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, another O-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 japonica cells. 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 Fe(2+), Cu(2+), Co(2+), Zn(2+), or Ni(2+), 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, an N-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 related O-methyltransferases is discussed.
Morishige
T
T
Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Tsujita
T
T
Yamada
Y
Y
Sato
F
F
D000470:Alkaloids / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007546:Isoquinolines / Q000378:metabolism*; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D059828:Plant Cells; D010944:Plants / Q000201:enzymology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000470:Alkaloids; D018076:DNA, Complementary; D007546:Isoquinolines; D008780:Methyltransferases; C412574:S-adenosyl-L-methionine 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase