Reference "Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin"

Reference ID

9012

Title

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

Source

Plant molecular biology, 1998, vol. 36, pp. 393-405

Authors (5)

Bak-S	Kahn-R-A
Nielsen-H-L	Moller-B-L
Halkier-B-A

Abstract

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.