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 carboxyla se/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodaka raensis KOD1, The gene encoded a protein consisting of 444 amino acid resid ues, corresponding in size to the large subunit of previously reported Rubi scos, Rubisco of P. kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% simil arity 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 enz yme was not a member of either type. Active site residues identified from t ype I and type II Rubiscos were conserved. We expressed the gene in Escheri chia coli, and we obtained a soluble protein with the expected molecular ma ss and N-terminal amino acid sequence. Purification of the recombinant prot ein revealed that Pk-Rubisco was an L-8 type homo-octamer. Pk-Rubisco showe d highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, a nd a tau value of 310 at 90 degrees C, both higher than any previously char acterized Rubisco, The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degrees C, Northern blot a nalysis demonstrated that the gene was transcribed in P. kodakaraensis KOD1 . Furthermore, Western blot analysis with cell-free extract of P. kodakarae nsis KOD1 clearly indicated the presence of Pk-Rubisco in the native host c ells.