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-methyltransfera se (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to re ticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-me thyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferas e (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 japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, in dicative that each cDNA encodes a different enzyme. 4'-OMT was purified usi ng recombinant protein, and its enzymological properties were characterized . It had enzymological characteristics similar to those of 6-OMT; the activ e enzyme was the dimer of the subunit, no divalent cations were required fo r 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)-laudanos oline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methyl ated norlaudanosoline and laudanosoline equally. Further characterization o f 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 related O-methyltran sferases is discussed.