Molecular Cloning and Characterization of CoclaurineN-Methyltransferase from Cultured Cells of Coptis japonica

Abstract

S-Adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5-and 3-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity. S-Adenosyl-L-methionine (AdoMet)1 :coclaurine N-methyltransferase (CNMT) (1-3) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids (Fig. 1). This enzyme is thought to be important because N-methylation of coclaurine strongly enhances the 4Ј-O-methylation activity of 3Ј-hydroxy-N-methylcoclaurine 4Ј-O-methyltransferase and enables the sequential metabolic conversion of substrates (4, 5). Furthermore, the enzymatic activity at this important step is rather low relative to the entire biosynthetic pathway (1, 6 -8). Thus, we purified CNMT and characterized its properties. Previous studies have clearly indicated that CNMT is non-stereospecific and has broad substrate specificity; Coptis enzyme methylated even simple dihydroxyisoquinoline alkaloids.Whereas several O-methyltransferases have been characterized at the molecular level (4, 9 -12), there have been very few molecular studies of N-methyltransferases in secondary metabolite biosynthesis in plants (13,14). The differences in the primary structures, including the S-adenosyl-L-methionine binding site of NMTs and OMTs reported to date, suggest that molecular isolation of CNMT based on the structural similarity of methyltransferases would be very difficult. Thus, we adapted the conventional strategy to isolate cDNA based on the amino acid sequence of purified enzyme. Whereas our purified CNMT fraction still contained two protein bands of about 45 kDa, careful inspection of the chromatographic behavior of the proteins and enzyme activity suggested that the slow moving 45-kDa polypeptide would encode CNMT (1). Based on this observation, we determined the N-terminal amino acids, isolated the corresponding cDNA fragment, and finally the fulllength cDNA. The nucleotide sequence of cDNA suggested that the deduced amino acid sequence showed some simi...

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

“…The glycine-rich sequence containing Gly-72 and Gly-74 ((E/ D)XGXGXG), often referred to as motif I (Fig. 3), is highly conserved in many AdoMet-dependent methyltransferases and is also present in TNMT, CNMTs, and MtPcaA (6,51,53). Structure and sequence conservation shows that MtPcaA exhibits a structural fold most similar to the small molecule subclass of methyltransferases (53).…”

“…The recently reported molecular cloning of cDNAs encoding CNMT from P. somniferum (8), T. flavum (29), and C. japonica (6) provided query sequences to identify a full-length cDNA encoding opium poppy TNMT in an EST data base by in silico mining. We previously used this approach to identify cDNAs encoding opium poppy NCS isoforms with T. flavum NCS as the query sequence (4).…”

“…1). CNMT has been purified (28), and corresponding cDNAs have been isolated from Coptis japonica (6), Thalictrum flavum (29), and P. somniferum (8). TNMT protein has been isolated from Eschscholzia californica and Corydalis vaginans cell cultures (30) and purified from Sanguinaria canadensis cell cultures (31).…”

Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications

“…3). 49,50) Since the biosynthetic enzymes in the berberine biosynthetic pathway are highly expressed in C. japonica cells that produce large amounts of berberine, several biosynthetic enzymes in the benzylisoquinoline alkaloid pathway, including scoulerine 9-O-methyltransferase (SMT), 55,56) norcoclaurine 6-O-methyltransferase (6OMT)/3 0 -hydroxy-N-methylcoclaurine 4 0 -O-methyltransferase (4 0 OMT), 57) and coclaurine N-methyltransferase (CNMT), 58,59) have been purified, and their enzymological properties have been determined. The isolation of their cDNAs based on the amino acid sequences of purified enzymes has provided molecular tools for investigating enzymes that are structurally similar but difficult to purify by ordinary column chromatography (e.g., 6OMT and 4 0 OMT).…”

Improvement of Reticuline Productivity from Dopamine by Using EngineeredEscherichia coli

“…THP is then converted to reticuline by three sequential methylation steps. [4][5][6] An Escherichia coli strain harboring this biosynthetic pathway has successfully produced reticuline from dopamine, although its conversion efficiency was less than 1.3%: 33 mM (11 mg/L) of reticuline was produced from 5 mM dopamine (note that one molecule of reticuline was synthesized from two molecules of dopamine).…”