Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.
O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4'-OMT (64'-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.
Morishige
Takashi
T
Kyoto University, Japan.
Tamakoshi
Masanori
M
Takemura
Tomoya
T
Sato
Fumihiko
F
D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D001599:Berberine / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D039323:Coptis / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000737:chemistry / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000470:Alkaloids; D018076:DNA, Complementary; D007546:Isoquinolines; D001599:Berberine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C039109:isoquinoline