷元受体具有一定的广谱性。 通过对asm21基因敲除,获得了asm21的突变株BLQ16,分别以安丝菌素酰胺N-糖苷P-3(AGP-3)和proansamitocin (PA)作为底物,用突变株BLQ16和HGF051(安丝菌素聚酮合成基因asmB缺失突变株)的无细胞提取物进行做活性测定,在HGF051的无细胞提取物进行反应体系中,分别以AGP-3和PA作为底物,检测到了AGP-3和PA氨甲酰化的产物,而在BLQ16无细胞提取物进行反应体系的反应产物中没有检测到氨甲酰化的产物,从体内验证了Asm21 的二重催化功能;克隆了asm21的基因,构建了在链霉菌中的表达系统,在变铅青链霉菌Streptomyces lividans T7中进行异源表达,同样分别以AGP-3和PA作为底物,对pJTU3205/S. lividans T7表达的总蛋白进行活性检测,检测到了AGP-3和PA氨甲酰化的产物,从体外证明了Asm21 的二重催化功能。
其他摘要Recently, several amide N-glycosides of ansamitocin were isolated from the solid fermentation products of Actinosynnema pretiosum ssp. aurantum. Part of glycosyl was normal glucose, and the other glycosyl was that 4-OH or 6-OH of glycosyl was replaced by carbamoyl. Glycosyltransferase gene asm25 and carbamoyltransferase gene asm21 are two genes involved in the post-PKS modifications of ansamitocin from the Actinosynnema pretiosum ssp. aurantum ATCC 31565. N-glycosyltransferase Asm25 was characterized through heterologous expression and in vitro catalysis. Analysis of the gene cluster for ansamitocin biosynthesis, only one gene (asm21) coding carbamoyltransferase was found, which was confirmed to carbamoylate the 7-hydroxyen on the ansamitocin according to report. From above points, we speculated that Asm21 was able to carbamoylate amide N-glycosides of ansamitocin besides carbamoylation of the 7-hydroxyen, possessing dual catalysis function. Through gene inactivation, complementation, and in vitro catalysis with total proteins, Asm21 was proved to perform carbamoylation both on the PKS backbone and the glucose moiety of ansamitocinosides. Active soluble Asm25 protein was obtained from asm25-expressing E. coli by solubilization from inclusion bodies. Its optimal reaction conditions including temperature, pH, metal ion requirement, and Km/Kcat were determined. Asm25 was optimally active at 37 °C and showed optimal activity at pH 7.5 in 50 mM Na-acetate/citric acid buffer (from pH 4.0 to 7.0) and 50 mM Tris-HCl buffer (from pH 7.0 to 9.0). Its activity was improved with the addition of Li+, Zn2+ or Mn2+ at a concentration of 0.1 mM. Negligible effects on the catalytic activity were observed as the concentrations of Li+ and Mn2+ increased to 10 mM. On the other hand, the activity was partially inhibited by 0.1 mM Hg2+ or Cu2+ and almost inhibited by 1 mM Hg2+, Zn2+ or Cu2+. The Vmax and Km values were determined by classical initial rate experiments and calculated from Lineweaver-Burk plots: Km for PND-3 and UDP-Glc were 17.8 M and 87.3 M, respectively, with a kcat of 1.3 ×10-2 • s-1. Asm25 showed also broad substrate specificity towards other ansamycins and synthetic indolin-2-ones. Mutant of asm21, BLQ-16, was obtained by gene inactivation. Using ansamitocinoside P-3 (AGP-3) and proansamitocin (PA) as substrates, dual catalysis of Asm21 was proved in vivo with the cell-free extracts of BLQ16 and asmB mutant HGF051, and proved in vitro with heterologous expressed Asm21 from Streptomyces lividans T7.
页数162
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/304
专题昆明植物所硕博研究生毕业学位论文
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赵沛基. 安丝菌素生物合成中酰胺N-糖基化和氨甲酰化的后修饰[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2008.
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