| Budget Amount *help |
¥31,100,000 (Direct Cost: ¥31,100,000)
Fiscal Year 2000: ¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 1999: ¥21,100,000 (Direct Cost: ¥21,100,000)
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| Research Abstract |
We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P.kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90
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degreesC, both higher than any previously characterized Rubisco. Northern blot analysis demonstrated that the gene was transcribed in P.kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P.kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells. In order to investigate the existence of small subunits in native Pk-Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk-Rubisco was detected when the cell-free extracts of KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk-Rubiscos exhibited identical mobilities on native-PAGE.These results indicated that native Pk-Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk-Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk-Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk -Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos. Less
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