title: Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.
abstract: The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.
authors.affiliation: From the Department of Microbiology, The Ohio State University, Columbus, Ohio 43210-1292 and.
authors.forename: Sriram
authors.initials: S
authors.lastname: Satagopan
authors.affiliation: UCLA-Department of Energy (DOE) Institute for Genomics and Proteomics, UCLA, Los Angeles, California 90095-1570.
authors.forename: Sum
authors.initials: S
authors.lastname: Chan
authors.affiliation: UCLA-Department of Energy (DOE) Institute for Genomics and Proteomics, UCLA, Los Angeles, California 90095-1570.
authors.forename: L Jeanne
authors.initials: LJ
authors.lastname: Perry
authors.affiliation: From the Department of Microbiology, The Ohio State University, Columbus, Ohio 43210-1292 and tabita.1@osu.edu.
authors.forename: F Robert
authors.initials: FR
authors.lastname: Tabita
mesh_terms: D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012241:Rhodopseudomonas / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001704:Biopolymers; D004269:DNA, Bacterial; D012273:Ribulose-Bisphosphate Carboxylase
keywords: Carbon Fixation; Enzyme Catalysis; Enzyme Structure; Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco); Site-directed Mutagenesis