Rubisco
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ABSTRACT:
PROVIDER: PRJEB94415 | ENA |
REPOSITORIES: ENA
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Project description:Heterologous expression of hornwort Rubisco from Anthoceros agrestis in Escherichia coliwith expression of Rubisco large and small subunits, chaperonins 60alpha/beta and chaperonin 20 as well as a suite of assembly factors, Raf1/Raf2/RbcX2/BSD2/RbcX1. Rubisco kinetics show differences in Rubisco assembled with and without RbcX1/2.
2024-11-04 | PXD052489 | Pride
Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.
2023-06-13 | PXD027027 | Pride
Project description:Multiple studies have shown Rubisco to be subject to Lys-acetylation at various residues; however, conflicting reports exist about the biological significance of these post-translational modifications. One aspect of the Lys-acetylation that has not been addressed in plants generally, or with Rubisco specifically, is the stoichiometry at which these Lys-acetylation events occur. As a method to ascertain which Lys-acetylation sites on Arabidopsis Rubisco might be of regulatory importance, we purified Rubisco from leaves in both the day and night-time and performed independent mass-spectrometry based methods to determine the stoichiometry of Rubisco Lys-acetylation events.
2020-09-28 | PXD019268 | Pride
Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the core rate-limiting enzyme of photosynthetic carbon metabolism, is regulated by acetylation, yet its upstream acetyltransferases remain largely uncharacterized. Here, we identified a novel soybean acetyltransferase GmGNAT10, which interacts with Rubisco and other photosynthetic enzymes via IP-MS/MS interactomics. Quantitative acetylomics under fluctuating light revealed dramatic genotypic differences in acetylation levels of key RbcL lysine residues (K23, K175, K236) between wild-type and GmGNAT10 mutants. Our findings expand the mechanistic understanding of chloroplast non-histone acetylation in fine-tuning photosynthetic carbon metabolism.
2026-04-28 | PXD077705 |
Project description:Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. Its relatively low catalytic constant as well as its tendency to also accept O2 as a substrate have made it a common target of enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library, without prior experimental knowledge, by coupling the phylogenetically-guided model EVmutation with “in silico evolution”. Starting with this targeted mutagenesis library, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. We identified an amino acid exchange which increased the thermostability of Gallionella Rubisco and conveyed resilience to detrimental exchanges. The establishment of this platform is a first step towards high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts. DIA proteomics was used to compare relative quantities of the native and heterologous RuBisCO, as well as the proteome at large at different gas conditions and with or without the heterologous enzyme.
2025-08-04 | PXD059631 | Pride
Project description:Cyanobacterial Form II Rubisco screening platform
| PRJEB79580 | ENA
Project description:Lateral gene transfer shapes the distribution of RuBisCO among Candidate Phyla Radiation bacteria and DPANN archaea
| PRJEB29721 | ENA
Project description:Form II Rubisco screening in cyanobacterium Synechocystis sp. PCC 6803
| PRJEB79633 | ENA
Project description:In the model green alga Chlamydomonas (Chlamydomonas reinhardtii), the synthesis of several chloroplast-encoded photosynthetic subunits is feedback-regulated by the assembly state of the respective protein complex. This regulation is known as control by epistasy of synthesis (CES) and matches protein synthesis with the requirements of protein complex assembly in photosystem II (PSII), the cytochrome b6f complex (Cyt b6f), photosystem I (PSI), ATP synthase and Rubisco . In embryophytes, however, CES was only described to coordinate synthesis of the large and small subunits of Rubisco, raising the question if additional CES mechanisms exist in land plants or if stoichiometric photosynthetic protein accumulation is only achieved by the wasteful degradation of excess subunits. We systematically examined suitable tobacco and Arabidopsis mutants with assembly defects in PSII, PSI, Cyt b6f complex, ATP synthase, NDH (NAD(P)H dehydrogenase-like) complex and Rubisco for feedback regulation. Thereby, we validated the CES in Rubisco and uncovered translational feedback regulation in PSII, involving psbA, psbB, psbD and psbH and in Cyt b6f, connecting PetA and PetB protein synthesis. Remarkably, some of these feedback regulation mechanisms are not conserved between the green alga and embryophytes. Our data do not provide any evidence for CES in PSI, ATP synthase or NDH complex assembly in embryophytes. In addition, our data disclose translational feedback regulation adjusting PSI levels with PSII accumulation. Overall, we discovered commonalities and differences in assembly-dependent feedback regulation of photosynthetic complexes between embryophytes and green algae.
2025-07-01 | GSE286011 | GEO
Project description:Future-proofing crop productivity by exploiting natural variation in Rubisco – an in silico analysis.
| PRJEB34312 | ENA