Catalytic promiscuity of -methyltransferases from leading to the structural diversity of benzylisoquinoline alkaloids.

-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, , 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 -methyltransferases in the formation of BIA diversity. Seven genes encoding -methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an -methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4'- and 6--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, ()-scoulerine. Two -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--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 -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 -methyltransferases in producing BIA diversity in and genetic elements for producing BIAs by metabolic engineering and synthetic biology.