RESUMO
Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.
Assuntos
Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Cinética , Dióxido de Carbono/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/metabolismo , Cianobactérias/enzimologia , Cianobactérias/genética , Bactérias/enzimologia , Bactérias/metabolismo , Bactérias/genéticaRESUMO
The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.
Assuntos
Diatomáceas , Príons , Ribulose-Bifosfato Carboxilase/genética , Microscopia Crioeletrônica , Condensados Biomoleculares , Diatomáceas/genéticaRESUMO
How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Rhodobacter sphaeroides/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Reagentes de Ligações Cruzadas/química , Medição da Troca de Deutério , Estabilidade Enzimática , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Rhodobacter sphaeroides/genética , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Relação Estrutura-Atividade , Fatores de Tempo , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/genéticaRESUMO
CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast-carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form-II and form-II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s-1 -sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.
Assuntos
Mineração de Dados , Bases de Dados de Ácidos Nucleicos , Ribulose-Bifosfato Carboxilase , Isoenzimas/classificação , Isoenzimas/genética , Ribulose-Bifosfato Carboxilase/classificação , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
The CO2-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IAC clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.
Assuntos
Proteínas de Bactérias , Ribulose-Bifosfato Carboxilase , Synechococcus , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono , Família Multigênica , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/enzimologia , Synechococcus/genética , Synechococcus/metabolismo , Ativador de Plasminogênio TecidualRESUMO
The world's population may reach 10 billion by 2050, but 10% still suffer from food shortages. At the same time, global warming threatens food security by decreasing crop yields, so it is necessary to develop crops with enhanced resistance to high temperatures in order to secure the food supply. In this review, the role of Rubisco activase as an important factor in plant heat tolerance is summarized, based on the conclusions of recent findings. Rubisco activase is a molecular chaperone determining the activation of Rubisco, whose heat sensitivity causes reductions of photosynthesis at high temperatures. Thus, the thermostability of Rubisco activase is considered to be critical for improving plant heat tolerance. It has been shown that the introduction of thermostable Rubisco activase through gene editing into Arabidopsis thaliana and from heat-adapted wild Oryza species or C4Zea mays into Oryza sativa improves Rubisco activation, photosynthesis, and plant growth at high temperatures. We propose that developing a universal thermostable Rubisco activase could be a promising direction for further studies.
Assuntos
Arabidopsis , Oryza , Termotolerância , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Ativador de Plasminogênio Tecidual , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Fotossíntese/fisiologia , Arabidopsis/genética , Oryza/metabolismo , Segurança AlimentarRESUMO
Aquatic autotrophs that fix carbon using ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) frequently expend metabolic energy to pump inorganic carbon towards the enzyme's active site. A central requirement of this strategy is the formation of highly concentrated Rubisco condensates (or Rubiscondensates) known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes, respectively. Recent data indicate that these condensates form by the mechanism of liquid-liquid phase separation. This mechanism requires networks of weak multivalent interactions typically mediated by intrinsically disordered scaffold proteins. Here we comparatively review recent rapid developments that detail the determinants and precise interactions that underlie diverse Rubisco condensates. The burgeoning field of biomolecular condensates has few examples where liquid-liquid phase separation can be linked to clear phenotypic outcomes. When present, Rubisco condensates are essential for photosynthesis and growth, and they are thus emerging as powerful and tractable models to investigate the structure-function relationship of phase separation in biology.
Assuntos
Dióxido de Carbono , Ribulose-Bifosfato Carboxilase , Dióxido de Carbono/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Plastídeos/metabolismo , Fotossíntese , Carbono/metabolismoRESUMO
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.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acidithiobacillus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Acidithiobacillus/genética , Acidithiobacillus/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Ativação Enzimática , Ensaios Enzimáticos , Microscopia Eletrônica , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/ultraestrutura , Mutagênese Sítio-Dirigida , Multimerização Proteica , Estrutura Secundária de Proteína , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/ultraestruturaRESUMO
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 â¼2-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.
Assuntos
Oryza/enzimologia , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Domínios Proteicos , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
CO2 enters the biosphere via the slow, oxygen-sensitive carboxylase, Rubisco. To compensate, most microalgae saturate Rubisco with its substrate gas through a carbon dioxide concentrating mechanism. This strategy frequently involves compartmentalization of the enzyme in the pyrenoid, a non-membrane enclosed compartment of the chloroplast stroma. Recently, tremendous advances have been achieved concerning the structure, physical properties, composition and in vitro reconstitution of the pyrenoid matrix from the green alga Chlamydomonas reinhardtii. The discovery of the intrinsically disordered multivalent Rubisco linker protein EPYC1 provided a biochemical framework to explain the subsequent finding that the pyrenoid resembles a liquid droplet in vivo. Reconstitution of the corresponding liquid-liquid phase separation using pure Rubisco and EPYC1 allowed a detailed characterization of this process. Finally, a large high-quality dataset of pyrenoidal protein-protein interactions inclusive of spatial information provides ample substrate for rapid further functional dissection of the pyrenoid. Integrating and extending recent advances will inform synthetic biology efforts towards enhancing plant photosynthesis as well as contribute a versatile model towards experimentally dissecting the biochemistry of enzyme-containing membraneless organelles.
Assuntos
Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Microalgas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Ribulose-Bifosfato Carboxilase/químicaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/enzimologia , Modelos Moleculares , Mutação , Subunidades Proteicas , Ribulose-Bifosfato Carboxilase , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoid-like aggregation for Rubisco derived from a higher plant.
Assuntos
Proteínas de Algas/metabolismo , Arabidopsis/metabolismo , Chlamydomonas reinhardtii/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
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.
Assuntos
Proteínas de Plantas/metabolismo , Rodófitas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Regulação Alostérica/fisiologia , Cinética , Chaperonas Moleculares/metabolismo , Fotossíntese/genética , Fotossíntese/fisiologia , Proteínas de Plantas/genética , Plastídeos/genética , Ribulose-Bifosfato Carboxilase/antagonistas & inibidores , Ribulosefosfatos/metabolismoRESUMO
Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO2-inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical ß-CAs. Our results identify the LCIB family as a previously unidentified group of ß-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.
Assuntos
Dióxido de Carbono/química , Anidrases Carbônicas/química , Chlamydomonas reinhardtii/enzimologia , Fotossíntese , Proteínas de Plantas/química , Ar , Carbono/metabolismo , Domínio Catalítico , Cloroplastos/enzimologia , Clonagem Molecular , Íons , Microalgas/enzimologia , Conformação Molecular , Mutação , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Água/química , Zinco/químicaRESUMO
To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-ß4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function.
Assuntos
Agave/enzimologia , Ácidos Carboxílicos/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Temperatura , Motivos de Aminoácidos , Sequência de Aminoácidos , Estabilidade Enzimática , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Ribulose-Bifosfato Carboxilase/químicaRESUMO
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a â¼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.
Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada/métodos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Multimerização Proteica , Ribulose-Bifosfato Carboxilase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fotossíntese , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/enzimologia , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/enzimologia , Synechocystis/genética , Synechocystis/metabolismoRESUMO
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-Å crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Rhodobacter sphaeroides/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Dióxido de Carbono/metabolismo , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Modelos Moleculares , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Ribulosefosfatos/metabolismo , Ribulosefosfatos/farmacologia , Relação Estrutura-AtividadeRESUMO
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal ß-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The ß-hairpins of four RbcS subunits form an eight-stranded ß-barrel that protrudes into the central solvent channel of the RbcL core complex. The two ß-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal ß-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco.
Assuntos
Chaperonina 60/metabolismo , Fotossíntese/genética , Ribulose-Bifosfato Carboxilase/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chaperonina 60/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-raf/metabolismo , Rhodobacter sphaeroides/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Ribulosefosfatos/química , Ribulosefosfatos/metabolismoRESUMO
The key photosynthetic, CO2-fixing enzyme Rubisco forms inactivated complexes with its substrate ribulose 1,5-bisphosphate (RuBP) and other sugar phosphate inhibitors. The independently evolved AAA+ proteins Rubisco activase and CbbX harness energy from ATP hydrolysis to remodel Rubisco complexes, facilitating release of these inhibitors. Here, we discuss recent structural and mechanistic advances towards the understanding of protein-mediated Rubisco activation. Both activating proteins appear to form ring-shaped hexameric arrangements typical for AAA+ ATPases in their functional form, but display very different regulatory and biochemical properties. Considering the thermolability of the plant enzyme, an improved understanding of the mechanism for Rubisco activation may help in developing heat-resistant plants adapted to the challenge of global warming.
Assuntos
Dióxido de Carbono/metabolismo , Fotossíntese/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Biotecnologia/métodos , Biotecnologia/tendências , Conformação Proteica , Fosfatos Açúcares/metabolismo , TemperaturaRESUMO
CO2 fixation in most unicellular algae relies on the pyrenoid, a biomolecular condensate, which sequesters the cell's carboxylase Rubisco. In the marine diatom Phaeodactylum tricornutum, the pyrenoid tandem repeat protein Pyrenoid Component 1 (PYCO1) multivalently binds Rubisco to form a heterotypic Rubisco condensate. PYCO1 contains prion-like domains and can phase-separate homotypically in a salt-dependent manner. Here we dissect PYCO1 homotypic liquid-liquid phase separation (LLPS) by evaluating protein fragments and the effect of site-directed mutagenesis. Two of PYCO1's six repeats are required for homotypic LLPS. Mutagenesis of a minimal phase-separating fragment reveals tremendous sensitivity to the substitution of aromatic residues. Removing positively charged lysines and arginines instead enhances the propensity of the fragment to condense. We conclude that PYCO1 homotypic LLPS is mostly driven by π-π interactions mediated by tyrosine and tryptophan stickers. In contrast π-cation interactions involving arginine or lysine are not significant drivers of LLPS in this system.