The UGT74L2 of Andrographis paniculata catalyzes phloretin to produce trilobatin and its enzymatic study
10.16438/j.0513-4870.2022-0936
- VernacularTitle:催化根皮素生成三叶苷的来源于穿心莲的糖基转移酶UGT74L2及其酶学研究
- Author:
Shu-fu SUN
1
,
2
;
Yu-ping TAN
3
;
Yin-yin JIANG
3
;
Ke-ke ZHANG
3
;
Jian YANG
3
;
Liang-ping ZHA
1
;
Jin-fu TANG
3
Author Information
1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
2. State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
3. State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
- Publication Type:Research Article
- Keywords:
italic>Andrographis paniculata;
trilobatin;
glycosyltransferase;
enzyme kinetics;
molecular docking simulation
- From:
Acta Pharmaceutica Sinica
2023;58(3):789-799
- CountryChina
- Language:Chinese
-
Abstract:
The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca2+, Mn2+ and Co2+) showed inhibitory effect on the activity of UGT74L2, while Mg2+ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the Km value was 29.84 μmol·L-1, the kcat was 0.02 s-1, and the kcat·Km-1 was 572.6 mol-1·s-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.
