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A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco.

ABSTRACT: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.

SUBMITTER: Scales JC

PROVIDER: S-EPMC3923112 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A single point mutation in the C-terminal extension of wheat Rubisco activase dramatically reduces ADP inhibition via enhanced ATP binding affinity.

Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca) is a AAA+ enzyme that uses ATP to remove inhibitors from the active site of Rubisco, the central carboxylation enzyme of photosynthesis. Rca ? and ? isoforms exist in most higher plant species, with the ? isoform being identical to the ? form but having an additional 25-45 amino acids at the Rca C terminus, known as the C-terminal extension (CTE). Rca is inhibited by ADP, and the extent of ADP sensitivity of the Rca complex can be modulated by the CTE of the ? isoform, particularly in relation to a disulfide bond structure that is specifically reduced by the redox-regulatory enzyme thioredoxin-f. Here, we introduced single point mutations of Lys-428 in the CTE of Rca-? from wheat (Triticum aestivum) (TaRca2-?). Substitution of Lys-428 with Arg dramatically altered ADP inhibition, independently of thioredoxin-f regulation. We determined that the reduction in ADP inhibition in the K428R variant is not due to a change in ADP affinity, as the apparent constant for ADP binding was not altered by the K428R substitution. Rather, we observed that the K428R substitution strongly increased ATP substrate affinity and ATP-dependent catalytic velocity. These results suggest that the Lys-428 residue is involved in interacting with the ?-phosphate of ATP. Considering that nucleotide-dependent Rca activity regulates Rubisco and thus photosynthesis during fluctuating irradiance, the K428R substitution could potentially provide a mechanism for boosting the performance of wheat grown in the dynamic light environments of the field.

| S-EPMC6879333 | biostudies-literature

Probing the rice Rubisco-Rubisco activase interaction via subunit heterooligomerization.

Project description:During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO2-fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ?2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO2-fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO2 assimilation rate, growth, and yield.

| S-EPMC6883792 | biostudies-literature

Molecular mechanism of Rubisco activase: Dynamic assembly and Rubisco remodeling.

Project description:Ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) enzyme is the limiting step of photosynthetic carbon fixation, and its activation is regulated by its co-evolved chaperone, Rubisco activase (Rca). Rca removes the intrinsic sugar phosphate inhibitors occupying the Rubisco active site, allowing RuBP to split into two 3-phosphoglycerate (3PGA) molecules. This review summarizes the evolution, structure, and function of Rca and describes the recent findings regarding the mechanistic model of Rubisco activation by Rca. New knowledge in these areas can significantly enhance crop engineering techniques used to improve crop productivity.

| S-EPMC9951593 | biostudies-literature

Small oligomers of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase are required for biological activity.

Project description:Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μM, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2-4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.

| S-EPMC3711324 | biostudies-literature

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco.

Project description:The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1-4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.

| S-EPMC7705312 | biostudies-literature

Characterization and expression of Rubisco activase genes in Ipomoea batatas.

Project description:Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl-RCAs-RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.

| S-EPMC3824211 | biostudies-literature

Characterization of the heterooligomeric red-type rubisco activase from red algae.

Project description:The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.

| S-EPMC5150372 | biostudies-literature

Arabidopsis Plant Natriuretic Peptide Is a Novel Interactor of Rubisco Activase.

Project description:Plant natriuretic peptides (PNPs) are a group of systemically acting peptidic hormones affecting solute and solvent homeostasis and responses to biotrophic pathogens. Although an increasing body of evidence suggests PNPs modulate plant responses to biotic and abiotic stress, which could lead to their potential biotechnological application by conferring increased stress tolerance to plants, the exact mode of PNPs action is still elusive. In order to gain insight into PNP-dependent signalling, we set out to identify interactors of PNP present in the model plant Arabidopsis thaliana, termed AtPNP-A. Here, we report identification of rubisco activase (RCA), a central regulator of photosynthesis converting Rubisco catalytic sites from a closed to an open conformation, as an interactor of AtPNP-A through affinity isolation followed by mass spectrometric identification. Surface plasmon resonance (SPR) analyses reveals that the full-length recombinant AtPNP-A and the biologically active fragment of AtPNP-A bind specifically to RCA, whereas a biologically inactive scrambled peptide fails to bind. These results are considered in the light of known functions of PNPs, PNP-like proteins, and RCA in biotic and abiotic stress responses.

| S-EPMC7823470 | biostudies-literature

Effects of Overproduction of Rubisco Activase on Rubisco Content in Transgenic Rice Grown at Different N Levels.

Project description:It has been reported that overproduction of Rubisco activase (RCA) in rice (Oryza sativa L.) decreased Rubisco content, resulting in declining photosynthesis. We examined the effects of RCA levels on Rubisco content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and Rubisco small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%-20% decrease in Rubisco content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, Rubisco content did not change in RCA-HI lines. Conversely, in plants with 50%-60% increased RCA content (RCA-MI lines), Rubisco levels remained unchanged, regardless of N concentration. Such effects on Rubisco content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, Rubisco levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on Rubisco content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in Rubisco content, depending on the degree of RCA production.

| S-EPMC7084177 | biostudies-literature

Protein oligomerization monitored by fluorescence fluctuation spectroscopy: self-assembly of rubisco activase.

Project description:A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us to quantitatively consider the relevance of any proposed intermediate form, and K(d) values can be estimated even when several oligomeric species coexist. We have tested this method on the AAA+ ATPase Rubisco activase (Rca). Rca self-association regulates the CO(2) fixing activity of the enzyme Rubisco, directly affecting biomass accumulation in higher plants. However, the elucidation of its assembly pathway has remained challenging, precluding a detailed mechanistic investigation. Here, we present the first, to our knowledge, thermodynamic characterization of oligomeric states of cotton ?-Rca complexed with Mg·ADP. We find that the monomer is the dominating species below 0.5 micromolar. The most plausible model supports dissociation constants of ?4, 1, and 1 micromolar for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equilibria, in line with the coexistence of four different oligomeric forms under typical assay conditions. Large aggregates become dominant above 40 micromolar, with continued assembly at even higher concentrations. We propose that under some conditions, ADP-bound Rca self-associates by forming spiral arrangements that grow along the helical axis. Other models such as the stacking of closed hexameric rings are also discussed.

| S-EPMC3433602 | biostudies-literature

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