红花黄酮类糖基转移酶基因CtUGT49功能表征及酶学特性分析  

Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius

作　　者：蔡鑫博 刘楠 李佳[1] 刘荣 骆云峰 张逸风[3] 王家典[1] 吴晓毅[1] 黄璐琦[3] CAI Xin-bo;LIU Nan;LI Jia;LIU Rong;LUO Yun-feng;ZHANG Yi-feng;WANG Jia-dian;WU Xiao-yi;HUANG Lu-qi(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;School of Chinese Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]天津中医药大学中药学院,天津301617 [3]中国中医科学院,中药资源中心,道地药材品质保障与资源持续利用全国重点实验室,北京100700

出　　处：《中国中药杂志》2023年第24期6624-6634,共11页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2020YFA0908000);中央本级重大增减支项目(2060302);山西德元堂横向课题(DYTKY180725)。

摘　　要：红花作为传统的活血化瘀中药,具有抗肿瘤、抗炎、免疫调节等药理活性。黄酮糖苷是红花植物的主要生物活性组分,糖基转移酶作为活性糖苷化合物生物合成的下游后修饰酶,具有重要的研究意义。该研究基于红花转录组数据分析,挖掘到一条糖基转移酶基因CtUGT49,对其进行了系统的生物信息学分析和酶学性质考察。该基因开放阅读框(ORF)长度为1416 bp,编码471个氨基酸残基,相对分子质量约为52 kDa。系统进化分析表明,该糖基转移酶属于UGT73家族。体外酶促结果显示,CtUGT49可催化柚皮素查尔酮生成樱桃苷和南酸枣苷;催化根皮素生成的主产物为根皮苷和三叶苷,并且具有良好的底物宽泛性。通过蛋白异源表达与纯化得到较为纯净的重组蛋白CtUGT49后,进行CtUGT49催化柚皮素查尔酮的酶学性质考察。结果显示,CtUGT49催化反应的最适pH为7.0,最适温度为37℃,反应8 h后,底物转化率达到最高。采用米氏方程计算CtUGT49的酶活常数Km=209.90μmol·L^(-1),kcat=48.36 s^(-1)。该研究发现的新颖糖基转移酶CtUGT49对于丰富糖基化工具酶库具有重要意义,且能为进一步解析红花黄酮糖苷的糖基化过程奠定基础。Carthami Flos,as a traditional blood-activating and stasis-resolving drug,possesses anti-tumor,anti-inflammatory,and immunomodulatory pharmacological activities.Flavonoid glycosides are the main bioactive components in Carthamus tinctorius.Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds.This study reported a flavonoid glycosyltransferase CtUGT49 from C.tinctorius based on the transcriptome data,followed by bioinformatic analysis and the investigation of enzymatic properties.The open reading frame(ORF)of the gene was 1416 bp,encoding 471 amino acid residues with the molecular weight of about 52 kDa.Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family.According to in vitro enzymatic results,CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin,and catalyze phloretin to phlorizin and trilobatin,exhibiting good substrate versatility.After the recombinant protein CtUGT49 was obtained by heterologous expression and purification,the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated.The results showed that the optimal pH value for CtUGT49 catalysis was 7.0,the optimal temperature was 37℃,and the highest substrate conversion rate was achieved after 8 h of reaction.The results of enzymatic kinetic parameters showed that the Km value was 209.90μmol·L^(-1) and kcat was 48.36 s^(-1 )calculated with the method of Michaelis-Menten plot.The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C.tinctorius.

关 键 词：红花 糖基转移酶 功能鉴定 酶学性质 

分 类 号：R285[医药卫生—中药学]