Wang Rui-Shan, Wang Sheng, Liang Jiu-Wen, Li Tan, Zhou Li, Zhan Zhi-Lai, Wan Xiu-Fu, Kang Chuan-Zhi, Guo Lan-Ping
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China.
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China Guangdong Provincial Research Center on Good Agricultural Practice & Comprehensive Agricultural Development Engineering Technology of Cantonese Medicinal Materials, College of Traditional Chinese Medicine, Guangdong Pharmaceutical University Guangzhou 510006, China.
Zhongguo Zhong Yao Za Zhi. 2021 Jan;46(1):86-93. doi: 10.19540/j.cnki.cjcmm.20200827.101.
Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.
咖啡酸及其低聚物是中药紫草的主要水溶性活性成分。这些化合物具有多种生物活性，如抗菌、抗氧化、心血管保护、肝脏保护、抗肝纤维化、抗病毒和抗癌活性。植物中的苯丙烷类途径负责咖啡酸及其低聚物的生物合成。糖基化可以改变苯丙烷类的溶解度、稳定性和潜在毒性，还会影响其区室化和生物活性。鉴于去糖基化在苯丙烷类稳态调节中发挥的重要作用，推测咖啡酸及其低聚物的生物合成受相关尿苷二磷酸糖基转移酶（UGTs）的控制。通过对新疆紫草转录组的数据挖掘，我们克隆了15个全长推定UGT基因。使用原核系统进行重组表达后，对推定UGT的粗酶液进行体外对咖啡酸和迷迭香酸的糖基化活性检测。在超高效液相色谱-质谱分析中，AeUGT_01、AeUGT_02、AeUGT_03、AeUGT_04和AeUGT_10能够使咖啡酸和/或迷迭香酸糖基化，产生不同的单糖基化和/或双糖基化产物。所鉴定的UGT彼此亲缘关系较远，在系统发育树中分为不同的分支。基于每个鉴定的UGT与其所在分支中的成员表现出底物或催化相似性的观察结果，我们推测对咖啡酸和/或迷迭香酸的糖基化能力在不同分支中是独立进化的。从新疆紫草中鉴定咖啡酸和迷迭香酸UGT有助于更深入了解咖啡酸低聚物的生物合成及其调控。此外，这些UGT可用于咖啡酸和迷迭香酸的区域特异性糖基化，以生产具有潜在治疗应用价值的生物活性化合物。
