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Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency 1 . The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)—a complex of eight large (RbcL) and eight small (RbcS) subunits—and carbonic anhydrase 2 – 4 . As HCO 3 − can diffuse through the proteinaceous carboxysome shell but CO 2 cannot 5 , carbonic anhydrase generates high concentrations of CO 2 for carbon fixation by Rubisco 6 . The shell also prevents access to reducing agents, generating an oxidizing environment 7 – 9 . The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM 10 , which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain 8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain 11 . It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS 2 – 4 . Here we have reconstituted the Rubisco–CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid–liquid phase separation in eukaryotes 12 , 13 . Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC1 14 . Understanding carboxysome biogenesis will be important for efforts to engineer CO 2 -concentrating mechanisms in plants 15 – 19 . The structure of a Rubisco–CcmM complex sheds light on the formation of carboxysomes in cyanobacteria.

Rubisco is the primary CO 2 -fixing enzyme of the biosphere 1 , yet it has slow kinetics 2 . The roles of evolution and chemical mechanism in constraining its biochemical function remain debated 3 , 4 . Engineering efforts aimed at adjusting the biochemical parameters of rubisco have largely failed 5 , although recent results indicate that the functional potential of rubisco has a wider scope than previously known 6 . Here we developed a massively parallel assay, using an engineered Escherichia   coli 7 in which enzyme activity is coupled to growth, to systematically map the sequence–function landscape of rubisco. Composite assay of more than 99% of single-amino acid mutants versus CO 2 concentration enabled inference of enzyme velocity and apparent CO 2 affinity parameters for thousands of substitutions. This approach identified many highly conserved positions that tolerate mutation and rare mutations that improve CO 2 affinity. These data indicate that non-trivial biochemical changes are readily accessible and that the functional distance between rubiscos from diverse organisms can be traversed, laying the groundwork for further enzyme engineering efforts. A massively parallel assay developed to map the essential photosynthetic enzyme rubisco showed that non-trivial biochemical changes and improvements in CO 2 affinity are possible, signposting further enzyme engineering efforts to increase crop yields.

Photosynthetic carbon assimilation enables energy storage in the living world and produces most of the biomass in the biosphere. Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is responsible for the vast majority of global carbon fixation and has been claimed to be the most abundant protein on Earth. Here we provide an updated and rigorous estimate for the total mass of Rubisco on Earth, concluding it is ≈0.7 Gt, more than an order of magnitude higher than previously thought. We find that >90% of Rubisco enzymes are found in the ≈2 × 1014 m² of leaves of terrestrial plants, and that Rubisco accounts for ≈3% of the total mass of leaves, which we estimate at ≈30 Gt dry weight. We use our estimate for the total mass of Rubisco to derive the effective time-averaged catalytic rate of Rubisco of ≈0.03 s−1 on land and ≈0.6 s−1 in the ocean. Compared with the maximal catalytic rate observed in vitro at 25 °C, the effective rate in the wild is ≈100-fold slower on land and sevenfold slower in the ocean. The lower ambient temperature, and Rubisco not working at night, can explain most of the difference from laboratory conditions in the ocean but not on land, where quantification of many more factors on a global scale is needed. Our analysis helps sharpen the dramatic difference between laboratory and wild environments and between the terrestrial and marine environments.

The mechanistic basis of tolerance to heat stress was investigated in Oryza sativa and two wild rice species, Oryza meridionalis and Oryza australiensis. The wild relatives are endemic to the hot, arid Australian savannah. Leaf elongation rates and gas exchange were measured during short periods of supraoptimal heat, revealing species differences. The Rubisco activase (RCA) gene from each species was sequenced. Using expressed recombinant RCA and leaf-extracted RCA, the kinetic properties of the two isoforms were studied under high temperatures. Leaf elongation was undiminished at 45°C in O. australiensis. The net photosynthetic rate was almost 50% slower in O. sativa at 45°C than at 28°C, while in O. australiensis it was unaffected. Oryza meridionalis exhibited intermediate heat tolerance. Based on previous reports that RCA is heat-labile, the Rubisco activation state was measured. It correlated positively with leaf elongation rates across all three species and four periods of exposure to 45°C. Sequence analysis revealed numerous polymorphisms in the RCA amino acid sequence from O. australiensis. The O. australiensis RCA enzyme was thermally stable up to 42°C, contrasting with RCA from O. sativa, which was inhibited at 36°C. We attribute heat tolerance in the wild species to thermal stability of RCA, enabling Rubisco to remain active.

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein–protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.Structural and binding studies show that a repeated peptide motif in the N-terminal domain of CsoS2 mediates multivalent interactions with assembled Rubisco to facilitate its encapsulation into the carboxysome.

Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO2 and NH3 and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to ‘supercharge’ photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO2 can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco’s inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.

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 (kcat c) 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.

A well-known case of evolutionary adaptation is that of ribulose-1,5-bisphosphate carboxylase (RubisCO), the enzyme responsible for fixation of CO ₂ during photosynthesis. Although the majority of plants use the ancestral C ₃ photosynthetic pathway, many flowering plants have evolved a derived pathway named C ₄ photosynthesis. The latter concentrates CO ₂, and C ₄ RubisCOs consequently have lower specificity for, and faster turnover of, CO ₂. The C ₄ forms result from convergent evolution in multiple clades, with substitutions at a small number of sites under positive selection. To understand the physical constraints on these evolutionary changes, we reconstructed in silico ancestral sequences and 3D structures of RubisCO from a large group of related C ₃ and C ₄ species. We were able to precisely track their past evolutionary trajectories, identify mutations on each branch of the phylogeny, and evaluate their stability effect. We show that RubisCO evolution has been constrained by stability-activity tradeoffs similar in character to those previously identified in laboratory-based experiments. The C ₄ properties require a subset of several ancestral destabilizing mutations, which from their location in the structure are inferred to mainly be involved in enhancing conformational flexibility of the open-closed transition in the catalytic cycle. These mutations are near, but not in, the active site or at intersubunit interfaces. The C ₃ to C ₄ transition is preceded by a sustained period in which stability of the enzyme is increased, creating the capacity to accept the functionally necessary destabilizing mutations, and is immediately followed by compensatory mutations that restore global stability.

Summary The efforts of engineering plant ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) with the goal of improving plant photosynthetic efficiency and crop yield have existed for long. However, the directed evolution of plant Rubisco has not been widely explored because its biogenesis in a heterologous host such as Escherichia coli remains challenging. Recent breakthroughs in developing the Arabidopsis five‐auxiliary‐chaperone package and optimizing the chaperone origins have enabled the functional assembly of several plant Rubisco large subunits with their native or other plant small subunits in E. coli. But tedious and unpredictable optimization of chaperone origins might still be required for the assembly of another plant Rubisco. Here, we identified several residues at the N terminus of the large subunit that were critical for Rubisco assembly in E. coli by comparative sequential and structural analysis of cyanobacterial and plant Rubiscos. These residues in cyanobacterial Rubisco showed intensive molecular interactions with other residues within this and neighbouring large subunits. The replacement of these residues of plant Rubisco by their cyanobacterial counterparts, in combination with co‐expression of the six auxiliary chaperones, enabled/improved the assembly of Rubiscos from Flaveria bidentis, Spinacia oleracea, Nicotiana tabacum and Arabidopsis thaliana in E. coli. These chimeric plant Rubiscos exhibited similar carboxylation kinetics as their native enzyme, indicating they can serve as a starting point for molecular engineering to identify those activity‐improving amino acid substitutions. This work may facilitate the development of a universal biogenesis platform for plant Rubiscos, where only some N‐terminal residues of a plant Rubisco are replaced by the cyanobacterial ones, whereas no complex chaperone optimization is needed.

Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an ancient protein critical for CO2-fixation and global biogeochemistry. Form-I RuBisCO complexes uniquely harbor small subunits that form a hexadecameric complex together with their large subunits. The small subunit protein is thought to have significantly contributed to RuBisCO's response to the atmospheric rise of O2 ∼2.5 billion years ago, marking a pivotal point in the enzyme's evolutionary history. Here, we performed a comprehensive evolutionary analysis of extant and ancestral RuBisCO sequences and structures to explore the impact of the small subunit's earliest integration on the molecular dynamics of the overall complex. Our simulations suggest that the small subunit restricted the conformational flexibility of the large subunit early in its history, impacting the evolutionary trajectory of the Form-I RuBisCO complex. Molecular dynamics investigations of CO2 and O2 gas distribution around predicted ancient RuBisCO complexes suggest that a proposed “CO2-reservoir” role for the small subunit is not conserved throughout the enzyme's evolutionary history. The evolutionary and biophysical response of RuBisCO to changing atmospheric conditions on ancient Earth showcase multi-level and trackable responses of enzymes to environmental shifts over long timescales.