title: Purification and characterization of S-adenosyl-L-methionine: norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells.
abstract: 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-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]qu inolizinium (berberine, the end-product of the biosynthetic pathway in which norcoclurine 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-adenosylhomocysteine versus S-adenosyl-L-methionine and (R,S)-norlaudanosoline were 2.1 mM and 0.18 mM, respectively.
authors.affiliation: Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Sato
authors.forename: T
authors.initials: T
authors.lastname: Tsujita
authors.forename: Y
authors.initials: Y
authors.lastname: Katagiri
authors.forename: S
authors.initials: S
authors.lastname: Yoshida
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010946:Plants, Medicinal / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D017886:Durapatite; D008780:Methyltransferases; C090040:S-adenosyl-L-methionine-norcoclaurine 6-O-methyltransferase