Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure

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.