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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...

Section: Discussionmentioning

confidence: 99%

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

Choi¹,

Morishige

Shitan

et al. 2002

146

mentioning

confidence: 88%

“…Although plant class I OMTs share only 15% sequence identity with their animal counterparts, the cation dependence, important catalytic residues, and topology are all conserved between the plant and animal enzymes (23). The structural similarities of plant class I OMTs with animal catechol OMTs are in contrast to the apparent differences between class I and class II OMTs of plants, as recently elaborated by crystal structures of two alfalfa class II OMTs, which are involved in methylation of flavonoids and caffeic acid (24,25).Mesembryanthemum crystallinum, the common ice plant, is a member of the herbaceous family Aizoaceae of the Caryophyl-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”

mentioning

confidence: 92%

A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

Ibdah

Zhang

Schmidt

et al. 2003

114

149

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-L-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg 2؉ -dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg 2؉ -dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant Omethyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.Methylation by S-adenosyl-L-methionine (AdoMet) 1 dependent O-methyltransferases (OMTs) (EC 2.1.1) is a common modification in natural product biosynthesis. Site-specific O-methylation modulates the physiological properties of such compounds and reduces the chemical reactivity of phenolic hydroxyl groups (1). In plants, O-methylation is also required for monolignol biosynthesis and accounts for the structural differences and properties of lignin, which next to cellulose is the most prominent polymer on earth (2).Plant OMTs can be categorized into two major classes (3). Class I includes a group of low molecular weight (23,000 to 27,000) and Mg 2ϩ -dependent OMTs, whereas class II consists of higher molecular weight OMTs of about 38,000 to 43,000 that do not require Mg 2ϩ for catalytic activity. Prominent class II members include caffeic acid, flavonoid, coumarin, and alkaloid OMTs (4 -6). Within the class I OMTs, a group of small caffeoyl coenzyme A OMTs (CCoAOMTs) have been suggested to be key enzymes in the biosynthesis of monolignols, the precursors of gymnosperm and angiosperm lignins (7,8). In angiosperms, this task may also be performed by class II OMTs that are specific for caffeic acid, caffeyl aldehyde, or caffeyl alcohol (COMT) (9 -11). This apparent redundancy results in a cell-and tis...

“…On the other hand, results did not change when data bases were scanned using the sequence of each domain independently. Fold recognition using the mGenThreader method (20 -23) gave rise to high confidence results for two proteins of known three-dimensional structure, chalcone O-methyltransferase and isoflavone O-methyltransferase from Medicago sativa (32). Although the primary sequences of these proteins are not very closely related, their secondary structures correlate very well with the one predicted for MtmMII (Fig.…”

Section: Resultsmentioning

confidence: 49%

MtmMII-mediated C-Methylation during Biosynthesis of the Antitumor Drug Mithramycin Is Essential for Biological Activity and DNA-Drug Interaction

Rodrı́guez

Quirós

Salas

2004

The antitumor drug mithramycin consists of a polyketide chromophore glycosylated with a trisaccharide and a disaccharide. Two post-polyketide methylations take place during mithramycin biosynthesis. One of these methylations has been shown to be very relevant for biological activity, that is the introduction of a methyl group at aromatic C-7. We have purified to 282-fold the MtmMII methyltransferase involved in this reaction. The protein is a monomer, and results from kinetic studies were consistent with a model for the enzyme acting via a compulsory order mechanism. The enzyme showed high substrate specificity and was unable to operate on structurally closely related molecules. Structural predictions suggest that the molecule is integrated by two domains, an essentially all ␣-amino domain and an ␣/␤-carboxyl domain displaying a variation of a Rossmann-fold containing the cofactor binding site. Although 7-demethyl-mithramycin did not show any biological activity, it was able to reach the nucleus of eukaryotic cells, with subsequent binding to DNA. Mithramycin and 7-demethylmithramycin were able to form similar complexes with Mg 2؉ , although their respective DNA binding isotherms were very different. The dinucleotide binding model fit well the isotherms recorded for both compounds, predicting that the C-7 methyl group was essential for high affinity binding to specific GC and CG sequences. Considering previous structural studies, we propose that this effect is performed by positioning the group in the floor of the minor groove, allowing the interaction with the third sugar moiety of the trisaccharide, D-mycarose, which is involved in sequence selectivity.