Structure-Function Studies on the Sulfate Activating Enzymes

硫酸盐活化酶的结构-功能研究

• 批准号: 0515352
• 负责人: Irwin Segel
• 金额: --
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0515352&HistoricalAwards=false
• 关键词: Structure; Function; Studies; Sulfate; Activating

This research project focuses on the structural features underlying the catalytic and regulatory properties of the first sulfate activating enzyme, ATP sulfurylase. ATP sulfurylase plays multiple roles in nature: (a) In fungi, yeasts, most heterotrophic bacteria, algae, and higher plants, this enzyme catalyzes the first intracellular reaction in the reductive assimilation of inorganic sulfate into organic molecules. In these organisms, APS is the active sulfate precursor of cysteine, methionine, etc. In all organisms, PAPS serves as the sulfuryl donor for sulfate ester biosynthesis. (b) In anaerobic sulfate reducing bacteria (e.g., Desulfovibrio), ATP sulfurylase forms APS solely to serve as the terminal electron acceptor of heterotrophic metabolism. (c) In certain chemo- and photolithotrophic bacteria (e.g., Aquifex, Chromatium), ATP sulfurylase catalyzes the last reaction in the oxidation of reduced inorganic sulfur compounds to sulfate i.e., the physiological reaction is in the opposite direction compared to that in sulfate assimilators. Sulfate (sulfuric acid) produced by sulfur chemolithotrophs is believed to be responsible for shaping many terrestrial caverns and for the acidic runoff from pyrite-containing mines. A long-term goal of this project is to identify the structural features that optimize the catalytic and regulatory properties of each class of homooligomeric ATP sulfurylase for the physiologically relevant task. Immediate efforts will focus on the homohexameric enzyme of Penicillium chrysogenum, which is allosterically inhibited by PAPS, the product of the APS kinase-catalyzed reaction. Experiments are proposed to determine (a) the mechanism by which the APS kinase-like regulatory domain communicates with the catalytic domain, (b) the structural basis for the high selectivity of the catalytic and allosteric sites for sulfate/sulfonucleotide over the more common phosphorous analogs, and (c) the functional properties of several chimeric forms including one that contains true APS kinase as the C-terminal domain and may channel APS between sites.Broader Impacts: A cross section of California students will receive training in the methods of molecular biology (cloning, site-directed mutagenesis, expression and purification of mutant and chimeric enzymes), enzymology (steady-state kinetics, equilibrium ligand binding, computer-assisted simulation and analysis of data), and protein chemistry (x-ray crystallography, chemical modification of target residues). The experiences help to prepare students for advanced academic programs or for entry-level positions in biotechnology firms. Students from underrepresented groups participate in our research as part of the Biology Undergraduate Scholars Program (BUSP) and Minority Research Participation in the Physical Sciences Program (MRPPSP).

该研究项目侧重于第一个硫酸盐激活酶ATP硫酸盐酶的催化和调节性能的基础。 ATP硫化物在自然界中起多种作用：（a）在真菌，酵母，大多数异养细菌，藻类和较高植物中，这种酶在将无机硫酸盐无机硫酸盐还原为有机分子中催化了第一个细胞内反应。在这些生物体中，AP是半胱氨酸，蛋氨酸等的活性硫酸盐前体。在所有生物体中，PAPs都是硫酸盐酯生物合成的硫酸盐供体。 （b）在厌氧硫酸盐还原细菌（例如Desulfovibrio）中，ATP硫酶形成APS仅作为异营代谢的末端电子受体。 （c）在某些化学和光致营养细菌（例如Aquifex，染色体）中，ATP硫酶催化了氧化降低的无机硫化合物对硫酸盐的氧化的最后一个反应，即与硫酸硫酸盐相比，生理反应在相反的方向上是相反的。据信，硫酸盐（硫酸）被认为是造成许多陆生洞穴和含含黄铁矿矿山的酸性径流的原因。该项目的一个长期目标是确定针对生理上相关任务的每类家族类ATP硫酸盐群的催化和调节性能的结构特征。即时的努力将集中在甲基氏菌的同兆列酶酶上，该酶在PAP上被短构抑制，这是APS激酶催化反应的产物。提出了实验来确定（a）APS激酶样调节结构域与催化结构域通信的机制，（b）硫酸/硫酸盐/硫酸盐核苷酸对较常见的磷酸化模拟和（c）的功能性属性的高度选择性的结构基础，包括更常见领域和可能在站点之间引导AP。公民影响：加利福尼亚州的一个横截面将接受分子生物学方法（克隆，定位定位的诱变，突变和嵌合酶的表达和纯化），酶学，酶学，稳态动力学，平衡结合，计算机化和分析的晶体学和分析，启用了分析和分析，启用了分析，启用了分析，启用了分析，启用了均匀的晶体学和分析。修改目标残留物）。这些经验有助于为学生准备高级学术课程或生物技术公司的入门级职位。作为生物学本科学者计划（BUSP）和少数群体研究参与物理科学计划（MRPPSP）的一部分，来自代表性不足的小组的学生参加了我们的研究。