Biochemistry and Molecular Biology of Beta-Glucosidases and Alpha-Hydroxynitrile Lyases Involved in Cyanogenesis

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9630935 Poulton Rosaceous stone fruits (Prunus spp.) are among many commercially important crops that cause cyanide poisoning in humans and livestock through release of hydrogen cyanide from cyanogenic glycosides by endogenous beta-glucosidases and alpha-hydroxynitrile lyases. Black cherry (P. serotina) seeds are highly cyanogenic because they are a rich source of the cyanogenic diglucoside (R)-amydalin and its catabolic enzymes amygdalin hydrolase (AH), prunasin hydrolase (PH) and (R)-mandelonitrile lyase (MDL). In undamaged seeds, large-scale amygdalin hydrolysis is precluded by tissue level compartmentation, since the two beta-glucosidases are located exclusively in the procambium, whereas amygdalin occurs with MDL within the cotyledonary parenchyma cells. Rapid cyanogenesis ensues when tissue disruption breaches critical permeability barriers, allowing amygdalin access to its catabolic enzymes. The objective of this proposal is to gain a better understanidng of the biochemistry and molecular biology of AH, PH and MDL. The Prunus cyanogenic system is complex in that all three enzymes exist as multiple forms, the exact nature and physiological significance of which require clarification. Whether individual isoforms of each catabolic enzyme carry out defferent functions or exhibit differential expression, either temporally or spatially, remains largely unclear. The proposed studies are designed to lead to an improved understanding of the spatial and temporal regulation of cyanogenesis in plants, thereby contributing toward the long-term goal of reducing the toxicity of cyanogenic plants to animals including man. Furthermore, this research will generate important information about the functioning of beta-glucosidases and FAD-containing alpha-hydroxynitrile lyases, thus facilitating future efforts to engineer the substrate specificities of these enzymes for biotechnological purposes.