Heterodimeric O ‐methyltransferases involved in the biosynthesis of noscapine in opium poppy

Summary Noscapine biosynthesis in opium poppy involves three characterized O ‐methyltransferases ( OMT s) and a fourth responsible for the 4ʹ‐methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes ( OMT 2 and OMT 3 ). Neither OMT 2 nor OMT 3 form stable homodimers, but yield a substrate‐specific heterodimer when their genes are co‐expressed in Escherichia coli . The only substrate, 4ʹ‐ O ‐desmethyl‐3‐ O‐ acetylpapaveroxine, is a seco ‐berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4ʹ‐ O ‐methylation leading to the formation of narcotine hemiacetal. In the absence of 4ʹ‐ O ‐methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4ʹ‐hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1‐hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6 OMT , which shares 81% amino acid sequence identity with OMT 3, also formed a functionally similar heterodimer with OMT 2. Suppression of OMT 2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT 3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT 2: OMT 3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT 2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate‐binding domain, although site‐directed mutagenesis revealed OMT 2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.