Like many enzymes, the biogenesis of the multi-subunit CO 2 -fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/ oxygenase (Rubisco) in different organisms requires molecular chaperones. When expressed in Escherichia coli, the large (L) subunits of the Rubisco from the archaeabacterium Methanococcoides burtonii assemble into functional dimers (L 2 ). However, further assembly into pentamers of L 2 (L 10 ) occurs when expressed in tobacco chloroplasts or E. coli producing RuBP. The biosynthesis and maintenance of functional enzymes is highly dependant on the assistance of molecular chaperones. Both during and after protein translation, numerous chaperone associations and disassociations prevent nascent peptides, newly synthesized peptides, and even unfolded mature proteins from misfolding into biologically nonfunctional products (1). The trait shared by the diverse array of molecular chaperones is their involvement in noncovalently assisting the folding/unfolding and assembly/disassembly of macromolecular structures.The role of chaperonins in protein folding was demonstrated by showing that Escherichia coli chaperonin GroEL (Cpn60) and its co-chaperonin GroES (Cpn10) promote assembly of the photosynthetic CO 2 -fixing enzyme Form II ribulose-1,5-bisphosphate (RuBP) 2 carboxylase/oxygenase (Rubisco, EC 4.1.1.39) from the bacterium Rhodospirillum rubrum (2). R. rubrum Rubisco comprises two 50-kDa large (L) subunits that assemble head-to-tail into a dimeric (L 2 ) structure (3). Because of its simple structure, high level of expression in E. coli, and innate capacity to reassemble in vitro, this L 2 Rubisco remains a common substrate for examining chaperone and chaperonin function (2, 4, 5). This contrasts with the more common, but structurally more complex, Form I Rubiscos found in higher plants, algae, cyanobacteria, and most photoand chemolithoauto-trophic proteobacteria. Form I Rubiscos share a hexadecameric structure comprising four L 2 catalytic dimers arranged around a 4-fold axis and eight 13-15-kDa small (S) subunits that cap both ends of the (L 2 ) 4 core and influence catalysis indirectly (3). Along with their more advanced structure, the Form I enzymes have more complex assembly needs that can involve Rubisco-specific chaperones whose requirements can limit and prevent their assembly in both prokaryotic hosts (6) and foreign chloroplasts (7-9).Despite their different subunit composition and amino acid sequence, the overall fold of the core L 2 units in all Rubiscos are alike (3). Each L 2 contains two active (catalytic) sites located at the interface between the C-domain of one L and the N-domain of the other. Consistent with a common multi-step catalytic chemistry, the identity and position of catalytically significant residues are highly conserved (10, 11). Productive binding of the five-carbon substrate, RuBP, in the active site requires a two-step activation process: the binding of nonsubstrate CO 2 to a conserved lysine to form a lysyl-carbamate and its stabilization by Mg 2ϩ binding. The bo...
Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.
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