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

参与氰生成的 β-葡萄糖苷酶和 α-羟基腈裂解酶的生物化学和分子生物学

• 批准号: 9723302
• 负责人: Jonathan Poulton
• 金额: $ 27万
• 依托单位: University of Iowa
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723302&HistoricalAwards=false
• 关键词: Biochemistry; Molecular; Biology; Beta; Glucosidases

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.

Poulton Rosaceous核果（Prunus spp.）是许多商业上重要的作物之一，其通过内源性β-葡糖苷酶和α-羟基腈裂解酶从生氰糖苷中释放氰化氢而引起人和牲畜的氰化物中毒。 黑樱桃种子是一种高度生氰的种子，因为它们富含生氰二葡萄糖苷（R）-苦杏仁苷及其分解代谢酶苦杏仁苷水解酶（AH）、扁桃酸水解酶（PH）和（R）-扁桃酸裂解酶（MDL）。 这项研究的一个主要目的是提高我们的理解的空间和时间调节植物中的氰，从而有助于减少这些植物的毒性，包括man. The李的动物氰系统的长期目标是复杂的，所有三种酶存在多种形式，确切的性质和生理意义，其中需要澄清。 每个分解代谢酶的个体亚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，在很大程度上仍然不清楚。 为了解决这些问题，额外的AH，PH和MDL基因将获得通过RT-PCR和筛选黑樱桃cDNA和基因组文库。 对这些基因进行测序将提供关于其结构的基本信息，并且还鉴定独特的区域，所述独特的区域用作通过北方分析、核糖核酸酶保护测定和原位杂交分析单个基因表达的同种型特异性探针。 该项目的第二个目标是产生关于β-葡糖苷酶和含FAD的β-羟基腈裂解酶的功能的重要信息，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，通过结构域交换和定点突变来鉴定赋予这些水解酶底物特异性的氨基酸残基。 许多重要的食用植物，包括樱桃、木薯、利马豆和高粱，在摄入或机械损坏时会释放出呼吸道毒素氰化氢。 每年，这种氰生成现象导致包括人类在内的许多动物急性和慢性氰化物中毒。本研究的一个主要目的是更好地了解控制氰化物产生的因素，从而有助于实现降低这些植物毒性的长期目标。 编码黑樱桃中释放氰化物的三种酶的基因将被分离和鉴定。 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每个基因在该物种中何时何地活跃。 第二个目标是研究β-葡糖苷酶（一种分解含糖化合物的酶）如何区分它们可能作用的底物分子。 这里研究的两种黑樱桃酶是β-葡萄糖苷酶。 虽然这些酶彼此密切相关，但它们降解不同的底物，这可能是由于它们的蛋白质结构存在细微变化。 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。 这将有助于未来的努力，工程的底物特异性，这些和其他β-葡糖苷酶的生物技术目的。