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: Since 64′-OMT was a newly constructed chimeric enzyme, we carefully examined the reaction product. HPLC and liquid chromatography-mass spectrography (LC/MS) analyses indicated that 64′-OMT product was mono-methylated at the A-ring of norlaudanosoline, but these analyses did not identify the position of methylation, i.e. 6- or 7-OH. Since rat-liver catechol O-methyltransferase (COMT) has been shown to methylate norlaudanosoline to produce both 6- and 7-O-methylated products,14) we compared the 64′-OMT product with 6-O-mono-methylated and 7-O-mono-methylated norlaudanosoline prepared with rat-liver COMT. LC/MS analysis of the 64′-OMT and rat-liver COMT products and authentic 6-O-methylnorlaudanosoline clearly indicated that 64′-OMT methylated the 6-OH group of norlaudanosoline (Fig. 4), which showed that the N-terminal half of 6-OMT determined the regio-specificity of 64′-OMT.