Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure
N. Maeda et al., Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure, J MOL BIOL, 293(1), 1999, pp. 57-66
We previously reported the presence of a highly active, carboxylase-specifi
c ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hypertherm
ophilic archaeon, Pyrococcus kodakaraensis KOD1. In this study, structural
analysis of Pk-Rubisco has been performed. Phylogenetic analysis of Rubisco
s indicated that archaeal Rubiscos, including Pk-Rubisco, were distinct fro
m previously reported type I and type II enzymes in terms of primary struct
ure. In order to investigate the existence of small subunits in native Pk-R
ubisco, immunoprecipitation and native-PAGE experiments were performed. No
specific protein other than the expected large subunit of Pk-Rubisco was de
tected when the cell-fr ee extracts of P. kodakaraensis KOD1 were immunopre
cipitated with poly clonal antibodies against the recombinant enzyme. Furth
ermore, native and recombinant Pk-Rubiscos exhibited identical mobilities o
n native-PAGE. These results indicated that native Plc-Rubisco consisted so
lely of large subunits. Electron micrographs of purified recombinant Pk-Rub
isco displayed pentagonal ring-like assemblies of the molecules. Crystals o
f Plc-Rubisco obtained from ammonium sulfate solutions diffracted X-rays be
yond 2.8 Angstrom resolution. The self-rotation function of the diffraction
data showed the existence of 5-fold and 2-fold axes, which are located per
pendicularly 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 st
ructure. This is the first report of a decameric assembly of Rubisco, which
is thought to belong to neither type I nor type II Rubiscos. (C) 1999 Acad
emic Press.