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

玫瑰核果（Prunus spp.）是许多重要的商业作物之一，通过内源性β -葡萄糖苷酶和α -羟基腈裂解酶释放氰化氢，导致人类和牲畜氰化物中毒。黑樱桃（p.s ertina）种子富含致氰二糖苷(R)-苦杏仁苷及其分解代谢酶苦杏仁苷水解酶（AH）、prunasin水解酶（PH）和(R)-扁桃核苷裂解酶（MDL），因此具有较高的致氰性。本研究的一个主要目的是提高我们对植物中氰生成的时空调控的理解，从而有助于减少这些植物对包括人类在内的动物的毒性的长期目标。李树的产氰系统是复杂的，因为所有三种酶都以多种形式存在，其确切性质和生理意义需要澄清。每种分解代谢酶的个体同工型是否在时间或空间上执行不同的功能或表现出差异表达，在很大程度上仍不清楚。为了解决这些问题，我们将通过RT-PCR以及黑樱桃cDNA和基因组文库的筛选获得更多的AH、PH和MDL基因。对这些基因进行测序将提供有关其结构的基本信息，并确定独特的区域，用于通过Northern分析、核糖核酸酶保护试验和原位杂交分析单个基因表达的同种异构体特异性探针。该项目的第二个目标是产生关于-葡萄糖苷酶和含有fad的α -羟基腈裂解酶功能的重要信息，从而促进未来为生物技术目的设计这些酶的底物特异性的努力。在毕赤酵母表达系统中表达重组AH和PH后，赋予这些水解酶底物特异性的氨基酸残基将通过结构域交换和位点导向突变进行鉴定。许多重要的食用植物，包括樱桃、木薯、利马豆和高粱，在被摄入或受到机械损伤时，会释放出呼吸毒性氰化氢。每年，这种氰化现象造成包括人在内的许多动物急性和慢性氰化物中毒病例。这项研究的一个主要目的是更好地了解控制氰化物生产的因素，从而有助于实现减少这些植物毒性的长期目标。对黑樱桃中三种释放氰化物酶的编码基因进行了分离和鉴定。对它们DNA序列的分析将揭示出独特的区域，这些区域将用于研究该物种中每个基因在何时何地活跃。第二个目标是研究β -葡萄糖苷酶（一种分解含糖化合物的酶）如何区分它们可能起作用的底物分子。这里研究的两种黑樱桃酶是-葡萄糖苷酶。虽然彼此密切相关，但这些酶降解不同的底物，可能是由于它们的蛋白质结构的微妙变化。从上述DNA序列开始，本研究将使用分子方法来阐明这些结构差异。这将有助于未来为生物技术目的设计这些和其他β -葡萄糖苷酶的底物特异性。

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.