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Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

Morishige T, Tamakoshi M, Takemura T, Sato F - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

Bottom Line: Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT.Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity.The creation of OMTs with novel reactivity is discussed.

View Article: PubMed Central - PubMed

Affiliation: Kyoto University, Japan.

ABSTRACT
O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4'-OMT (64'-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.

Show MeSH
Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 6-OMT, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase; 4′-OMT, S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine.
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fig01: Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 6-OMT, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase; 4′-OMT, S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine.

Mentions: Methyltransferases are crucial for directing intermediates to specific biosynthetic pathways in plant secondary metabolism.1) We have been studying the O-methyltransferases (OMTs) in isoquinoline alkaloid biosynthesis, since these alkaloids include pharmaceutically important alkaloids such as analgesic morphinan alkaloids (e.g. morphine), anti-microbial berberine alkaloids (e.g. berberine), and anti-microbial benzophenanthridine alkaloids (e.g. sanguinarine). In the biosynthetic pathway of berberine, three OMTs [S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT);2–4) S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′-OMT)4,5) and S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase (SMT)6–8)] have been isolated and characterized (Fig. 1). Each OMT shows strict substrate specificity, despite the structural similarities of the various substrates. Isolation of the cDNA of OMT from cultured Coptis japonica cells4) and successful expression with other biosynthetic genes in Escherichia coli clearly indicated that the reaction specificities of OMTs determine the biosynthetic pathway of reticuline, an important intermediate in berberine biosynthesis.9)


Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

Morishige T, Tamakoshi M, Takemura T, Sato F - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 6-OMT, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase; 4′-OMT, S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066540&req=5

fig01: Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 6-OMT, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase; 4′-OMT, S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine.
Mentions: Methyltransferases are crucial for directing intermediates to specific biosynthetic pathways in plant secondary metabolism.1) We have been studying the O-methyltransferases (OMTs) in isoquinoline alkaloid biosynthesis, since these alkaloids include pharmaceutically important alkaloids such as analgesic morphinan alkaloids (e.g. morphine), anti-microbial berberine alkaloids (e.g. berberine), and anti-microbial benzophenanthridine alkaloids (e.g. sanguinarine). In the biosynthetic pathway of berberine, three OMTs [S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT);2–4) S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′-OMT)4,5) and S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase (SMT)6–8)] have been isolated and characterized (Fig. 1). Each OMT shows strict substrate specificity, despite the structural similarities of the various substrates. Isolation of the cDNA of OMT from cultured Coptis japonica cells4) and successful expression with other biosynthetic genes in Escherichia coli clearly indicated that the reaction specificities of OMTs determine the biosynthetic pathway of reticuline, an important intermediate in berberine biosynthesis.9)

Bottom Line: Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT.Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity.The creation of OMTs with novel reactivity is discussed.

View Article: PubMed Central - PubMed

Affiliation: Kyoto University, Japan.

ABSTRACT
O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4'-OMT (64'-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.

Show MeSH