2000 Fiscal Year Final Research Report Summary
Efficient carbon dioxide fixation by a Rubisco from hyperthermophilic archaea
| Project/Area Number |
11305059
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
生物・生体工学
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
IMANAKA Tadayuki Grad.Sch.Engineering, KYOTO UNIVERSITY Prof., 工学研究科, 教授 (30029219)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
EZAKI Satoshi Grad.Sch.Engineering, KYOTO UNIVERSITY Assis. Prof., 工学研究科, 助手 (20291429)
ATOMI Haruyuki Grad.Sch.Engineering, KYOTO UNIVERSITY Assoc. Prof., 工学研究科, 助教授 (90243047)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Keywords | Rubisco / carbon dioxide fixation / archaeon / archaea / Calvin cycle |
|---|
| 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
… More
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
|
