Plant Molecular Biology

, Volume 36, Issue 3, pp 393–405 | Cite as

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

  • Søren Bak
  • Rachel Alice Kahn
  • Hanne Linde Nielsen
  • Birger Lindberg Møller
  • Barbara Ann Halkier


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-α-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.

cyanogenic glucosides cytochrome P450 E. coli expression oxime reconstitution PCR 


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Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Søren Bak
    • 1
  • Rachel Alice Kahn
    • 1
  • Hanne Linde Nielsen
    • 1
  • Birger Lindberg Møller
    • 1
  • Barbara Ann Halkier
    • 1
  1. 1.Plant Biochemistry Laboratory, Department of Plant BiologyThe Royal Veterinary and Agricultural UniversityFrederiksberg C, CopenhagenDenmark

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