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Structure and functioin of 2-oxoacid : ferredoxin oxidoreductase

2-含氧酸的结构和功能：铁氧还蛋白氧化还原酶

基本信息

批准号：
18580088
负责人：
WAKAGI Takayoshi
金额：
$ 2.48万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580088/
关键词：
thermophile archaea oxidoreductase affinity label フェレドキシン 2-オキソ酸

项目摘要

[1] Tb determine three-dimensional structure of PKOR from Sulfolobus tokodaii, a thermoacidophilic archaon, gene expression system was constructed. Using this system, recombinant PKOR was produced, purified and crystallized. X-ray diffraction upto 3 angstrom was achieved. In our former effort, PKOR crystal diffracted upto 5-6 angstrom, so the present analysis was greatly improved. Molecular replacement analysis was carried out using PDB data of a model molecule, POR from Desulfovibrio africanus,. However we could not find any solution. This is probably the similarity of the two enzymes is too poor Trials to obtain a seleno-Met derivative of PKOR were not successful so far, but the crystallizing condition is further being investigated.[2] Tb determine the amino acid residue that interacts with coenzyme-A, NBD-fluoride was used as an affinity-labeling reagent of PKOR. NBD-F inactivated the enzyme dependent on its concentration, which was protected by CoA. NBD-F was incorporated into b-su … More bunit of PKOR, with concomitant increase of fluorescence. NBD-labeled b-subunit was isolated and digested by lysyl endopeptidase to obtain a mixture of polypeptides that starts at the position next to Lys in the b-subunit. The mixture was applied to reverse phase HPLC to isolate a fluorescent, polypeptide. Amino acid sequence determination of the fluorescent peptide fraction revealed that it was a mixture of two polypeptides, which contain Lys-125 and Lys-173. Since these Lys residues are the candidates that reacted with NBD-F, two mutant PKORs were constructed : b-Lys125Ala, and b-Lys173Ala. Kinetic analysis revealed that Lys125 was the residue that reacted with NBD-F, and this residue is responsible for binding CoA in the PKOR.[3] Sulfolobus tokodaii genome contains several 2-oxoacid:ferredoxin oxidoreductase genes, among which IOR genes were selected and subjected to expression in E. coli. The IOR is (a dieter of) a heterodimer and the genes were overlapped, expression vector carrying two subunit genes tandemly and separately after T7 promoter. So far, however, no expression of the IOR activity was monitored in recombinant E. coli. Culture condition, as well as strains of host E. coli, is further being investigated. Less

[1]构建了耐高温嗜酸古细菌Tb确定tokodaii PKOR三维结构的基因表达系统。利用该系统对重组PKOR进行了生产、纯化和结晶。获得了高达3埃的X射线衍射谱。在我们以前的工作中，PKOR晶体的衍射角达到了5-6埃，因此我们的分析方法得到了很大的改进。利用非洲脱硫弧菌模型分子POR的PDB数据进行了分子置换分析。然而，我们找不到任何解决方案。这可能是两种酶的相似性太差，到目前为止还没有成功获得PKOR的硒-蛋氨酸衍生物，但结晶条件正在进一步研究中。[2]Tb确定了与辅酶-A相互作用的氨基酸残基，以NBD-氟化物作为PKOR的亲和标记剂。NBD-F对该酶的失活依赖于其浓度，并受CoA的保护。Nbd-F并入b-su…PKOR单位越多，荧光强度越强。分离NBD标记的b亚基，用赖氨酰内切酶消化，得到从b亚基中Lys旁边的位置开始的多肽混合物。将该混合物应用于反相高效液相色谱分离荧光多肽。氨基酸序列测定表明，该荧光肽是由含有Lys-125和Lys-173的两个多肽组成的混合物。由于这些Lys残基是与NBD-F反应的候选者，因此构建了两个突变型PKOR：B-Lys125Ala和b-Lys173Ala。动力学分析表明，Lys125是与NBD-F反应的残基，该残基负责与PKOR中的CoA结合。[3]tokodaii基因组中含有几个2-氧代酸：铁氧还蛋白氧化还原酶基因，其中IOR基因被筛选并在大肠杆菌中表达。IOR是异源二聚体，基因重叠，T7启动子后分别携带两个亚单位基因的表达载体。然而，到目前为止，还没有在重组大肠杆菌中检测到IOR活性的表达。培养条件以及宿主大肠杆菌的菌株正在进一步调查中。较少