title: Molecular cloning and characterization of coclaurine N-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.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Kum-Boo
authors.initials: KB
authors.lastname: Choi
authors.forename: Takashi
authors.initials: T
authors.lastname: Morishige
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Shitan
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Yazaki
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C430217:S-adenosyl-L-methionine coclaurine N-methyltransferase