Molecular cloning of columbamine O‐methyltransferase from cultured Coptis japonica cells

Abstract

To identify all of the O-methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica cells, we sequenced 1014 cDNA clones isolated from high-alkaloidproducing cultured cells of C. japonica. Among them, we found all three reported O-methyltransferases and an O-methyltransferase-like cDNA clone (CJEST64). This cDNA was quite similar to S-adenosyl-L-methionine:coclaurine 6-O-methyltransferase and S-adenosyl-L-methionine:isoflavone 7-O-methyltransferase. As S-adenosyl-L-methionine:columbamine O-methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O-methyltransferase. The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…Similarly, (S)-sinactine is formed from (S)-scoulerine by (1) the addition of a methylenedioxy bridge by cheilanthifoline synthase, also a member of the CYP719A subfamily (Díaz Chávez et al 2011;Ikezawa et al 2009), followed by (2) 2-O-methylation of cheilanthifoline by an enzyme not yet characterized from opium poppy. A protoberberine 2-O-methyltransferase, columbamine O-methyltransferase, has been reported from meadow rue (Coptis japonica) (Morishige et al 2002). Alternatively, cheilanthifoline can be oxidized to stylopine by another CYP719A, stylopine synthase (SPS) (Ikezawa et al 2007;Díaz Chávez et al 2011).…”

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

“…All known OMTs involved in BIA metabolism exhibit extensive amino acid homology and form a distinctive clade with respect to OMTs involved in other metabolic pathways, such as flavonoid metabolism . Characterized OMTs involved in BIA biosynthesis include norcoclaurine 6-O-methyltransferase (6OMT; Sato et al, 1994;Morishige et al, 2000;Ounaroon et al, 2003), 39-hydroxy-N-methylcoclaurine 49-O-methyltransferase (49OMT; Morishige et al, 2000;Ziegler et al, 2005), reticuline 7-O-methyltransferase (7OMT; Ounaroon et al, 2003), norreticuline 7-O-methyltransferase (N7OMT; Pienkny et al, 2009), scoulerine-9-O-methyltransferase (SOMT; Takeshita et al, 1995), and columbamine O-methyltransferase (CoOMT; Morishige et al, 2002). Functional homologs for all enzymes except SOMT and CoOMT have been identified in opium poppy (Facchini and De Luca, 2008).…”

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

“…Several S-adenosyl-L-methionine (AdoMet)-dependent Omethyltransferases involved in the biosynthesis of a variety of plant secondary metabolites have been characterized at the molecular level (7,11,12,19,20) and structural levels (21)(22)(23). However, few studies have focused on the molecular biochemical characterization of AdoMet-dependent N-methyltransferases (24 -27).…”