| Abstrak/Abstract |
California poppy (Eschscholzia californica), a member of the
Papaveraceae family, produces many biologically active
benzylisoquinoline alkaloids (BIAs), such as sanguinarine,
macarpine and chelerythrine. Sanguinarine biosynthesis
has been elucidated at the molecular level, and its biosynthetic
genes have been isolated and used in synthetic biology
approaches to produce BIAs in vitro. However, several genes
involved in the biosynthesis of macarpine and chelerythrine
have not yet been characterized. In this study, we report the
isolation and characterization of a novel O-methyltransferase
(OMT) involved in the biosynthesis of partially characterized
BIAs, especially chelerythrine. A search of the RNA
sequence database from NCBI and PhytoMetaSyn for the
conserved OMT domain identified 68 new OMT-like sequences,
of which the longest 22 sequences were selected
based on sequence similarity. Based on their expression in
cell lines with different macarpine/chelerythrine profiles, we
selected three OMTs (G2, G3 and G11) for further characterization.
G3 expression in Escherichia coli indicated Omethylation
activity of the simple benzylisoquinolines,
including reticuline and norreticuline, and the protoberberine
scoulerine with dual regio-reactivities. G3 produced 7-Omethylated,
30-O-methylated and dual O-methylated products
from reticuline and norreticuline, and 9-O-methylated
tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine
from scoulerine. Further enzymatic analyses
suggested that G3 is a scoulerine-9-O-methyltransferase for
the biosynthesis of chelerythrine in California poppy. In the
present study, we discuss the physiological role of G3 in BIA
biosynthesis |
