Abstract
Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) 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 R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 and 13 μm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT 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 N. nucifera.
Highlights
Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits
Fifteen alkaloids were identified based on their retention times and collisioninduced dissociation (CID) spectra compared with available authentic standards or previously published data. 1-Benzylisoquinoline and aporphine alkaloids were prevalent in the leaves, whereas bisbenzylisoquinolines were most abundant in the embryos
We have shown that two cDNAs from sacred lotus efficiently catalyze the regiospecific 6-O-methylation of norcoclaurine to coclaurine (NnOMT1) and 7-O-methylation of coclaurine and N-methylcoclaurine to norarmepavine and armepavine, respectively (NnOMT5)
Summary
Alkaloids corresponding to the leaf (folded and unfolded developmental stages), rhizome, root, and embryo (lotus plumule) of two N. nucifera cultivars, referred as Pink and White because of their distinct floral coloration, were extracted and analyzed by mass spectrometry (MS) (Fig. 2, b and c, Table S1). Reaction products corresponding to the 6-O-methylation of norcoclaurine and norlaudanosoline (coclaurine (m/z 286) and 6-O-methylnorlaudanosoline (m/z 302), respectively) and the 7-O-methylation of N-methylcoclaurine (armepavine (m/z 314)), were unambiguously identified by comparison with available authentic standards. O-methyltransferase activity, and OMT gene expression were measured in leaves, rhizome, roots, and embryos for two sacred lotus varieties. Alkaloid content was determined for norcoclaurine, coclaurine, N-methylcoclaurine, and armepavine, for all of which authentic standards were available (Fig. S6) In both the Pink and White varieties, 1-benzylisoquinoline intermediates were predominantly detected in the leaves (Fig. 6a, Table S5). In the White variety, norcoclaurine was the most abundant alkaloid, with folded leaves accumulating 7-, 70-, and 1400-fold higher levels compared with the embryo, rhizome, and root, respectively. The levels of all five NnOMT transcripts were similar in the embryos of both varieties
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