Purification and characterization of coclaurine N-methyltransferase from cultured Coptis japonica cells

• Published in: Phytochemistry (Oxford) 2001-04, Vol.56 (7), p.649-655
• Main Authors: Choi, Kum-Boo, Morishige, Takashi, Sato, Fumihiko
• Format: Article
• Language: English
• Subjects: Alkaloid biosynthesis; Alkaloids - metabolism; Analytical, structural and metabolic biochemistry; Berberine; Biological and medical sciences; Cells, Cultured; Chromatography, Gel; Chromatography, Ion Exchange; Coclaurine N-methyltransferase; Coptis japonica; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Isoquinoline alkaloid; Isoquinolines - metabolism; Kinetics; Metabolism; Methyltransferases - chemistry; Methyltransferases - isolation & purification; Methyltransferases - metabolism; Molecular Weight; Plant physiology and development; Plants, Medicinal - cytology; Plants, Medicinal - enzymology; Ranunculaceae; S-Adenosyl- l-methionine; Stereoisomerism; Substrate Specificity; Transferases
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(00)00481-7

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 M r of 160 kDa (gel-filtration chromatography) and a subunit M r 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 Co 2+, Cu 2+ or Mn 2+ 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 K m values of 0.38 and 0.65 mM.