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).

该研究项目重点研究第一种硫酸盐活化酶 ATP 硫酸化酶的催化和调节特性背后的结构特征。 ATP 硫酸化酶在自然界中发挥着多种作用：（a）在真菌、酵母、大多数异养细菌、藻类和高等植物中，该酶催化无机硫酸盐还原同化为有机分子的第一个细胞内反应。在这些生物体中，APS 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 充当硫酸酯生物合成的硫酰基供体。 (b) 在厌氧硫酸盐还原菌（例如脱硫弧菌）中，ATP 硫酸化酶形成 APS 仅作为异养代谢的末端电子受体。 (c) 在某些化能和光能营养细菌（例如 Aquifex、Chromatium）中，ATP 硫酸化酶催化还原性无机硫化合物氧化成硫酸盐的最后一个反应，即与硫酸盐同化器中的生理反应方向相反。据信，由硫化能自养生物产生的硫酸盐（硫酸）是形成许多陆地洞穴以及含黄铁矿矿山的酸性径流的原因。该项目的长期目标是确定优化每类同聚 ATP 硫酸化酶催化和调节特性的结构特征，以完成生理相关任务。目前的研究重点将集中在产黄青霉的同型六聚体酶上，该酶受到 PAPS（APS 激酶催化反应的产物）的变构抑制。实验旨在确定 (a) APS 激酶样调节结构域与催化结构域通讯的机制，(b) 硫酸盐/磺基核苷酸的催化和变构位点相对于更常见的磷类似物的高选择性的结构基础，以及 (c) 几种嵌合形式的功能特性，包括含有真正的 APS 激酶作为 C 末端的嵌合形式 更广泛的影响：加州学生将接受分子生物学方法（克隆、定点诱变、突变体和嵌合酶的表达和纯化）、酶学（稳态动力学、平衡配体结合、计算机辅助模拟和数据分析）和蛋白质化学（X 射线晶体学、化学分析）方法的培训。 目标残基的修饰）。这些经验有助于学生为高级学术课程或生物技术公司的入门级职位做好准备。来自代表性不足群体的学生参与我们的研究，作为生物学本科学者计划 (BUSP) 和物理科学少数群体研究参与计划 (MRPPSP) 的一部分。