Cyanogenic
glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic
glucosides are α-hydroxynitriles, and their hydrolysis releases toxic
hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf
nitrogen to the biosynthesis of the cyanogenic monoglucoside,
prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in
prunasin content, as well as the presence of the cyanogenic diglucoside
amygdalin in flower buds and flowers. The identification and biochemical characterization of the
prunasin biosynthetic
enzymes revealed a unique
enzyme configuration for
prunasin production in E. cladocalyx This result indicates that a multifunctional
cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of
l-phenylalanine into its corresponding
aldoxime,
phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of
phenylacetaldoxime to the α-hydroxynitrile,
mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the
dehydration of
phenylacetaldoxime, an unusual CYP reaction. The resulting
phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form
mandelonitrile. The final glucosylation step to yield
prunasin is catalyzed by a
UDP-
glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic
glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic
glucosides in plants.