title: Purification and characterization of coclaurine N-methyltransferase from cultured Coptis japonica cells.
abstract: S-Adenosyl-L-methionine (SAM): coclaurine N-methyltransferase (CNMT), which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. was purified 340-fold from Coptis japonica cells in 1% yield to give an almost homogeneous protein. The purified enzyme, which occurred as a homotetramer with a native Mr of 160 kDa (gel-filtration chromatography) and a subunit Mr of 45 kDa (SDS-polyacrylamide gel electrophoresis), had an optimum pH of 7.0 and a pI of 4.2. Whereas (R)-coclaurine was the best substrate for enzyme activity, Coptis CNMT had broad substrate specificity and no stereospecificity CNMT methylated norlaudanosoline, 6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. The enzyme did not require any metal ion. p-Chloromercuribenzoate and iodoacetamide did not inhibit CNMT activity, but the addition of Co2+, Cu2+ or Mn2+ at 5 mM severely inhibited such activity by 75, 47 and 57%, respectively. The substrate-saturation kinetics of CNMT for norreticuline and SAM were of the typical Michaelis-Menten-type with respective Km values of 0.38 and 0.65 mM.
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan.
authors: K B
authors: KB
authors: Choi
authors: T
authors: T
authors: Morishige
authors: F
authors: F
authors: Sato
mesh_terms: D000470:Alkaloids / Q000378:metabolism; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007546:Isoquinolines / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D010946:Plants, Medicinal / Q000166:cytology / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000470:Alkaloids; D007546:Isoquinolines; C004690:coclaurine; D008780:Methyltransferases; C430217:S-adenosyl-L-methionine coclaurine N-methyltransferase