Structure and functioin of 2-oxoacid : ferredoxin oxidoreductase

[1]为了确定嗜热嗜酸古菌Sulfolobus tokodaii PKOR的三维结构，构建了基因表达系统。使用该系统，生产、纯化和结晶重组PKOR。实现了高达3埃的X射线衍射。在我们以前的工作中，PKOR晶体的衍射可达5-6埃，因此本分析有很大改进。使用来自非洲脱硫弧菌的模型分子POR的PDB数据进行分子置换分析。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的缘故。迄今为止，获得PKOR的硒-Met衍生物的试验还没有成功，但结晶条件正在进一步研究中。[2]为了确定与辅酶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的浓度依赖性地使酶失活，这受到CoA的保护。NBD-F被并入b-su ...更多信息 b单位的PKOR，伴随着荧光增强。分离NBD标记的b-亚基，并通过赖氨酰内肽酶消化，以获得在b-亚基中与Lys相邻的位置处开始的多肽的混合物。将混合物应用于反相HPLC以分离荧光多肽。荧光肽组分的氨基酸序列测定表明，它是两种多肽的混合物，其中含有Lys-125和Lys-173。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体PKOR：b-Lys 125 Ala和b-Lys 173 Ala。动力学分析表明，Lys 125是与NBD-F反应的残基，该残基负责结合PKOR中的CoA。[3]硫化叶菌基因组中含有多个2-酮酸：铁氧还蛋白氧化还原酶基因，从中筛选出IOR基因并在大肠杆菌中表达。杆菌IOR为异源二聚体，基因重叠，表达载体在T7启动子后分别串联携带两个亚基基因。然而，到目前为止，在重组大肠杆菌中没有监测到IOR活性的表达。杆菌对培养条件、寄主E.大肠杆菌，正在进一步研究中。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