S-Adenosyl-L-methionine:scoulerine-9--̀methyltransferase from cultured Coptis japonica cells

S-Adenosyl-L-methionine: scoulerine-9-O-methyltransferase (SMT: EC 2.1.1.-), the enzyme which catalyses the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine, was purified to homogeneity from cultured Coptis japonica cells and its enzymological properties were characterized. The purified SMT had an apparent pI of 4.7, a native molecular mass of 120-140 kDa (gel filtration) and a subunit molecular mass of 41 kDa (SDS-polyacrylamide gel electrophoresis). The enzyme did not require a divalent cation for activity, and the addition of Ca2+, Cu2+ and Mn2+ at 1 mM inhibited enzyme activity by 64, 72 and 59%, respectively. p-Chloromercuribenzoate inhibited the activity by 41% at 0.5 mM, whereas N-methylmaleimide and iodoacetamide were without effect even at 5 mM. Berberine and palmatine (end products of the biosynthetic pathway of which SMT catalyses an intermediate step) also inhibited the activity by 70% at 5 mM. SMT was specific for the methylation of scoulerine. Substrate-saturation kinetics of the purified enzyme for (R,S)-scoulerine and SAM were typical Michaelis-menten type with Km values of 0.1 and 0.17 mM, respectively. The enzyme was inhibited by S-adenosylhomocysteine and its Ki value for S-adenosylhomocysteine versus SAM was 0.17 mM.