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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.

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

“…Bifunctional hydroxylase/dehydrase has been reported previously, e.g. CYP71E1 is a cytochrome P450-dependent hydroxylase/dehydrase involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (33). To our knowledge, JadH is the first example of a bifunctional FADdependent oxygenase/dehydrase in polyketide biosynthesis.…”

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

“…Since only a subset of P450 enzymes function in E. coli, the widely used heterologous bacterial system was not necessarily a feasible approach. [39][40][41]. Our demonstration that these members of the CYP97 clan function in E. coli indicates that this system will be valuable in further dissecting the structural basis for ring specificity and other molecular applications.…”

“…In E. coli, a bacterium which lacks P450 enzymes, flavodoxin: NADPH flavodoxin reductase can serve as a replacement for the natural redox partner to facilitate function of some plant P450 enzymes but not others [63,65]. For example, sorghum CYP71E1, required for cyanogenic glycoside biosynthesis, could both function in vivo in E. coli using the E. coli flavodoxin: NADPH flavodoxin reductase or reconstituted in vitro with a plant NADPH cytochrome P450 reductase [40]. In comparison, CYP79B2, an enzyme required for indole glucosinolate biosynthesis in Arabidopsis, functioned in E. coli only with support of the plant reductase [66] as found in other cases [39].…”

Camalexin is synthesized from indole-3-acetaldoxime, a key branching point between primary and secondary metabolism in Arabidopsis

“…Interestingly, S-alkyl thiohydroximate intermediates that are produced in vitro by the oximemetabolizing CYP83 enzymes in the glucosinolate pathway undergo internal cyclization to form a thiazoline ring with structural similarity to the thiazol ring in camalexin (15). CYP83A1 and CYP83B1 belong phylogenetically to the CYP71 family (31), as does the oxime-metabolizing enzyme in the biosynthesis of cyanogenic glucosides (32). This relationship suggests that the oxime-metabolizing enzyme in camalexin biosynthesis may belong to the CYP71 family.…”