Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.
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 degreesC, both higher than any previously characterized Rubisco. The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC. 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.
Ezaki
S
S
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Maeda
N
N
Kishimoto
T
T
Atomi
H
H
Imanaka
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004269:DNA, Bacterial; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid