Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.
S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.
Takeshita
N
N
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Fujiwara
H
H
Mimura
H
H
Fitchen
J H
JH
Yamada
Y
Y
Sato
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant; D004926:Escherichia coli / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000166:cytology / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D008780:Methyltransferases; C093217:S-adenosyl-L-methionine - scoulerine-9-O-methyltransferase