Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids

Abstract

Abstract O-methyltransferases (OMTs) play essential roles in producing structural diversity and improving biological property 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 OMTs in the formation of BIAs diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an OMT (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 OMTs (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase), and 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, through integrated phylogenetic, molecular docking analysis, and site directed mutation, it suggested that residues at sites of 172, 306, 313 and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of 6-position and to expand substrate scopes. This work provides insight of O-methyltransferases in producing BIAs diversity in C. yanhusuo, and genetic elements for producing BIAs by metabolic engineering and synthetic biology.