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.
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Mentions: Enzyme activity was measured with [3H]AdoMet using laudanosoline and norlaudanosoline as substrates. While all constructs showed detectable activity, the activities differed (data not shown). We used LC/MS analysis to determine the regio-specificity of chimeric OMTs, since 4′- and 6-O-methylation of laudanosoline showed different fragmentation patterns; i.e., the 4′-OMT-type reaction produces a fragment of m/z = 178 and the 6-OMT-type gives a fragment of m/z = 192 (Fig. 6). The chimeric OMTs, i.e., 64′-17, 64′-2, 4′6-2, 4′6-3 and 4′6-1 OMTs, produced 4′-OMT-type products, whereas 64′-13 and 4′6-19 OMTs produced 6-OMT-type products. These results indicated that the region between the 34th and 125th amino acids is crucial for the determination of regio-specificity, whereas some other region might be involved in substrate recognition.