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Article|23 Jun 2022|OPEN
Identification and characterization of two Isatis indigotica O-methyltransferases methylating C-glycosylflavonoids 
Yuping Tan1,2 , Jian Yang1 , Yinyin Jiang1 , Shufu Sun1,3 , Xiaoyan Wei1,4 , Ruishan Wang1 , Junling Bu1 , Dayong Li5 , Liping Kang1 , Tong Chen1 and Juan Guo1 , Guanghong Cui1 , Jinfu Tang1 , , Luqi Huang,1,2 ,
1State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
2School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 117004, China
3School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
4College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China
5National Engineering Research Center for Vegetables, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Science, Beijing 100097, China
*Corresponding author. E-mail:,

Horticulture Research 9,
Article number: uhac140 (2022)
Views: 99

Received: 30 Dec 2021
Accepted: 14 Jun 2022
Published online: 23 Jun 2022


Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3′-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3′-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3′-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.