| Abstrak/Abstract |
The most prominent alkaloid of Chelidonium majus is dihydrocoptisine,
revealing the characteristic benzophenanthridine
skeleton. To date, any informationon on the
enzymes responsible for its biosynthesis and the related
genes in C. majus is lacking. Based on sequence similarities
to the corresponding methylenedioxy bridge-forming Cyt
P450 enzymes involved in isoquinoline alkaloid biosynthesis
in Eschscholzia californica, genes for a cheilanthifoline
synthase and a stylopine synthase from C. majus were isolated,
sequenced and heterologously expressed in yeast.
The activity of the heterologously expressed Cyt P450 enzymes
was determined in situ as well as on the basis of
microsomal fractions. It was shown that cheilanthifoline
synthase (c8931) converts scoulerine into cheilanthifoline,
the latter subsequently being converted to stylopine by the
action of a stylopine synthase (c1128). Based on the wellknown
instability of stylopine, it can be assumed that
in vivo—under the acidic conditions in the vacuole—this
alkaloid is converted to dihydrocoptisine, which accumulates
in C. majus leaves. Both methylenedioxy bridge-forming
Cyt P450 enzymes from C. majus are characterized by
their high substrate specificity. Apart from their genuine
substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline
synthase and stylopine synthase do not accept other
substrates tested; the only alternative substrate identified
was scoulerine, which is converted by stylopine synthase to
yield minor amounts of nandinine. Quantitative real-time
PCR revealed that the expression of cheilanthifoline
synthase and stylopine synthase genes is very similar in
both roots and leaves from C. majus, although the alkaloid
accumulation patterns in these organs are quite
different. |
