Lysine Biosynthesis in Yeast

酵母中赖氨酸的生物合成

• 批准号: 6777896
• 负责人: PAUL F COOK
• 金额: $ 26.34万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777896
• 关键词: NAD(P)H dehydrogenase; Saccharomyces cerevisiae; X ray crystallography; acidity /alkalinity; aminoacid biosynthesis; catalyst; chemical kinetics; crystallization; enzyme activity; enzyme mechanism; fluorescent dye /probe; fungal proteins; gene expression; ligands; lysine; mutant; oxoacid lyase; protein binding; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): main infectious agents in this regard are the opportunistic pathogens Candida albicans (responsible for candidiasis) and AspergiUus fumigatus (responsible for aspergillosis), and Cryptococcus neoformans (responsible for cryptococcosis). The aminoadipate pathway for the biosynthesis of lysine in fungi is for the most part unique to these organisms. The seven-enzyme pathway has been studied to a limited extent, but all of the reactions have mechanistic precedence in other systems. The PI initially proposes to study the regulated homocitrate synthase and the saccharopine dehydrogenases, which catalyze the final two steps in the pathway. Each of the enzymes will be studied to determine their mechanism with respect to transition state structure. Studies will be conducted according to the following specific aims. 1) The gene for the lysine-regulated homocitrate synthase has been obtained, expressed, purified and characterized. The genes for the glutamate- and lysine-forming saccharopine dehydrogenases have been obtained by PCR from the Saccharomyces cerevisiae genome, and are being subcloned into the pQE expression vector (Qiagen). They will then be expressed in Escherichia coli, and purified. 2) Each of the three enzymes will be crystallized in the absence and presence of appropriate ligands. One of the enzymes, saccharopine dehydrogenase (Lglutamate forming) from Magnaporthe grisea has been crystallized and had its structure solved in the presence of substrates. 3) Each of the three enzymes will be studied with respect to their kinetic, and catalytic mechanism using a variety of techniques including steady state and presteady state kinetic studies, measurement of pH-rate profiles and isotope effects, and site-directed mutagenesis. The only enzyme that has been studied mechanistically to any extent is the final enzyme in the pathway, saccharopine dehydrogenase (L-lysine forming), and these data will provide a starting point for further, more complete studies. 4) Site-directed mutagenesis will be carried out initially on saccharopine dehydrogenase (Lglutamate forming). Three residues have been selected as possible catalytic residues, and these will be changed and the resulting mutants will be characterized. Additionally, possible catalytic and binding residues in the homocitrate synthase have been identified by multiple sequence alignment, and alanine scanning mutagenesis will be applied to these.

描述（由申请人提供）： 在这方面，主要的传染剂是机会性病原体白色念珠菌（负责 念珠菌病）和曲霉菌（负责曲霉病）和加密环球球菌 （负责隐球菌病）。真菌中赖氨酸生物合成的氨基化途径在很大程度上是这些生物体所特有的。已经对七酶途径进行了有限的研究，但是所有反应在其他系统中具有机械优先级。 PI最初提议研究调节的同二酸合酶和糖含量脱氢酶，从而催化途径的最后两个步骤。将研究每种酶，以确定其在过渡态结构方面的机制。研究将根据以下特定目的进行。 1）已获得，表达，纯化和表征赖氨酸调节的同核酸合酶的基因。谷氨酸和赖氨酸形成的糖含量脱氢酶的基因是从酿酒酵母基因组中获得的，并被亚克隆到PQE表达载体（QIAGEN）中。然后，它们将在大肠杆菌中表达并净化。 2）在不存在和存在适当的配体的情况下，三种酶中的每种都将结晶。其中一种酶，糖含量脱氢酶（LGLUTAMATE形成）来自Magnaporthe Grisea，已结晶，并在存在底物的情况下解决了其结构。 3）将研究三种酶的每种动力学和催化机制，使用各种技术，包括稳态和质疑状态动力学研究，测量pH率谱和同位素效应以及定向诱变。唯一在任何程度上进行了机械研究的酶是途径中的最终酶，糖charopine脱氢酶（L-赖氨酸形成），这些数据将为进一步，更完整的研究提供一个起点。 4）定点诱变最初将在糖charopine脱氢酶（LGLUTAMATE形成）上进行。选择了三个残基作为可能的催化残基，这些残基将被更改，并将所得突变体进行表征。另外，已经通过多个序列比对鉴定了同叶酸酯合酶中可能的催化和结合残基，并且丙氨酸扫描诱变将应用于这些诱变。