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Article|06 Jul 2022|OPEN
Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids
Junling Bu1 ,† , Xiuhua Zhang1 ,† , Qishuang Li1 , Ying Ma1 , Zhimin Hu1 , Jian Yang1 , Xiuyu Liu2 , Ruishan Wang1 , Xiang Jiao3 , Tong Chen1 , Changjiangsheng Lai1 , Guanghong Cui1 , Jinfu Tang1 , Yu Kong4 , Lei Yang4 , Sheng Lin5 , Yun Chen3 , Juan Guo1 , and Luqi Huang,1 ,
1State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16 South Side Street, Dongzhimen, Beijing 100700, China
2School of Pharmacy, Henan University of Chinese Medicine, No. 156 Jinshuidong Road, Zhengzhou 450008, China
3Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE41296, Gothenburg, Sweden
4Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China
5Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China
*Corresponding author. E-mail: guojuan@wbgcas.cn,huanglq@cacms.cn
Both authors contributed equally to the study.

Horticulture Research 9,
Article number: uhac152 (2022)
doi: https://doi.org/10.1093/hr/uhac152
Views: 80

Received: 28 Jan 2022
Accepted: 27 Jun 2022
Published online: 06 Jul 2022

Abstract

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4′- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.