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[1]胡佳伟 李伟 张倩 刘展 乔崇 张国林 罗应刚**.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[J].应用与环境生物学报,2021,27(03):1-10.[doi:10.19675/j.cnki.1006-687x.2020.07033]
 HU Jiawei,LI Wei,ZHANG Qian,et al.Glycosyltransferase ASP OleD-catalyzed regio-selective O-glycosylation of epimedin C to synthesize acuminatoside*[J].Chinese Journal of Applied & Environmental Biology,2021,27(03):1-10.[doi:10.19675/j.cnki.1006-687x.2020.07033]
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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()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
27卷
期数:
2021年03期
页码:
1-10
栏目:
研究论文
出版日期:
2021-06-25

文章信息/Info

Title:
Glycosyltransferase ASP OleD-catalyzed regio-selective O-glycosylation of epimedin C to synthesize acuminatoside*
作者:
胡佳伟12 李伟1 张倩12 刘展12 乔崇12 张国林1 罗应刚1**
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
Author(s):
HU Jiawei12 LI Wei1 ZHANG Qian12 LIU Zhan12 QIAO Cong12 ZHANG Guolin1 & LUO Yinggang1**
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing, China
关键词:
黄酮醇糖苷糖基转移酶AcuminatosideEpimedin C酶催化合成
Keywords:
Flavonal glycosides Glycosyltransferase Acuminatoside Epimedin C Enzymatic synthesis
DOI:
10.19675/j.cnki.1006-687x.2020.07033
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--L-rhamnopyranosyl-(12)--L-rhamnopyranoside,在C-7位含O--D-glucopyranosyl-(12)--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。本研究基于糖基转移酶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,ASP OleD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-羟基区域选择性的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,可为Acuminatoside的进一步研究与开发奠定基础。
Abstract:
Acuminatoside, a flavonal glycoside isolated from the aerial part of Epimedzum acuminatum Franch, features with an O--L-rhamnopyranosyl-(12)--L-rhamnopyranoside at C-3and an O--D-glucopyranosyl-(12)--D-glucopyranoside at C-7. Despite that acuminatoside was shown to have remarkable anti-prostate cancer effect, the further research and development of acuminatoside have been limited by its low content in the plant and the low extraction yield. While epimedin C, another flavonal glycoside isolated from the plants of the Epimedzum genus, is commercially available in a large amount. Structurally viewing, regio-selective introduction of a -D-glucopyranosyl to 2-OH of the 7-O--D-glucopyranosyl of epimedin C will generate 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 glucopyranosyl group. Three amino acid residues, A242V/S132F/P67T, were mutated to afford ASP OleD that was evidenced to be more promiscuous and efficient than wild type OleD. The codon-optimized ASP OleD was synthesized, authenticated, overexpressed in Escherichia coli, and purified to homogeneity. HPLC-DAD, UPLC-HRMS, and NMR data analyses revealed that epimedin C was regio-selectively O-glycosylated at 2-OH of 7-O--D-glucopyranosyl group by ASP OleD, which generated acuminatoside efficiently. This study provides a green, convenient, efficient, and regio-selective enzymatic method for synthesis of acuminatoside.

备注/Memo

备注/Memo:

收稿日期 Received: 2020-07-20 接受日期 Accepted: 2020-08-24
中国科学院战略生物资源服务网络计划(KFJ-BRP-008)、国家重大新药专项计划(2018ZX0971001-001-006)和国家自然科学基金项目(21861142007)资助
**通讯作者 Corresponding author (E-mail: yinggluo@cib.ac.cn)
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更新日期/Last Update: 2020-09-10