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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。ArsRs是As（III）反应性转录抑制因子，调节砷解毒基因的表达。我们已经确定了三个同源的ArsRs从三种不同的细菌，但显然已经演变出不同的砷结合位点在不同的位置在每个蛋白质。第一个是来自质粒R773的充分表征的ArsR。该阻遏物中的结合位点是由DNA结合结构域中的三个共线性半胱氨酸残基Cys 32、Cys 34和Cys 37组成的S3位点。第二种是来自氧化亚铁酸硫杆菌的ArsR，氧化亚铁酸硫杆菌是一种用于金矿开采业的硫酸生产细菌。初步EXAFS结果表明，在这个ArsR的网站是一个混合的S/O/N网站，建议S2网站和羟基配体。该阻遏物具有C-末端邻位半胱氨酸对残基，其可形成As（III）结合位点。第三个ArsR来自谷氨酸棒杆菌，其用于生产谷氨酸。这ArsR有一个N-末端邻位半胱氨酸对和第三个半胱氨酸附近的DNA结合域，我们假设形成一个S3网站的As（III）。结合As（III）的三种阻遏物的EXAFS将区分这些可能性。缺乏半胱氨酸残基的突变体ArsR，单独和组合，将用于识别每个位点的特异性配体。ArsD是一种新型的As（III）金属伴侣。它有三个邻位半胱氨酸对和几个其他半胱氨酸。Cys 12、13和18已通过诱变鉴定为分子伴侣活性所需。这些半胱氨酸残基中的ArsD突变体，单独和组合，将用于表征As（III）结合位点。ArsM是一种新发现的As（III）-SAM甲基转移酶，其将As（III）甲基化为多种物种。将研究保守残基Cys 30和31在As（III）结合位点中的参与。

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.