Isolation and Characterization of Reticuline N-Methyltransferase Involved in Biosynthesis of the Aporphine Alkaloid Magnoflorine in Opium Poppy

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.

An N-methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production

“…The PaNMT pH optimum of 9.0 is in agreement with the more alkaline pH optima determined for recombinant BIA NMTs, which range from 7.0 to 9.0 (24,27,28). Given that enzymes likely evolve for efficient catalysis under conditions in their subcellular environment (37), a high pH optimum might suggests that PaNMT functions in a relatively alkaline environment, such as can be found in peroxisomes, the mitochondrial matrix, or plastidial stroma (38).…”

“…Knowing that (pseudo)ephedrine pathway intermediates are reported to accumulate at substantially higher levels in mature E. sinica stems (12), and given the typical confinement of specialized metabolic pathways to specific cell types or subcellular compartments, the concentration of PaNMT substrates could be high enough to ensure that the low substrate affinities of the enzyme do not impede pathway flux. Relative to the phenylalkylamine substrates, PaNMT showed a much higher affinity for SAM (140 M; Table 1), which was comparable with K m values reported for recombinant PsRNMT (160 M) (24) and CjCNMT (390 M) (27). Other BIA NMTs had substantially better affinity for SAM, with K m values between 43 (TfC-NMT) and 1.2 M (ScTNMT) (28,29,33,34).…”

“…In contrast, the most closely related and functionally characterized enzymes to PaNMT showed higher affinities for their respective substrates. For example, recombinant CjCNMT, PsTNMT, PsRNMT, and TfPavNMT were reported to display K m values between 0.6 and 160 M (24,(27)(28)(29). In contrast, enzymes similar to PaNMT in terms of substrate profile exhibit comparably low substrate affinities.…”

“…To compare with PaNMT, three previously characterized BIA NMTs (23,24) were heterologously expressed in E. coli, affinity-purified ( Fig. S5), and subjected to the same determination of substrate range (Fig.…”

Gloriosa superba and Colchicum autumnale multi-tissue transcriptome analysis for colchicine pathway and rhizome development candidate gene identification

“…PNMT is also considered a possible alternative reference sequence due to its capability of adding a methyl group to (S)-tetrahydropapaverine where the nitrogen is present, which is similar to the colchicine pathway mechanism. These putative transcripts also share homology with reticuline NMT involved in biosynthesis of the aporphine alkaloid magnoflorine in opium poppy roots [78].…”