Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.
Bottom Line: 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.
Affiliation: Kyoto University, Japan.
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.
Mentions: The regio-specificities of chimeric enzymes were further characterized with another substrate, i.e., 3′-hydroxy-N-methylcoclaurine (Fig. 7). As expected, 4′-OMT and 4′-OMT-type chimeric enzymes (64′-17, 64′-2, 4′6-1, 4′6-2, 4′6-3 OMTs) showed 4′-O-methylation activity to produce reticuline. Unexpectedly, however, 6-OMT showed a weak but evident peak that was distinct from that of the 4′-O-methylation product. This novel peak was speculated to be a 3′-O-methylation product from the fragment signal of m/z 192 (data not shown). Interestingly, other 6-OMT-type enzymes, 64′-OMT and 4′6-19 OMT, showed both 3′-O-methylation and 4′-O-methylation activities, whereas another 6-OMT-type chimeric enzyme, 64′-13OMT, showed only 4′-O-methylation activity.