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

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。该项目的总体目标是表征参与砷解毒的蛋白质中的砷结合位点。 ArsR 是 As(III) 响应性转录抑制因子，可调节砷解毒基因的表达。我们已经从三种不同的细菌中鉴定出三种同源的 ArsR，但显然在每种蛋白质的不同位置进化出了不同的砷结合位点。第一个是来自质粒 R773 的已充分表征的 ArsR。该阻遏物中的结合位点是由 DNA 结合域中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，这是一种用于金矿开采行业的产硫酸细菌。初步 EXAFS 结果表明，该 ArsR 中的位点是混合的 S/O/N 位点，表明有 S2 位点和羟基配体。该阻遏蛋白具有 C 端邻近半胱氨酸对残基，可形成 As(III) 结合位点。第三个ArsR来自谷氨酸棒杆菌，用于生产谷氨酸。该 ArsR 有一个 N 端邻近半胱氨酸对和靠近 DNA 结合结构域的第三个半胱氨酸，我们假设它形成 As(III) 的 S3 位点。结合 As(III) 的三个阻遏蛋白中的每一个的 EXAFS 将区分这些可能性。缺乏半胱氨酸残基的突变体 ArsR，单独或组合，将用于鉴定每个位点的特定配体。 ArsD 是一种新型 As(III) 金属伴侣。它具有三个邻近的半胱氨酸对和几个其他半胱氨酸。 Cys12、13 和 18 已通过诱变鉴定为伴侣活性所需。这些半胱氨酸残基中的 ArsD 突变体（单独或组合）将用于表征 As(III) 结合位点。 ArsM 是一种新鉴定的 As(III)-SAM 甲基转移酶，可将 As(III) 甲基化为多种物种。将研究 As(III) 结合位点中保守残基 Cys30 和 31 的参与。