Functional characterization and key residues engineering of a regio-promiscuity O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera


 Lotus (Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study, we identified the regiopromiscuity of the O-methyltransferase designated as NnOMT6 isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O, aporphine skeleton 6-O, phenylpropanoid 3-O, and protoberberine 2-O. We further probed the key residues for affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that the residues D316, N130, L135, N176A, D269, and E328 were critical for BIA O-methyltransferase activities; furthermore, N323A, a mutant of NnOMT6, demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6. To the best of our knowledge, the present is the first to report the O-methyltransferase activity of aporphine skeleton without benzyl moiety substitutions in N. nucifera. The study findings provided biocatalysts for the semisynthesis of related medical compounds and gave insights into protein engineering to strengthen the O-methyltransferase activity in plants.