Purification and Characterization of S‐adenosyl‐l‐methionine:norcoclaurine 6‐O‐Methyltransferase from Cultured Coptis japonica Cells

• Published in: European journal of biochemistry Vol. 225; no. 1; pp. 125 - 131
• Main Authors: Sato, Fumihiko, Tsujita, Tetsuya, Katagiri, Yasuyuki, Yoshida, Shigeru, Yamada, Yasuyuki
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.10.1994
• Subjects: Amino Acid Sequence; Cells, Cultured; Chromatography; Chromatography, Ion Exchange; Durapatite; Kinetics; Methyltransferases - chemistry; Methyltransferases - isolation & purification; Methyltransferases - metabolism; Molecular Sequence Data; Molecular Weight; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Plants, Medicinal - enzymology; Substrate Specificity
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1994.00125.x

S‐adenosyl‐l‐methionine:norcoclaurine 6‐O‐methyltransferase (norcoclaurine 6‐O‐methyltransferase), which catalyzes the transfer of the S‐methyl group of S‐adenosyl‐l‐methionine to the 6‐hydroxyl group of 1,2,3,4‐tetrahydro‐1‐[(4‐hydroxyphenyl)methyl]‐6,7‐isoquinolinediol (norcoclaurine), was purified from cultured Coptis japonica cells and its enzymic properties were characterized. Purified norcoclaurine 6‐O‐methyltransferase had apparent pI 4.7, a native molecular mass of 95 kDa (determined by gel filtration) and subunit molecular mass of 40 kDa (SDS/PAGE). The enzyme did not require a divalent cation for activity, and the addition of Fe2+, Cu2+, Co2+, Zn2+, Mn2+, or Ni2+ at 5 mM severely inhibited enzyme activity. Neither p‐chloromercuribenzoate, N‐methylmaleimide nor iodoacetamide inhibited enzyme activity at 1 mM. 5, 6‐Dihydro‐9, 10‐dime‐thoxybenzo[g]‐1,3‐benzodioxolo[5,6‐a]quinolizinium (berberine, the end‐product of the biosynthetic pathway in which norcoclaurine 6‐O‐methyltransferase catalyzes an intermediate step) also inhibited the activity by 50% at 10 mM. Norcoclaurine 6‐O‐methyltransferase methylated both (S)‐norcoclaurine and (R)‐norcoclaurine and (R,S)‐norlaudanosoline. Further characterization of substrate‐saturation kinetics and product inhibition of the purified enzyme indicated that norcoclaurine 6‐O‐methyltransferase follows a bi‐bi ping‐pong mechanism with Km, values of 2.23 mM and 3.95 mM for (R,S)‐norlaudanosoline and S‐adenosyl‐l‐methionine, respectively, while Ki values for S‐adenosyl‐homocysteine versus S‐adenosyl‐l‐methionine and (R,S)‐norlaudanosoline were 2.1 mM and 0.18 mM, respectively.