The UDP-glucose:p-Hydroxymandelonitrile-O-Glucosyltransferase That Catalyzes the Last Step in Synthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor

Abstract

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (BzMc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with phydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

“…The protein extracts were subjected to immunoblotting and probed with a specific antibody against UGT85B1 (Jones et al, 1999). The immunoblotting analysis confirmed that each of the mutant proteins produced a discrete protein band with the expected M r .…”

“…Mandelonitrile was utilized as an acceptor molecule instead of p-hydroxymandelonitrile because mandelonitrile is more stable than p-hydroxymandelonitrile. UGT85B1 has previously been shown to glucosylate the two compounds at comparable rates (Reay and Conn, 1974;Jones et al, 1999). Product formation was assessed by thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS) and showed that UDP-Glc, but not UDP-Xyl or UDP-GlcUA, served as sugar donors for UGT85B1 (Fig.…”

“…This observation has been substantiated by metabolome and transcriptome analyses of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the entire dhurrin pathway (Tattersall et al, 2001;Jørgensen et al, 2005;Kristensen et al, 2005), as well as by confocal laser-scanning microscopy studies showing that the three enzymes CYP79A1, CYP71E1, and UGT85B1 catalyzing dhurrin formation are physically associated within a tight metabolon . In contrast, UGT85B1 exhibits a rather broad specificity toward acceptors in vitro, a key determinant for glycosylation being the presence of a sterically unmasked acceptor hydroxyl group (Jones et al, 1999;Hansen et al, 2003 …”

“…The final step in plant natural product synthesis is often a glycosylation reaction that serves to stabilize, solubilize, and provide a safe storage form of potentially toxic aglycones. In Sorghum bicolor, the GT UGT85B1 catalyzes glucosylation of Tyr-derived p-hydroxymandelonitrile to yield the cyanogenic glucoside dhurrin (Jones et al, 1999). In planta, neither p-hydroxymandelonitrile nor its degradation products are detectable, indicating that the glucosylation reaction is highly efficient and channeled (Møller and Conn, 1980).…”

“…The major clade is vastly expanded and specific to plants, and is generally thought to be involved in biosynthesis of the vast array of glycosidic plant natural products. As documented for UGT85B1, plant UGTs involved in natural product synthesis generally exhibit a broad substrate specificity with respect to the aglycon (sugar acceptor; Jones et al, 1999;Taguchi et al, 2000;Jackson et al, 2001;Lim et al, 2001aLim et al, , 2001bLim et al, , 2003aLim et al, , 2003bVogt, 2002;Hansen et al, 2003;Paquette et al, 2003). Most animal UGTs utilize UDP-GlcUA as a sugar donor, whereas plant UGTs typically utilize UDP-Glc.…”