Isolation and characterization of two -methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus ().

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus () is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant enantiomeric conformation have been isolated from , its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two -methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6--methyltransferase (6OMT) accepting both and substrates, whereas NnOMT5 is mainly a 7--methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that -methylation precedes BIA formation from 1-benzylisoquinoline intermediates. values for NnOMT1 and NnOMT5 were 20 and 13 μm for ()-norcoclaurine and ()--methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant .