糖基转移酶ASP OleD催化朝藿定C的区域选择性O-糖基化合成Acuminatoside  

Glycosyltransferase ASP OleD-catalyzed regioselective O-glycosylation of epimedin C to synthesize acuminatoside

作　　者：胡佳伟 李伟[1] 张倩 刘展 乔崇 张国林[1] 罗应刚[1] HU Jiawei;LI Wei;ZHANG Qian;LIU Zhan;QIAO Cong;ZHANG Guolin;LUO Yinggang(Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院成都生物研究所,成都610041 [2]中国科学院大学,北京100049

出　　处：《应用与环境生物学报》2021年第6期1427-1433,共7页Chinese Journal of Applied and Environmental Biology

基　　金：中国科学院战略生物资源服务网络计划项目(KFJ-BRP-008);国家重大新药专项计划项目(2018ZX0971001-001-006);国家自然科学基金项目(21861142007)资助。

摘　　要：Acuminatoside是从粗毛淫羊藿(Epimedzum acuminatum Franch)的地上部分中分离得到的黄酮醇四糖苷,即淫羊藿素(icaritin或anhydroicaritin)在C-3位含有O-a-L-rhamnopyranosyl-(1→2)-a-L-rhamnopyranoside,在C-7位含O-b-D-glucopyranosyl-(1→2)-b-D-glucopyranoside,该化合物具有潜在的治疗前列腺癌作用.但是从植物分离提取该化合物的收率极低(0.00008%-0.075%),这极大地限制了对Acuminatoside的深入研究、开发与综合利用.朝藿定C是大量易得的淫羊藿素三糖苷,在其C-7-葡萄糖基的2-羟基上引入另一分子葡萄糖基,即可得到Acuminatoside.基于糖基转移酶OleD催化O-糖基化的杂泛性,从NCBI数据库中获得该酶的编码基因,通过密码子优化,全基因合成具有3个氨基酸突变(A242V/S132F/P67T)的ASP OleD.将上述合成基因在大肠杆菌中进行可溶性表达,分离、纯化获得ASP OleD.以朝藿定C为底物,尿苷二磷酸葡萄糖(UDP-Glc)为糖基供体,ASPOleD为催化剂,发现一个新的酶催化反应.该酶催化反应的产物经HPLC-DAD、UPLC-HRMS及NMR数据分析确定为Acuminatoside.本研究实现了基于糖基转移酶OleD催化O-糖基化的杂泛性,在朝藿定C的C-7-葡萄糖基的2-羟基区域选择性地引入另一分子葡萄糖基,得到Acuminatoside,提供了一种绿色环保、操作简单、选择性高的酶催化转化合成Acuminatoside方法.Acuminatoside, a flavanol glycoside extracted from the aerial part of Epimedium acuminatum Franch,features an O-a-L-rhamnopyranosyl-(1→2)-a-L-rhamnopyranoside at C-3 and an O-b-D-glucopyranosyl-(1→2)b-β-D-glucopyranoside at C-7. Despite acuminatoside’s remarkable anti-prostate cancer effect, further research and development of it has been limited by its low concentration in E. acuminatum and its low extraction yield. Epimedin C, another flavanol glycoside extracted from plants of the Epimedium genus, is commercially available in large amounts. Regioselective introduction of a b-D-glucopyranosyl to 2-OH of the 7-O-b-D-glucopyranosyl of epimedin C generates acuminatoside. Herein, we optimized the codon usage of OleD, a glucopyranosyl transferase involved in the inactivation of macrocyclic glycoside antibiotic oleandomycin in Streptomyces antibioticus. OleD showed promiscuous O-glycosylation for various natural compounds, particularly for the 2-OH of the glucopyranosyl group.Three amino acid residues, A242 V/S132 F/P67 T, were mutated to attain ASP OleD, which was shown to be more promiscuous and efficient than the OleD wild type. The codon-optimized ASP OleD was synthesized, validated,overexpressed in Escherichia coli, and purified to homogeneity. HPLC-DAD, UPLC-HRMS, and NMR data analyses revealed that epimedin C was regioselectively O-glycosylated at 2-OH of the 7-O-b-D-glucopyranosyl group by ASP OleD, which generated acuminatoside efficiently. This study provides an environmentally friendly, convenient,efficient, and regioselective enzymatic method for the synthesis of acuminatoside.

关 键 词：黄酮醇糖苷 糖基转移酶 Acuminatoside Epimedin C 酶催化合成 

分 类 号：O629.13[理学—有机化学]