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 pi ducal 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.