Abstract
Benzylisoquinoline alkaloids are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties associated with numerous structural scaffolds and diverse functional group modification. N-Methylation is one of the most common tailoring reactions, yielding tertiary and quaternary pathway intermediates and products. Two N-methyltransferases accepting (i) early 1-benzylisoquinoline intermediates possessing a secondary amine and leading to the key branch-point intermediate (S)-reticuline and (ii) downstream protoberberines containing a tertiary amine and forming quaternary intermediates destined for phthalideisoquinolines and antimicrobial benzo[c]phenanthridines were previously characterized. We report the isolation and characterization of a phylogenetically related yet functionally distinct N-methyltransferase (NMT) from opium poppy (Papaver somniferum) that primarily accepts 1-benzylisoquinoline and aporphine substrates possessing a tertiary amine. The preferred substrates were the R and S conformers of reticuline and the aporphine (S)-corytuberine, which are proposed intermediates in the biosynthesis of magnoflorine, a quaternary aporphine alkaloid common in plants. Suppression of the gene encoding reticuline N-methyltransferase (RNMT) using virus-induced gene silencing in opium poppy resulted in a significant decrease in magnoflorine accumulation and a concomitant increase in corytuberine levels in roots. RNMT transcript levels were also most abundant in roots, in contrast to the distribution of transcripts encoding other NMTs, which occur predominantly in aerial plant organs. The characterization of a third functionally unique NMT involved in benzylisoquinoline alkaloid metabolism will facilitate the establishment of structure-function relationships among a large group of related enzymes.
Highlights
As with all BIAs, the biosynthesis of magnoflorine begins with the condensation of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde, which yields the first committed pathway intermediate (S)-norcoclaurine (Fig. 1)
Isolation and Phylogenetic Analysis—Opium poppy transcripts encoding two putative NMTs were identified in stem and root transcriptome databases based on their predicted amino acid sequence identity with coclaurine N-methyltransferase (CNMT) and tetrahydroprotoberberine cis-N-methyltransferase (TNMT) (51 and 61%, respectively)
We have shown that an uncharacterized cDNA from opium poppy with homology to known NMTs involved in BIA metabolism efficiently catalyzes the previously unreported N-methylation of both S and R conformers of the 1-benzylisoquinoline alkaloid reticuline yielding the corresponding enantiomers of tembetarine, a quaternary ammonium compound and putative intermediate in the biosynthesis of magnoflorine
Summary
As with all BIAs, the biosynthesis of magnoflorine begins with the condensation of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde, which yields the first committed pathway intermediate (S)-norcoclaurine (Fig. 1). We report the isolation and characterization of a cDNA encoding reticuline N-methyltransferase (RNMT) from opium poppy (Papaver somniferum), and we demonstrate a physiological role for the enzyme in the biosynthesis of magnoflorine in the plant.
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