Structure and functioin of 2-oxoacid : ferredoxin oxidoreductase

通过测定嗜热酸性古菌tokodaisulfolobus PKOR的三维结构，构建了PKOR基因表达体系。利用该体系制备、纯化和结晶重组PKOR。x射线衍射达到3埃。在我们之前的工作中，PKOR晶体衍射高达5-6埃，因此我们的分析得到了很大的改进。利用非洲Desulfovibrio africanus的POR模型分子的PDB数据进行分子替代分析。然而，我们找不到任何解决办法。这可能是两种酶的相似性太差，迄今为止，获得PKOR的硒- met衍生物的试验尚未成功，但结晶条件仍在进一步研究中为了测定与辅酶a相互作用的氨基酸残基，采用nbd -氟化物作为PKOR的亲和标记试剂。NBD-F在不同浓度下使酶失活，受辅酶a的保护。NBD-F掺入到b-su中，PKOR的单位数增加，荧光随之增加。nbd标记的b亚基被分离出来，并被赖氨酸内肽酶消化，得到从b亚基中赖氨酸旁边的位置开始的多肽混合物。采用反相高效液相色谱法分离荧光多肽。氨基酸序列测定表明，该荧光肽片段为含有Lys-125和Lys-173两种多肽的混合物。由于这些赖氨酸残基是与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，该残基负责结合PKOR中的CoAtokodaii Sulfolobus基因组包含多个2-氧酸：铁氧还蛋白氧化还原酶基因，选择其中IOR基因在大肠杆菌中表达。IOR是一个异源二聚体，基因重叠，表达载体在T7启动子后串联或分别携带两个亚基基因。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菌株正在进一步调查中。少

[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