Molecular Characterization of the S-Adenosyl-l-methionine:3′-Hydroxy-N-methylcoclaurine 4′-O-Methyltransferase Involved in Isoquinoline Alkaloid Biosynthesis in Coptis japonica *

S-Adenosyl-l-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′-OMT) catalyzes the conversion of 3′-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, anotherO-methyltransferase,S-adenosyl-l-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4′-OMT preparation from cultured Coptis japonicacells. Heterologously expressed proteins had 6-OMT or 4′-OMT activities, indicative that each cDNA encodes a different enzyme. 4′-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, or Ni²⁺, but none by the SH reagent. 4′-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, anN-methylated substrate, was a much better substrate for 4′-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4′-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two relatedO-methyltransferases is discussed.