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

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 almo...

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Bibliographic Details
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
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Summary: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.
ISSN:0031-9422
1873-3700
DOI:10.1016/S0031-9422(00)00481-7