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

Abstract

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.

The CYP80A and CYP80G Are Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in the Sacred Lotus (Nelumbo nucifera)

“…Two OMTs in N. nucifera were identified to supplement the synthesis of monobenzylisoquinoline alkaloids [14]. Subsequently, OMTs were isolated and identified, and high-yield mutants were obtained by modification [15]. Using digital gene expression technology, Yang et al [16] demonstrated that corytuberine synthase participates in the aporphine alkaloid biosynthetic pathway, and (S)-N-methylcoclaurine and (S)-reticuline are the precursors of bisbenzylisoquinoline as well as aporphine alkaloids, respectively.…”

Identification of One O-Methyltransferase Gene Involved in Methylated Flavonoid Biosynthesis Related to the UV-B Irradiation Response in Euphorbia lathyris

“…The process of methylation is catalyzed using a methyltransferase that transfers a methyl group from S-adenosyl-L-methionine to an appropriate substrate. O-methylation occurs as one vital step in many plants' secondary metabolic biosynthesis, which involves flavonoids [9,10], terpenoids [11,12], and alkaloids [13,14]. O-methyltransferase is responsible for the formation of the structural diversity of flavonoids through methylation in plants.…”