O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquino-line 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.

Junling Bu;Xiuhua Zhang;Qishuang Li;Ying Ma;Zhimin Hu;Jian Yang;Xiuyu Liu;Ruishan Wang;Xiang Jiao;Tong Chen;Changjiangsheng Lai;Guanghong Cui;Jinfu Tang;Yu Kong;Lei Yang;Sheng Lin;Yun Chen;Juan Guo;Luqi Huang

State 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;School of Pharmacy,Henan University of Chinese Medicine,No.156 Jinshuidong Road,Zhengzhou 450008,China;Department of Biology and Biological Engineering,Chalmers University of Technology,Kemiv?gen 10,SE41296,Gothenburg,Sweden;Shanghai Key Laboratory of Plant Functional Genomics and Resources,Shanghai Chenshan Botanical Garden,Shanghai,201602,China;Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing,Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China

园艺研究（英文）

[1]Junling Bu;Xiuhua Zhang;Qishuang Li;Ying Ma;Zhimin Hu;Jian Yang;Xiuyu Liu;Ruishan Wang;Xiang Jiao;Tong Chen;Changjiangsheng Lai;Guanghong Cui;Jinfu Tang;Yu Kong;Lei Yang;Sheng Lin;Yun Chen;Juan Guo;Luqi Huang-.Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids)[J].园艺研究（英文）,2022(01):441-454

promiscuity,benzylisoquinoline,benzylisoquino,BIAs,CyOMT2,CyOMT6,tetrahydroprotoberberines,scoulerine,CyOMT5,CyOMT7

Catalytic,methyltransferases,from,Corydalis,yanhusuo,leading,structural,diversity,alkaloids,play,essential,roles,producing,improving,biological,properties,plants,In,this,study,used,traditional,Chinese,medicine,analgesic,effects,its,active,compounds,was,employed,analyze,catalytic,characteristics,formation,Seven,genes,encoding,were,cloned,functionally,characterized,using,seven,potential,substrates,Specifically,highly,efficient,activity,both,methylations,found,perform,two,sequential,positions,intermediate,Two,wide,preferential,named,addition,results,integrated,phylogenetic,molecular,docking,analysis,directed,mutation,suggested,that,residues,sites,are,important,enzyme,related,Cys,proved,promote,expand,scopes,This,work,provides,insight,into,elements,by,metabolic,engineering,synthetic,biology

0.408716