Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells.
To identify all of the O-methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica cells, we sequenced 1014 cDNA clones isolated from high-alkaloid-producing cultured cells of C. japonica. Among them, we found all three reported O-methyltransferases and an O-methyltransferase-like cDNA clone (CJEST64). This cDNA was quite similar to S-adenosyl-l-methionine:coclaurine 6-O-methyltransferase and S-adenosyl-l-methionine:isoflavone 7-O-methyltransferase. As S-adenosyl-l-methionine:columbamine O-methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O-methyltransferase. The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.
Morishige
Takashi
T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Dubouzet
Emilyn
E
Choi
Kum-Boo
KB
Yazaki
Kazufumi
K
Sato
Fumihiko
F
D000595:Amino Acid Sequence; D001600:Berberine Alkaloids / Q000737:chemistry* / Q000378:metabolism*; D039323:Coptis / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020224:Expressed Sequence Tags; D008401:Gas Chromatography-Mass Spectrometry; D015723:Gene Library; D005822:Genetic Vectors; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001600:Berberine Alkaloids; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; C055786:columbamine; D008780:Methyltransferases; C470096:S-adenosyl-L-methionine columbamine O-methyltransferase, plant