XAS STUDIES OF NOVEL ARSENIC BINDING SITES IN AS (III)-RESPONSIVE TRANSCRIPTIONA

AS (III) 响应转录中新型砷结合位点的 XAS 研究

• 批准号: 7598285
• 负责人: BARRY P. ROSEN
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598285
• 关键词: Arsenic; Bacteria; Binding; Binding Sites; C-terminal; Computer Retrieval of Information on Scientific Projects Database; Corynebacterium glutamicum; Cysteine; DNA Binding Domain; Drug Metabolic Detoxication; Funding; Genes; Glutamic Acid; Goals; Gold; Grant; Hydroxyl Radical; Industry; Institution; Ligands; Location; Methyltransferase; Mining; Molecular Chaperones; Mutagenesis; N-terminal; Personal Satisfaction; Plasmids; Production; Proteins; Research; Research Personnel; Resources; Site; Source; Sulfuric Acids; Transcription Repressor/Corepressor; United States National Institutes of Health; mutant; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is characterization of arsenic binding sites in proteins involved in arsenic detoxification. ArsRs are As(III)-responsive transcriptional repressors that regulate expression of arsenic detoxification genes. We have identified three homologous ArsRs from three different bacteria but apparently have evolved different arsenic binding sites in different locations in each protein. The first is the well-characterized ArsR from plasmid R773. The binding site in this repressor is an S3 site composed of three co-linear cysteine residues, Cys32, Cys34 and Cys37 in the DNA binding domain. The second is an ArsR from Acidothiobacillus ferrooxidans, a sulfuric acid-producing bacterium used in the gold mining industry. Preliminary EXAFS results suggest that the site in this ArsR is a mixed S/O/N site, suggesting an S2 site and a hydroxyl ligand. This repressor has a C-terminal vicinal cysteine pair residues that may form an As(III) binding site. The third ArsR is from In Corynebacterium glutamicum, which is used for the production of glutamic acid. This ArsR has an N-terminal vicinal cysteine pair and a third cysteine near the DNA binding domain that we hypothesize forms an S3 site for As(III). EXAFS of each of the three repressors with bound As(III) will differentiate between these possibilities. Mutant ArsRs lacking cysteine residues, singly and in combination, will be used to identify the specific ligands in each site. ArsD is a novel As(III) metallochaperone. It has three vicinal cysteine pairs and several other cysteines. Cys12,13 and 18 have been identified by mutagenesis as required for chaperone activity. ArsD mutants in these cysteine residues, singly and in combination, will be used to characterize the As(III) binding site. ArsM is a newly identified As(III)-SAM methyltransferase that methylates As(III) to a variety of species. Participation of conserved residues Cys30 and 31 in As(III) binding site will be investigated.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的总体目标是确定参与砷解毒的蛋白质中砷结合部位的特征。ArsRs是AS(III)反应的转录抑制因子，调节砷解毒基因的表达。我们已经从三个不同的细菌中鉴定出三个同源的ArsR，但显然在每个蛋白质的不同位置进化了不同的砷结合部位。第一个是来自R773的具有良好特性的ArsR。该抑制子的结合部位是DNA结合区的三个共线半胱氨酸残基Cys32、Cys34和Cys37组成的S3位点。第二种是来自氧化亚铁硫杆菌的ArsR，这是一种用于黄金开采行业的硫酸产生菌。初步的EXAFS结果表明，该ArsR是一个混合的S/O/N位，可能是一个S2位和一个羟基配体。该抑制子具有C端邻近的半胱氨酸对残基，可形成As(III)结合部位。第三个ArsR来自谷氨酸棒状杆菌，用于生产谷氨酸。这个ArsR有一个N端邻近的半胱氨酸对和第三个半胱氨酸在DNA结合域附近，我们假设它形成了As(III)的S3位点。结合了As(III)的三个阻遏子的EXAFs将区分这些可能性。缺乏半胱氨酸残基的突变型ARSR将单独或组合用于鉴定每个位点的特定配体。ARSD是一个新的AS(III)金属配位体。它有三个邻近的半胱氨酸对和其他几个半胱氨酸。经突变鉴定，Cys12、13和18是伴侣活性所必需的。这些半胱氨酸残基中的ARSD突变体，无论是单独的还是组合的，都将被用来表征As(III)的结合部位。ArsM是一种新发现的(III)-SAM甲基转移酶，可与多种物种发生(III)甲基化。将研究保守残基Cys30和31在As(III)结合部位的参与情况。