| 1. |
- Ekeberg, Tomas, 1983-, et al.
(författare)
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Observation of a single protein by ultrafast X-ray diffraction
- 2024
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Ingår i: Light. - : Springer Nature. - 2095-5545 .- 2047-7538. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
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| 2. |
- Gunn, Laura, 1986-, et al.
(författare)
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A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) acts as a small subunit mimic
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292:16, s. 6838-6850
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) byproduct of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5 and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino-acid insertion near the C-terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues co-ordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco sub-group, named form IIIB.
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| 3. |
- Gunn, Laura, 1986-, et al.
(författare)
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The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:41, s. 25890-25896
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Tidskriftsartikel (refereegranskat)abstract
- Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhanced RsRubisco activation to 75% under elevated CO2 (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2 specificity impaired 30-50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.
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| 4. |
- Mao, Yuwei, et al.
(författare)
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The small subunit of Rubisco and its potential as an engineering target
- 2022
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 74:2, s. 543-561
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Tidskriftsartikel (refereegranskat)abstract
- Rubisco catalyses the first rate-limiting step in CO2 fixation and is responsible for the vast majority of organic carbon present in the biosphere. The function and regulation of Rubisco remain an important research topic and a longstanding engineering target to enhance the efficiency of photosynthesis for agriculture and green biotechnology. The most abundant form of Rubisco (Form I) consists of eight large and eight small subunits, and is found in all plants, algae, cyanobacteria, and most phototrophic and chemolithoautotrophic proteobacteria. Although the active sites of Rubisco are located on the large subunits, expression of the small subunit regulates the size of the Rubisco pool in plants and can influence the overall catalytic efficiency of the Rubisco complex. The small subunit is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. Here we review our current understanding of the role of the small subunit and our growing capacity to explore its potential to modulate Rubisco catalysis using engineering biology approaches.
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| 5. |
- Oh, Zhen Guo, et al.
(författare)
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Red Rubiscos and opportunities for engineering green plants
- 2022
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 74:2, s. 520-542
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Tidskriftsartikel (refereegranskat)abstract
- Nature's vital, but notoriously inefficient, CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limits the growth of photosynthetic organisms including crop species. Form I Rubiscos comprise eight catalytic large- and eight auxiliary small-subunits and can be classified into two distinct lineages - "red" and "green". While red-type Rubiscos (Form IC and ID) are found in Rhodophytes, their secondary symbionts and certain proteobacteria, green-type Rubiscos (Form IA and IB) exist in terrestrial plants, Chlorophytes, cyanobacteria and other proteobacteria. Eukaryotic red-type Rubiscos exhibit desirable kinetic properties, namely high specificity and high catalytic efficiency, with certain isoforms outperforming green-type Rubiscos. However, it is not yet possible to functionally express a high-performing red-type Rubisco in chloroplasts to boost photosynthetic carbon assimilation in green plants. Understanding the molecular and evolutionary basis for divergence between red- and green-type Rubiscos could help us to harness the superior CO2-fixing power of red-type Rubiscos. Here we review our current understanding about red-type Rubisco distribution, biogenesis and sequence-structure and present opportunities and challenges to utilising red-type Rubisco kinetics towards crop improvements.
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| 6. |
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| 7. |
- Sharwood, Robert, et al.
(författare)
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Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis
- 2016
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Ingår i: Nature Plants. - 2055-0278. ; 2:12
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Tidskriftsartikel (refereegranskat)abstract
- Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C3 and C4 species within the grass tribe Paniceae. The CO2 fixation rate (kccat) for Rubisco from the C4 grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kccat of Rubisco from NAD-ME species across all temperatures. The declining response of CO2/O2 specificity with increasing temperature was less pronounced for PCK and NADP-ME Rubisco, which would be advantageous in warmer climates relative to the NAD-ME grasses. Modelled variation in the temperature kinetics of Paniceae C3 Rubisco and PCK Rubisco differentially stimulated C3 photosynthesis relative to tobacco above and below 25 °C under current and elevated CO2. Amino acid substitutions in the large subunit that could account for the catalytic variation among Paniceae Rubisco are identified; however, incompatibilities with Paniceae Rubisco biogenesis in tobacco hindered their mutagenic testing by chloroplast transformation. Circumventing these bioengineering limitations is critical to tailoring the properties of crop Rubisco to suit future climates.
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| 8. |
- Valegård, Karin, et al.
(författare)
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Structure of Rubisco from Arabidopsis thaliana in complex with 2-carboxyarabinitol-1,5-bisphosphate
- 2018
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Arabidopsis thaliana is reported at 1.5 Å resolution. In light of the importance of A. thaliana as a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of an A. thaliana Rubisco crystal structure. A. thaliana Rubisco is an L8S8 hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits. A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot of A. thaliana Rubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate. A. thaliana Rubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understand A. thaliana Rubisco and the potential catalytic differences that could be conferred by alternative A. thaliana Rubisco small-subunit isoforms.
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| 9. |
- Zhou, Yu, et al.
(författare)
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Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth
- 2023
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Ingår i: NATURE PLANTS. - : Springer Nature. - 2055-0278. ; 9, s. 978-986
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Tidskriftsartikel (refereegranskat)abstract
- Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.7 angstrom. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides RsRubisco that, unlike GmRubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 RsRubisco chimaeras revealed that incorporating C329A and A332V substitutions from GmRubisco Loop 6 (corresponding to plant residues 328 and 331) into RsRubisco increased the carboxylation rate (kcatc) by 60%, the carboxylation efficiency in air by 22% and the CO2/O-2 specificity (Sc/o) by 7%. Plastome transformation of this RsRubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type RsRubisco. Our findings demonstrate the utility of RsRubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme's carboxylase potential.
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