Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55

Abstract

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are αhydroxynitriles, 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.www.plantphysiol.org on August 1, 2020 -Published by Downloaded from Metabolite data were analyzed in SigmaPlot (version 13.0) using oneway ANOVA. Normality (Kolmogorov-Smirnov) and equal variance (Brown-Forsythe) were tested to cutoffs of P > 0.02 and P > 0.04, respectively, and when assumptions were not met, the data were transformed by natural logarithm. Pearson correlation was used to analyze gene expression. PK, Svendsen I, Møller BL (2000) Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin: cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes. J Biol Chem 275: 1966-1975 Bak S, Kahn RA, Nielsen HL, Møller BL, Halkier BA (1998) 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. Plant Mol Biol 36: 393-405 Bassard JE, Møller BL, Laursen T (2017) Assembly of dynamic P450-mediated metabolons: order versus chaos. Curr Mol Biol Rep 3: 37-51 Bjarnholt N, Li B, D'Alvise J, Janfelt C (2014) Mass...

Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis)

“…Even though CYP71s are considered more promiscuous in nature (Kahn et al, 1999), we see a clear case of strict conservation of the functional homologs within the Prunus species (Supplemental Table S2). In contrast, the conversion of the oxime to the cyanohydrin was shown recently to be catalyzed by two separate P450 enzymes in E. cladocalyx (Hansen et al, 2018). This represents yet another example showing that cyanogenic glucoside biosynthesis has evolved independently in several plant lineages (Takos et al, 2011).…”

“…Some eucalypt species also produce prunasin and prunasin-derived diglucosides (Neilson et al, 2011). In Eucalyptus cladocalyx, the conversion of the oxime to the cyanohydrin was shown recently to be catalyzed by two separate P450 enzymes (EcCYP706C55 and EcCYP71B103; Hansen et al, 2018). This represents yet another example of repeated evolution of this chemical defense pathway (Takos et al, 2011).…”

Setting and Diffusing the Cyanide Bomb in Plant Defense

“…1). In this issue of Plant Physiology, reports elucidate distinct biosynthetic routes to mandelonitrile and its cyanogenic glucosides in almond (Thodberg et al, 2018) and sugar gum (Eucalyptus cladocalyx; Hansen et al, 2018).…”

“…Cyanohydrins are then typically glycosylated by UDP-Glc glycosyl transferases (UGTs) from the UGT85 family (Gleadow and Møller, 2014). A second report in this issue of Plant Physiology details the pathway for prunasin biosynthesis in sugar gum and reveals an unexpected reconfiguration of this pathway (Hansen et al, 2018).…”

“…Biosynthesis of the cyanogenic glycosides prunasin and amygdalin and their deployment as chemical defenses. The sugar gum pathway reported byHansen et al (2018) is shown in blue, and the almond pathway reported byThodberg et al (2018) is shown in green.…”