S. Ezaki et al., Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, J BIOL CHEM, 274(8), 1999, pp. 5078-5082
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.