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

Abstract

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.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The conversion order was inferred by the substrate preference of the relevant enzymes although recent evidence suggests that the pathway is not linear, but rather operates as a grid (Desgagné-Penix and Facchini 2012). (S)-Norcoclaurine is first converted by norcoclaurine-6-O-methyltransferase (6OMT) to (S)-coclaurine (Morishige et al 2000;Ounaroon et al 2003), which is accepted by coclaurine N-methyltransferase (CNMT) (Choi et al 2001). N-Methylcoclaurine is hydroxylated by (S)-N-methylcoclaurine 3′-hydroxylase (NMCH) (Pauli and Kutchan 1998;Frick et al 2007) to 3′-hydroxy-N-methylcoclaurine, which is converted by 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) (Morishige et al 2000) to (S)-reticuline.…”

“…(S)-Norcoclaurine is first converted by norcoclaurine-6-O-methyltransferase (6OMT) to (S)-coclaurine (Morishige et al 2000;Ounaroon et al 2003), which is accepted by coclaurine N-methyltransferase (CNMT) (Choi et al 2001). N-Methylcoclaurine is hydroxylated by (S)-N-methylcoclaurine 3′-hydroxylase (NMCH) (Pauli and Kutchan 1998;Frick et al 2007) to 3′-hydroxy-N-methylcoclaurine, which is converted by 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) (Morishige et al 2000) to (S)-reticuline. Interestingly, only NMCH has been reported to show strict substrate and stereoisomer specificity, accepting (S)-N-methylcoclaurine and not the corresponding (R)-N-methylcoclaurine or N-desmethyl compounds.…”

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

“…Generally, OMTs involved in BIA biosynthesis display diverse substrate specificities, with enzymes operating early in the pathway being more promiscuous. For example, 6OMT and 49OMT from C. japonica and opium poppy accept simple benzylisoquinoline and protoberberine substrates (Sato et al, 1994;Morishige et al, 2000;Ziegler et al, 2005). In contrast, N7OMT from opium poppy accepts only norreticuline (Pienkny et al, 2009), whereas 7OMT can catalyze the O-methylation of certain phenolic compounds (e.g.…”

“…(S)-Norcoclaurine is subsequently converted via several steps to the key branch point intermediate (S)-reticuline ( Fig. 1), from which numerous BIA structural subgroups are derived (Frenzel and Zenk, 1990;Pauli and Kutchan, 1998;Morishige et al, 2000;Choi et al, 2002). Rearrangement of the benzylisoquinoline backbone is achieved through carbon-carbon or carbon-oxygen coupling catalyzed by a variety of specific cytochromes P450 or by a FAD-linked oxidoreductase.…”

“…All known OMTs involved in BIA metabolism exhibit extensive amino acid homology and form a distinctive clade with respect to OMTs involved in other metabolic pathways, such as flavonoid metabolism . Characterized OMTs involved in BIA biosynthesis include norcoclaurine 6-O-methyltransferase (6OMT; Sato et al, 1994;Morishige et al, 2000;Ounaroon et al, 2003), 39-hydroxy-N-methylcoclaurine 49-O-methyltransferase (49OMT; Morishige et al, 2000;Ziegler et al, 2005), reticuline 7-O-methyltransferase (7OMT; Ounaroon et al, 2003), norreticuline 7-O-methyltransferase (N7OMT; Pienkny et al, 2009), scoulerine-9-O-methyltransferase (SOMT; Takeshita et al, 1995), and columbamine O-methyltransferase (CoOMT; Morishige et al, 2002). Functional homologs for all enzymes except SOMT and CoOMT have been identified in opium poppy (Facchini and De Luca, 2008).…”