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] TB从Sulfolobus Tokodaii唱出PKOR的三维结构，生产重组PKOR，纯化和结晶，PKOR晶体衍射为5-6 Angstrom，因此当前的分析得到了极大的改善Desulfovibrio Africanus的模型分子的PDB数据，我们找不到任何解决方案。氟化物是PKOR的亲和力标记试剂，将NBD-F置于B-SU中，并伴随着荧光的增加。相HPLC分离荧光肽的荧光，多肽氨基酸序列的测定显示，这是两个多肽173的混合物在pkor中与nbd-f的coa反应。活性在重组中进行了监测