S-ADENOSYL-L-METHIONINE - 3'-HYDROXY-N-METHYL-(S)-COCLAURINE-4'-O-METHYL TRANSFERASE, A REGIOSELECTIVE AND STEREOSELECTIVE ENZYME OF THE (S)-RETICULINE PATHWAY

A new enzyme SAM: 3′-hydroxy-N-methyl-(S)-coclaurine-4′-O-methyltransferase (4′-OMT) was found in cell cultures of several isoquinoline-containing plant species belonging to four plant families. The enzyme was purified ca 400-fold from Berberis koetineana cell cultures. The final preparation consisted of only two proteins, namely the 4′-OMT and SAM: norcoclaurine-6-O-methyltransferase. Separation of both enzymes by standard procedures failed. The 4′-OMT is a highly specific enzyme catalysing in the presence of SAM the transfer of a methyl group onto the C-4′ hydroxyl group of benzylisoquinoline alkaloids with absolute (S)-configuration at C-1 and possessing adjacent hydroxyl groups in the C-3′- and C-4′ position of ring C. The pH optimum of the enzyme is 8.3 and the natural substrate is 3′-hydroxy-N-methyl-(S)-coclaurine (Km=4.5 μM; Km SAM=30 μM). The enzyme is a single polypeptide (Mr 40 × 103 by SDS) and is potently inhibited by the product and to a lesser extent by other alkaloids. The enzyme has a half life of three days at room temperature and is stable for more than a year if stored at -20° (in 20% glycerol). © 1990.