THE ATP-COUPLED ARSENICAL PUMP OF ESCHERICHIA COLI

大肠杆菌的 ATP 耦合砷泵

• 批准号: 6240144
• 负责人: BARRY P. ROSEN
• 金额: $ 2.69万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240144
• 关键词: Escherichia coli; active sites; adenosine triphosphate; adenosinetriphosphatase; anions; antibacterial agents; arsenic; dimer; drug resistance; molecular biology; plasmids; protein structure function; receptor coupling; site directed mutagenesis

The overall goals of this research are elucidation of the molecular mechanisms of an ion pump whose genes are natural components of a bacterial resistance plasmid, and second, the role of this transport system in bacterial resistance to antibiotics and toxic compounds. The clinical resistance plasmid R773 carries the arsenical resistance (ars)operon, which produces resistance to arsenate, arsenite, and antimonite. The operon encodes an oxyanion-translocating ATPase which functions as an ATP-coupled extrusion pump for the toxic oxyanions. The catalytic subunit of the pump is the ArsA protein, an arsenite-stimulated ATPase. The specific aims of the project involve a structure-function analysis of the ArsA protein. In particular a) the role of residues within the two nucleotide binding sites will be examined by molecular genetic and biochemical approaches; b) the four cysteine residues will be mutagenized to examine their role in catalysis; c) the domains involved in dimerization and interaction with the other components of the pump will be determined. While plasmid-mediated antibiotic and heavy metal resistances which are due to energy-dependent efflux systems may be wide spread in nature, anion pumps appear to be rather rare. The arsenical efflux system provide a good model system for the study of transmissible bacterial antibiotic resistances. The plasmid encoded ars system also provides a bacterial model for the study of multidrug resistance in mammalian cells. The Ars ATPase exhibits structural and functional similarity to the P-glycoprotein, the protein which produces multiple drug resistance in tumor cells.

本研究的总体目标是阐明 其基因是细菌的天然成分的离子泵的机制 耐药质粒，第二，这种运输系统在 细菌对抗生素和有毒化合物的耐药性。 临床 抗性质粒R773携带砷抗性操纵子， 对砷酸盐、亚砷酸盐和亚锑酸盐产生抗性。 操纵子 编码氧阴离子转运ATP酶，其功能为ATP偶联的 有毒含氧阴离子的挤压泵 泵的催化子单元 是ArsA蛋白，一种亚砷酸盐刺激的ATP酶。 的具体目标 该项目涉及ArsA蛋白的结构-功能分析。 在 特别是a）两个核苷酸结合位点内残基的作用 将通过分子遗传学和生物化学方法进行检查; B） 四个半胱氨酸残基将被诱变，以检查它们在 催化; c）参与二聚化的结构域和与二聚体的相互作用; 将确定泵的其它部件。 而质粒介导的抗生素和重金属抗性， 依赖能量的外排系统可能在自然界中广泛传播，阴离子 水泵似乎相当罕见。 砷外排系统提供了一个良好的 用于研究传染性细菌抗生素的模型系统 阻力。 质粒编码的ars系统还提供了一种细菌 用于研究哺乳动物细胞中的多药耐药性的模型。 索马里再次解放联盟 ATP酶表现出与P-糖蛋白的结构和功能相似性， 在肿瘤细胞中产生多重耐药性的蛋白质。