A
cDNA encoding the multifunctional
cytochrome P450,
CYP71E1, involved in the biosynthesis of the cyanogenic
glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type
cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied. Three novel
cytochromes P450 (
CYP71E1,
CYP98, and
CYP99) in addition to a PCR fragment encoding sorghum
cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant
CYP71E1 heterologously expressed in Escherichia coli and sorghum
NADPH-
cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl
choline micelles identified
CYP71E1 as the
cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in
dhurrin biosynthesis. In accordance to the proposed pathway for
dhurrin biosynthesis
CYP71E1 catalyses the
dehydration of the
oxime to the corresponding
nitrile, followed by a C-hydroxylation of the
nitrile to produce p-hydroxymandelonitrile. In vivo administration of
oxime to E. coli cells results in the accumulation of the
nitrile, which indicates that the
flavodoxin/
flavodoxin reductase system in E. coli is only able to support
CYP71E1 in the
dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of
tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic
glucoside dhurrin. Reconstitution of both CYP79 and
CYP71E1 in combination with sorghum
NADPH-
cytochrome P450-reductase resulted in the conversion of
tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic
glucoside dhurrin. Isolation of the
cDNA for
CYP71E1 together with the previously isolated
cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic
glucoside levels in plants to optimize food safety and pest resistance.