Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria.
ABSTRACT: Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and ?-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.
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 ?-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 ?-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.
Project description: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 ?4-?4 loop, pore loop 1, and the presensor 1-? hairpin (PS1-?H). 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.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO2 molecule and binding of a Mg2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.
Project description: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 CO? 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 CO? 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.
Project description:Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The ?-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The ?-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.
Project description:Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO2 fixation machinery will need to take into consideration the requirement for Rubisco activases as well as their properties.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP-dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries.
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first major step of carbon fixation in the Calvin-Benson-Bassham (CBB) cycle. This autotrophic CO2 fixation cycle accounts for almost all the assimilated carbon on Earth. Due to the primary role that RubisCO plays in autotrophic carbon fixation, it is important to understand how its gene expression is regulated and the enzyme is activated. Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18' S along the Mid-Atlantic Ridge. It is 13,046 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. In silico analyses uncovered several putative LysR binding sites and promoter structures. Functions of some of these DNA motifs were experimentally confirmed. For example, according to mobility shift assays LysR1's binding ability to the intergenic region of lysR1 and cbbL appears to be intensified when CbbL or LysR2 are present. Binding of LysR2 upstream of cbbM appears to be intensified if CbbM is present. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR's binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression. A model summarizes the findings and predicts putative interactions of the different proteins influencing RubisCO gene regulation and expression.
Project description:Three genes, cbbX, cbbY, and cbbZ were found downstream from the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes of Rhodobacter sphaeroides. As in chemoautotrophic bacteria, cbbZ was shown to encode phosphoglycolate phosphatase (PGP), whereas the identities of cbbX and cbbY are not known. To determine the physiological function of the cbbXYZ gene products, we constructed R. sphaeroides strains in which the genes were inactivated and characterized the resultant mutant strains according to growth phenotype and levels of RubisCO and PGP. Only a mutation in cbbX resulted in a discernible phenotype, namely, impaired photoautotrophic growth. No PGP activity was observed in any of the mutants, suggesting that the three genes are transcriptionally linked. Studies with a spontaneous chemoautotrophic competent derivative of the CBBX mutant suggested that the cbbXYZ gene products are not essential for chemoautotrophic growth. PGP activity determined in the wild-type strain grown under a variety of growth conditions, and in various strains containing mutations in Calvin-Benson-Bassham cycle structural and regulatory genes, indicated that transcription of the cbb(I) operon influenced expression of the downstream cbbXYZ operon.
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.