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

点击翻译按钮获取中文摘要

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 rich source of the cyanogenic diglucoside ( R )- amygdalin and its catabolic enzymes amygdalin hydrolase (AH), prunasin hydrolase (PH) and ( R )-mandelonitile lyase (MDL). A major objective of this research is to improve our understanding of the spatial and temporal regulation of cyanogenesis in plants, thereby contributing toward the long-term goal of reducing the toxicity of these plants to animals including man. 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 different functions or exhibit differential expression, either temporally or spatially, remains largely unclear. To address these questions, additional AH, PH and MDL genes will be obtained by RT-PCR and screening of black cherry cDNA and genomic libraries. Sequencing these genes will provide basic information about their structure and also identify unique regions for use as isoform-specific probes in analyzing the expression of individual genes by Northern analyses, ribonuclease protection assays, and in situ hybridization. The second goal of this project is to generate important information about the functioning of beta-glucosidases and FAD-containing alph-hydroxynitrile lyases, thus facilitating future efforts to engineer the substrate specificities of these enzymes for biotechnological purposes. After expressing recombinant AH and PH in the Pichia pastoris expression system, amino acid residues that confer substrate specificity upon these hydrolases will be identified by domain swapping and site-directed mut agenesis. Many important food plants, including cherries, cassava, lima beans and sorghum, liberate the respiratory poison hydrogen cyanide when ingested or mechanically damaged. Each year, this phenomenon of cyanogenesis accounts for numerous cases of acute and chronic cyanide poisoning of animals including man. A major objective of this research is to better understand the factors that control cyanide production, thereby contributing toward the long-term goal of reducing the toxicity of these plants. Genes encoding three enzymes that release cyanide in black cherry will be isolated and characterized. Analysis of their DNA sequences will reveal unique regions that will be used to study where and when each gene is active within this species. A second objective is to investigate how beta-glucosidases, a class of enzymes which breaks down sugar-containing compounds, distinguish between substrate molecules upon which they can potentially act. Two of the black cherry enzymes under study here are beta-glucosidases. Although closely related to each other, these enzymes degrade different substrates, presumably due to subtle variations in their protein structures. Beginning with the above DNA sequences, this research will use molecular methods to elucidate these structural differences. This will facilitate future efforts to engineer the substrate specificities of these and other beta-glucosidases for biotechnological purposes.