Limits...
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

Related in: MedlinePlus

LC/MS analysis of the chimeric OMT reaction products using laudanosoline as a substrate. Upper panel; OMT reactions and their predicted ions in LC/MS analysis. Lower panels; Mass ion chromatogram of the reaction products. Product peaks with the same retention time and fragmentation ion as the 4′-OMT product (22 min, fragment of m/z = 178) are indicated by solid arrows, and those with the same parameters as 6-OMT (19 min, m/z = 192) are shown by open arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3066540&req=5

fig06: LC/MS analysis of the chimeric OMT reaction products using laudanosoline as a substrate. Upper panel; OMT reactions and their predicted ions in LC/MS analysis. Lower panels; Mass ion chromatogram of the reaction products. Product peaks with the same retention time and fragmentation ion as the 4′-OMT product (22 min, fragment of m/z = 178) are indicated by solid arrows, and those with the same parameters as 6-OMT (19 min, m/z = 192) are shown by open arrows.

Mentions: Enzyme activity was measured with [3H]AdoMet using laudanosoline and norlaudanosoline as substrates. While all constructs showed detectable activity, the activities differed (data not shown). We used LC/MS analysis to determine the regio-specificity of chimeric OMTs, since 4′- and 6-O-methylation of laudanosoline showed different fragmentation patterns; i.e., the 4′-OMT-type reaction produces a fragment of m/z = 178 and the 6-OMT-type gives a fragment of m/z = 192 (Fig. 6). The chimeric OMTs, i.e., 64′-17, 64′-2, 4′6-2, 4′6-3 and 4′6-1 OMTs, produced 4′-OMT-type products, whereas 64′-13 and 4′6-19 OMTs produced 6-OMT-type products. These results indicated that the region between the 34th and 125th amino acids is crucial for the determination of regio-specificity, whereas some other region might be involved in substrate recognition.


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)

LC/MS analysis of the chimeric OMT reaction products using laudanosoline as a substrate. Upper panel; OMT reactions and their predicted ions in LC/MS analysis. Lower panels; Mass ion chromatogram of the reaction products. Product peaks with the same retention time and fragmentation ion as the 4′-OMT product (22 min, fragment of m/z = 178) are indicated by solid arrows, and those with the same parameters as 6-OMT (19 min, m/z = 192) are shown by open arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: LC/MS analysis of the chimeric OMT reaction products using laudanosoline as a substrate. Upper panel; OMT reactions and their predicted ions in LC/MS analysis. Lower panels; Mass ion chromatogram of the reaction products. Product peaks with the same retention time and fragmentation ion as the 4′-OMT product (22 min, fragment of m/z = 178) are indicated by solid arrows, and those with the same parameters as 6-OMT (19 min, m/z = 192) are shown by open arrows.
Mentions: Enzyme activity was measured with [3H]AdoMet using laudanosoline and norlaudanosoline as substrates. While all constructs showed detectable activity, the activities differed (data not shown). We used LC/MS analysis to determine the regio-specificity of chimeric OMTs, since 4′- and 6-O-methylation of laudanosoline showed different fragmentation patterns; i.e., the 4′-OMT-type reaction produces a fragment of m/z = 178 and the 6-OMT-type gives a fragment of m/z = 192 (Fig. 6). The chimeric OMTs, i.e., 64′-17, 64′-2, 4′6-2, 4′6-3 and 4′6-1 OMTs, produced 4′-OMT-type products, whereas 64′-13 and 4′6-19 OMTs produced 6-OMT-type products. These results indicated that the region between the 34th and 125th amino acids is crucial for the determination of regio-specificity, whereas some other region might be involved in substrate recognition.

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
Related in: MedlinePlus