Lysine Biosynthesis in Yeast

描述(由申请人提供)： 这方面的主要感染源是条件致病菌白色念珠菌(负责 假丝酵母菌)和烟曲霉菌(导致曲霉病)，以及新生隐球菌 (对隐球菌病负责)。在真菌中，氨基己二酸酯合成赖氨酸的途径在很大程度上是这些生物所独有的。人们对七种酶途径的研究很有限，但所有的反应在其他体系中都有机理上的优先。PI最初建议研究受调控的高柠檬酸合成酶和糖胺脱氢酶，这两个酶催化该途径的最后两步。将对每种酶进行研究，以确定它们与过渡态结构相关的机制。将根据以下具体目标进行研究。1)获得了赖氨酸调节的高柠檬酸合成酶基因，并对其进行了表达、纯化和鉴定。利用聚合酶链式反应从酿酒酵母基因组中获得了谷氨酸和赖氨酸形成糖胺脱氢酶的基因，并将其亚克隆到pQE表达载体(Qiagen)中。然后它们将在大肠杆菌中表达，并进行纯化。2)这三种酶中的每一种都将在没有或存在合适的配体的情况下结晶。稻瘟病菌产生的糖胺脱氢酶(LGlu)是其中的一种酶，在有底物存在的情况下进行了结晶和结构解析。3)将使用各种技术来研究这三种酶的动力学和催化机理，包括稳态和稳态前的动力学研究，pH速率分布和同位素效应的测量，以及定点突变。唯一在一定程度上被机械研究的酶是该途径中的最后一种酶，糖胺脱氢酶(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)首先对糖胺脱氢酶(谷氨酸形成)进行定点突变。已经选择了三个残基作为可能的催化残基，这些残基将被改变，所产生的突变体将被表征。此外，高柠檬酸合成酶中可能的催化残基和结合残基已经通过多个序列比对确定，并将对这些残基进行丙氨酸扫描突变。

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.