title: Characterization of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability.
abstract: Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (β-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach α-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the α-isoform can act as a structural scaffold for hexamer formation by the β-isoform. Additionally, it is shown that a variant of the tobacco β-isoform acts in a similar fashion to the α-isoform of spinach, forming thermally stable hexamers in the presence of ATPγS. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface.
authors.affiliation: *Biomolecular Interactions Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand.
authors.forename: Jeremy R
authors.initials: JR
authors.lastname: Keown
authors.affiliation: *Biomolecular Interactions Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand.
authors.forename: Frederick Grant
authors.initials: FG
authors.lastname: Pearce
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D050505:Mutant Proteins; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D007527:Isoenzymes; D050505:Mutant Proteins; D010940:Plant Proteins; C054765:rca protein, plant; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium