| Budget Amount *help |
¥3,880,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
[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
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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
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