Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

• Published in: The Journal of biological chemistry 2014-08, Vol.289 (31), p.21433-21450
• Main Authors: Satagopan, Sriram, Chan, Sum, Perry, L.Jeanne, Tabita, F.Robert
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biopolymers - chemistry; Biopolymers - metabolism; Carbon Fixation; Crystallography, X-Ray; DNA, Bacterial - genetics; Enzyme Catalysis; Enzyme Structure; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure and Folding; Rhodopseudomonas - enzymology; Rhodopseudomonas - genetics; Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco); Ribulose-Bisphosphate Carboxylase - chemistry; Ribulose-Bisphosphate Carboxylase - genetics; Ribulose-Bisphosphate Carboxylase - metabolism; Sequence Homology, Amino Acid; Site-directed Mutagenesis; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.578625

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 (Ile165 and Met331) 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 (Ala47) 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. Background: Rubisco fixes atmospheric CO2 to organic carbon and sustains life on earth. Results: A form II Rubisco structure has been solved, and functional analysis was conducted with divergent residues. Conclusion: The unique structure combined with functional analysis can help better understand and improve Rubisco catalysis. Significance: This is the first high resolution structure of an activated transition-state analog (CABP)-bound form II Rubisco.