Lysine Biosynthesis in Yeast

描述（由申请人提供）： 在这方面的主要感染因子是机会性病原体白色念珠菌（Candida albicans）（负责 念珠菌病）和烟曲霉菌（引起曲霉病），以及新型隐球菌 （负责隐球菌病）。真菌中赖氨酸生物合成的氨基己二酸途径在很大程度上是这些生物体所特有的。七酶途径已经在有限的程度上进行了研究，但所有的反应在其他系统中具有机械优先性。PI最初建议研究受调节的高柠檬酸合酶和蔗糖嘌呤脱氢酶，它们催化途径中的最后两个步骤。将研究每种酶，以确定其过渡态结构的机制。将根据以下具体目标开展研究。1)赖氨酸调节的高柠檬酸合酶的基因已经获得，表达，纯化和表征。已通过PCR从酿酒酵母基因组获得形成谷氨酸和赖氨酸的糖嘌呤氧化酶的基因，并将其亚克隆到pQE表达载体（Qiagen）中。然后将它们在大肠杆菌中表达并纯化。2)三种酶中的每一种都将在不存在和存在适当配体的情况下结晶。稻瘟病菌的一种酶，即L-谷氨酸脱氢酶已被结晶，并在底物存在下解析了其结构。3)三种酶中的每一种都将使用各种技术研究其动力学和催化机制，包括稳态和presteady状态动力学研究，测量pH速率曲线和同位素效应，以及定点诱变。在任何程度上进行了机械研究的唯一酶是该途径中的最终酶，糖精脱氢酶（L-赖氨酸形成），这些数据将为进一步更完整的研究提供起点。4)首先对糖豆碱脱氢酶（L谷氨酸形成）进行定点诱变。已经选择了三个残基作为可能的催化残基，并且这些残基将被改变，并且所得突变体将被表征。此外，高柠檬酸合酶中可能的催化和结合残基已经通过多序列比对鉴定，并且丙氨酸扫描诱变将应用于这些。

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.