title: Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure.
abstract: We previously reported the presence of a highly active, carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. In this study, structural analysis of Pk -Rubisco has been performed. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. In order to investigate the existence of small subunits in native Pk -Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk -Rubisco was detected when the cell-free extracts of P. kodakaraensis KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk -Rubiscos exhibited identical mobilities on native-PAGE. These results indicated that native Pk -Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk -Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk -Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly 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 structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, 606-8501, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Maeda
authors.forename: K
authors.initials: K
authors.lastname: Kitano
authors.forename: T
authors.initials: T
authors.lastname: Fukui
authors.forename: S
authors.initials: S
authors.lastname: Ezaki
authors.forename: H
authors.initials: H
authors.lastname: Atomi
authors.forename: K
authors.initials: K
authors.lastname: Miki
authors.forename: T
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000648:ultrastructure; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008854:Microscopy, Electron; D010802:Phylogeny; D011233:Precipitin Tests; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D014961:X-Ray Diffraction
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase