Functional characterization and key residues engineering of a regiopromiscuity <i>O</i>-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in <i>Nelumbo nucifera</i>

Abstract

Lotus (<i>Nelumbo nucifera</i>), 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 <i>N. nucifera</i> have been isolated and characterized. In the present study we identified the regiopromiscuity of an <i>O</i>-methyltransferase, designated NnOMT6, isolated from <i>N. nucifera</i>; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-<i>O</i>/7-<i>O</i>, aporphine skeleton 6-<i>O</i>, phenylpropanoid 3-<i>O</i>, and protoberberine 2-<i>O</i>. We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that residues D316, N130, L135, N176A, D269, and E328 were critical for BIA <i>O</i>-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, this is the first study to report the <i>O</i>-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in <i>N. nucifera</i>. The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen <i>O</i>-methyltransferase activity in plants.