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

被引:32
作者
Choi, KB
Morishige, T
Sato, F [1 ]
机构
[1] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Sakyo Ku, Kyoto 6068502, Japan
[2] Kyoto Univ, Grad Sch Agr, Div Integrated Life Sci, Sakyo Ku, Kyoto 6068502, Japan
关键词
alkaloid biosynthesis; berberine; Coptis japonica; Ranunculaceae; isoquinoline alkaloid; S-adenosyl-L-methionine; coclaurine N-methyltransferase;
D O I
10.1016/S0031-9422(00)00481-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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 K-m values of 0.38 and 0.65 mM. (C) 2001 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:649 / 655
页数:7
相关论文
共 19 条