Liu Rong, Wu Xiaoyi, Jiang Zhouqian, Liu Xuan, Zhang Yifeng, Zhao Huan, Gao Jie, Gao Wei, Hu Yating, Huang Luqi
School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China.
National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing 100700, China.
J Nat Prod. 2024 Sep 27;87(9):2160-2169. doi: 10.1021/acs.jnatprod.4c00298. Epub 2024 Aug 27.
Notoginsenosides are important bioactive compounds from (Burk.) F. H. Chen, most of which have xylose in their sugar chains. However, the xylosyltransferases involved in the generation of notoginsenosides remain poorly understood, posing a bottleneck for further study of the biosynthesis of notoginsenosides. In this work, a new xylosyltransferase gene, (named ), was identified from , which has a distinct sequence and could catalyze the 2'- glycosylation of ginsenosides Rh1 and Rg1 to produce notoginsenosides R2 and R1, respectively. We first characterized the optimal conditions for the UGT57 activity and its enzymatic kinetic parameters, and then, molecular docking and site-directed mutagenesis were performed to elucidate the catalytic mechanism of UGT57. Combined with the results of site-directed mutagenesis, Glu26, Ser266, Glu267, Trp347, Ser348, and Glu352 in UGT57 were identified as the key residues involved in 2'- glycosylation of C-6 O-Glc, and UGT57 and UGT57 could significantly improve the catalytic activity of UGT57. These findings not only provide a new xylosyltransferase gene for augmenting the plant xylosyltransferase database but also identify the pivotal sites and catalytic mechanism of the enzyme, which would provide reference for the modification and application of xylosyltransferases in the future.
三七皂苷是五加科植物三七（Panax notoginseng (Burk.) F. H. Chen）中的重要生物活性化合物，其中大多数在其糖链中含有木糖。然而，参与三七皂苷生成的木糖基转移酶仍知之甚少，这成为三七皂苷生物合成进一步研究的瓶颈。在这项工作中，从三七中鉴定出一个新的木糖基转移酶基因（命名为UGT57），其具有独特的序列，可催化人参皂苷Rh1和Rg1的2'-糖基化反应，分别生成三七皂苷R2和R1。我们首先表征了UGT57活性的最佳条件及其酶动力学参数，然后进行分子对接和定点诱变以阐明UGT57的催化机制。结合定点诱变结果，确定UGT57中的Glu26、Ser266、Glu267、Trp347、Ser348和Glu352是参与C-6 O-Glc 2'-糖基化反应的关键残基，并且UGT57和UGT57可显著提高UGT57的催化活性。这些发现不仅为扩充植物木糖基转移酶数据库提供了一个新的木糖基转移酶基因，还鉴定了该酶的关键位点和催化机制，这将为未来木糖基转移酶的修饰和应用提供参考。
