Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy

Abstract

S-Adenosyl-L-methionine:tetrahydroprotoberberine cis-Nmethyltransferase (EC 2.1.1.122) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-Nmethylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K m values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 M, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat

“…Avenacins A-1 and B-1 are both acylated with N-methyl anthranilate, implicating anthranilate methylation in the avenacin biosynthetic pathway. Phylogenetic analysis shows that MT1 lies within a clade of methyltransferases that consists mainly of O-methyltransferase enzymes with roles in secondary metabolism ( Figure 2B; see Supplemental Data Set 1 online), although two characterized members of this clade (Limonium latifolium b-alanine-N-methyltransferase and Ruta graveolens anthranilate-N-methyltransferase) are known to have N-methyltransferase activity (Liscombe and Facchini, 2007;Rohde et al, 2008), consistent with a possible role for MT1 as an N-methyltransferase. Avenacins are synthesized in the epidermal cells of the root tip, and the expression of genes that encode the previously characterized steps in the biosynthetic pathway is restricted to these cells (Qi et al, 2006;Wegel et al, 2009;Mugford et al, 2009).…”

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…Promiscuity is a common theme among enzymes involved in plant-specialized metabolism and is one of the factors contributing to the great chemodiversity of plant secondary metabolites 41 . While broad substrate specificity of PsTNMT had been previously described 34 , we present the first experimental evidence of its acceptance of scoulerine and cheilanthifoline as substrates. Possible solutions for overcoming the problem of PsTNMT promiscuity in our system would include screening for orthologous plant enzymes with narrower substrate specificity, enzyme engineering 42 , substrate channelling and/or spatio-temporal sequestration of the reactions 43,44 .…”

“…These exact masses, and their fragmentation profiles, correspond to N-methylscoulerine and N-methylcheilanthifoline, respectively 36 . The compounds had been previously identified in opium poppy cell culture and were predicted to be a product of TNMT activity, an enzyme that had also been shown to methylate (S)-canadine 34 . To confirm that TNMT was responsible for the N-methylation of cheilanthifoline and scoulerine, we compared the Block 2 integrant strain harbouring CPR on a plasmid (strain GCY1101) with the Block 2 integrant strain harbouring both CPR and TNMT on a plasmid (strain GCY1127).…”

“…Stylopine is N-methylated to (S)-cis-N-methylstylopine by TNMT 34 . The next two biosynthetic steps are catalysed by the P450 hydroxylases (S)-cis-N-methylstylopine 14-hydroxylase (MSH) and protopine 6-hydroxylase (P6H).…”

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

“…Subsequently, (S)-canadine would be N-methylated by tetrahydroprotoberberine cis-N-methyltransferase to produce N-methylcanadine , which would be oxidized via several steps and 1-Omethylated at some point to yield noscapine. Previously, cDNAs encoding berberine bridge enzyme and tetrahydroprotoberberine cis-N-methyltransferase have been isolated from opium poppy (Facchini et al, 1996;Liscombe and Facchini, 2007), but their involvement in the noscapine pathway has not been validated. SOMT and canadine synthase cDNAs have only been isolated and characterized from berberine-producing species, including Coptis japonica (Takeshita et al, 1995;Ikezawa et al, 2003Ikezawa et al, , 2007.…”