Lysine Biosynthesis in Yeast

描述（由申请人提供）： 这方面的主要传染源是机会性病原体白色念珠菌（负责 念珠菌病）和烟曲霉（负责曲霉病）和新型隐球菌 （负责隐球菌病）。真菌中生物合成赖氨酸的氨基己二酸途径在很大程度上是这些生物体所独有的。七酶途径的研究程度有限，但所有反应在其他系统中都具有机械优先性。 PI 最初提议研究受调节的高柠檬酸合酶和糖苷脱氢酶，它们催化该途径的最后两个步骤。将研究每种酶以确定它们在过渡态结构方面的机制。研究将根据以下具体目标进行。 1) 赖氨酸调节的高柠檬酸合酶基因已获得、表达、纯化和表征。形成谷氨酸和赖氨酸的糖苷脱氢酶的基因已通过 PCR 从酿酒酵母基因组中获得，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纯化。 2) 三种酶中的每一种都会在适当配体存在和不存在的情况下结晶。其中一种酶，来自稻瘟病菌的糖碱脱氢酶（形成 L 谷氨酸）已被结晶，并在底物存在下解析了其结构。 3) 将使用多种技术来研究这三种酶的动力学和催化机制，包括稳态和前稳态动力学研究、pH 速率曲线和同位素效应的测量以及定点诱变。唯一在某种程度上进行过机械研究的酶是该途径中的最终酶，糖苷脱氢酶（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)首先对糖碱脱氢酶（L谷氨酸形成）进行定点诱变。已选择三个残基作为可能的催化残基，并且将改变这些残基并对所得突变体进行表征。此外，高柠檬酸合酶中可能的催化和结合残基已通过多重序列比对鉴定出来，丙氨酸扫描诱变将应用于这些残基。

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.