Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.

Structure of a Synthetic ß-Carboxysome Shell.

Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified ß-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.

Rubisco condensate formation by CcmM in ß-carboxysome biogenesis.

Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency1. The α- and ß-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-a complex of eight large (RbcL) and eight small (RbcS) subunits-and carbonic anhydrase2-4. As HCO3- can diffuse through the proteinaceous carboxysome shell but CO2 cannot5, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco6. The shell also prevents access to reducing agents, generating an oxidizing environment7-9. The formation of ß-carboxysomes involves the aggregation of Rubisco by the protein CcmM10, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain11. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS2-4. Here we have reconstituted the Rubisco-CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes12,13. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC114. Understanding carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants15-19.

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography.

Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.

Coupled chaperone action in folding and assembly of hexadecameric Rubisco.

Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-A density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.

Discoveries in Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase): a historical perspective.

Historic discoveries and key observations related to Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase), from 1947 to 2006, are presented. Currently, around 200 papers describing Rubisco research are published each year and the literature contains more than 5000 manuscripts on the subject. While trying to ensure that all the major events over this period are recorded, this analysis will inevitably be incomplete and will reflect the areas of particular interest to the authors.

Intact carboxysomes in a cyanobacterial cell visualized by hilbert differential contrast transmission electron microscopy.

Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.

[Distribution of Rubisco and RCA in Brassica chinensis chloroplasts and effect of TuMV-infection on their cellular localization].

The cellular localizations of Rubisco and Rubisco activase (RCA) in Chinese cabbage (Brassica chinensis L. cv. Suzhou) leaves were investigated by immunogold-labeling electron microscopy. The results showed that Rubisco and RCA were mainly located in chloroplasts of mesophyll, guard cell of stomatal apparatus and parenchyma of vascular bundle. A high density of gold particles were localized preferentially to the chloroplast stroma. In contrast, there were no specific binding of gold particles detected in the cytoplasm, vacuole, mitochondria and other organelles. As infected by turnip mosaic virus, the density of gold particles decreased lightly in the abnormal chloroplasts and dropped by much lower (58.44% and 64.67% as that in the health chloroplasts respectively) in swollen chloroplasts. This indicated that virus infection caused the decreases of Rubisco and RCA in host plant, which then affected plant photosynthesis.

Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure.

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.

Crystallization and X-ray analysis of a multienzyme complex containing RUBISCO and RuBP.

Single crystals of a multienzyme complex isolated from spinach leaves, and containing RUBISCO bound to the substrate RuBP have been grown and characterized. The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 173 A, b = 134 A and c = 112 A, and contain two enzyme complex molecules in the unit cell. Diffraction data to 2.5 A resolution have been collected on the sychrotron source at the photon factory in Japan. Initial structure determination has been carried out using the molecular replacement method. The RUBISCO molecule in the complex has the normal L8S8 subunit configuration, and difference electron density is clearly observed for the other component enzymes and the RuBP substrate.

Ribulose-1,5-bisphosphate carboxylase of thermophilic hydrogen-oxidizing microorganism Bacillus schlegelii.

Ribulose-1,5-bisphosphate carboxylase was isolated from thermophilic hydrogen-oxidizing Bacillus schlegelii. Molecular mass of the native enzyme is 560,000 and optimal reaction temperature is 70 degrees C. Km value for ribulose 1,5-bisphosphate is 0.27 mM. The carboxylase activity of the enzyme is dependent on Mg2+ with the optimum at 10 mM. The enzyme is an oligomer of L8S8 type with Mr of large subunits and small subunits of 56,000 and 14,000, respectively. Negatively stained enzyme has regular polygonal shape in top view, 12 nm in diameter, with central electron dense patch.

Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves.

The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of 14 subunits arranged with 72 point group symmetry, and oligomeric (L8S8) Rubisco was obtained.

D-ribulose-1,5-bisphosphate carboxylase/oxygenase: function-dependent structural changes.

The key carboxylating enzyme of the reductive pentose phosphate cycle, D-ribulose-1,5-bisphosphate carboxylase/oxygenase [RuBisCO] isolated from the chemolithoautotrophic, H2-oxidizing bacterium Alcaligenes eutrophus H16 has been analyzed by several different techniques that allow conclusions about structure and function-dependent structural changes. The techniques include a novel approach in which the enzyme was induced to form 2D-crystals suitable for electron microscopy in each of its three stable functional states: as active enzyme [Ea] (in the presence of Mg2+ and HCO3-); as inactivated enzyme [Eia] (in the absence of Mg2+ and HCO3-) and as enzyme locked in an in vitro transition state [CABP-E] (Ea fully saturated with the transition state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate [CABP-E]). In conjunction with X-ray crystallography, X-ray small angle scattering and other biophysical and biochemical data, the results obtained by electron microscopy support the idea that drastic configurational changes occur. Upon transition from Ea to the CABP-E the upper and lower L4S4 halves of the molecule consisting of eight large and eight small subunits (L8S8; MW = 536,000 Da) are assumed to be laterally shifted by as much as 3.6 nm relative to one another while the location of the small subunits on top of the large subunits, and relative to them, remains the same. For the Eia a similar sliding-layer configurational change in the range of 2-2.5 nm is proposed and in addition it is suggested that other configurational/conformational changes take place. The proposed structural changes are discussed with respect to the current model for the tobacco enzyme and correlated with data obtained for various other plant and (cyano) bacterial L8S8 RuBisCOs leading to speculations about structure-function relationships.