Structure-Function Studies on the Sulfate Activating Enzymes

该研究项目的重点是第一个硫酸盐活化酶ATP硫酸化酶的催化和调节特性的结构特征。 ATP硫酸化酶在自然界中起着多种作用：（a）在真菌、酵母、大多数异养细菌、藻类和高等植物中，这种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供体。(b)在厌氧硫酸盐还原菌（例如，ATP硫酸化酶形成APS，仅作为异养代谢的末端电子受体。(c)在某些化学营养型和营养型细菌（例如，Aquifex，Chromatium），ATP硫酸化酶催化还原的无机硫化合物氧化成硫酸盐的最后反应，即，其生理反应方向与硫酸盐同化物相反。由硫化学无机营养菌产生的硫酸盐（硫酸）被认为是形成许多陆地洞穴和含黄铁矿矿山酸性径流的原因。该项目的长期目标是确定优化生理相关任务的每一类同源寡聚ATP硫酸化酶的催化和调节特性的结构特征。近期的努力将集中在产黄青霉的同六聚体酶上，该酶被APS激酶催化反应的产物PAPS变构抑制。提出实验以确定（a）APS激酶样调节结构域与催化结构域通信的机制，（B）硫酸酯/磺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，和（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-终端域，并可在站点之间传输APS。更广泛的影响：加州的学生将接受分子生物学方法的培训（突变体和嵌合酶的克隆、定点诱变、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、靶残基的化学修饰）。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加我们的研究作为生物本科学者计划（BUSP）和少数民族研究参与物理科学计划（MRPPSP）的一部分。

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).