Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance

Abstract

Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO 2 -fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS] 8 ) and hexadecameric ([[LS] 8 ] 2 ) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50 -90% relative to the assembly of unlinked L and S into L 8 S 8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO 2 /O 2 specificity, carboxylation rate, and Michaelis constants for CO 2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L 8 S 8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.The catalytic efficiency of the photosynthetic CO 2 -fixing enzyme D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) has a central role in determining how efficiently plants use their resources of water, fertilizer nutrient, and light (1-3). As the primary port of entry of inorganic carbon into the biosphere, it is surprising that plant Rubisco is not very efficient, particularly at limiting CO 2 concentrations where the rate of catalytic turnover is less than one-thousandth that of many other plant enzymes (4). The tendency of Rubiscos to confuse CO 2 with the more abundant atmospheric gas, O 2 , encumbers photosynthesis in higher plants with both a requirement to invest large amounts of protein in Rubisco and also a requirement for an energy-intensive photorespiratory metabolism to recycle the oxygenated waste product. However, higher plant Rubisco is not the pinnacle of evolution as more efficient and specific forms are found in nature, particularly in a number of red algae (5). The benefits of replacing the Rubisco in C 3 crops with these natural variants have been modeled and show substantial improvements that come at no additional energy or resource cost (1, 2, 6). Against this background, it is no surprise efforts are continuing into understanding Rubisco's catalytic mechanism, how it ha...

Isoleucine 309 acts as a C 4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

“…The preparation and analysis of soluble leaf protein by SDS/PAGE, nondenaturing PAGE, and immunoblot analysis was performed as described (33). Total leaf genomic DNA was isolated using the DNeasy Plant Mini Kit and used to PCR amplify and sequence the transformed plastome region using primers 5′-CTATGGAATTCGAACCT-GAACTT-3′ (LSH) and 5′-GAGGTGTGATACTTGGCTTGATTC-3′ (LSE) (Fig.…”

“…Rates of rubisco 14 CO 2 fixation using soluble leaf protein extract were measured in 7-mL septum-capped scintillation vials in reaction buffer [50 mM Hepes-NaOH (pH 7.8), 10 mM MgCl 2 , 0.5 mM RuBP] containing varying concentrations of NaH 14 CO 3 (0-67 μM) and O 2 (0-25%) (vol/vol), accurately mixed with nitrogen using Wostoff gasmixing pumps as described (24,33). Assays (0.5 mL total volume) were started by the addition of activated leaf protein, and the Michaelis constants (K m ) for CO 2 (K C ) and O 2 (K O ) were determined from the fitted data.…”

The Catalytic Properties of Hybrid Rubisco Comprising Tobacco Small and Sunflower Large Subunits Mirror the Kinetically Equivalent Source Rubiscos and Can Support Tobacco Growth

“…A sample of lysate was taken for SDS-PAGE analysis as described (Whitney et al, 2001) and the remainder centrifuged (14,000g, 1 min, 4°C) and samples of the soluble protein taken for SDS-PAGE or incubated (activated) with NaHCO 3 and MgCl 2 (15 mM each) for 10 min and used to: measure Rubisco content in duplicate aliquots incubated with 10 or 30 mM of [ 14 C]CABP and the amount of Rubiscobound [ 14 C]CABP recovered by gel filtration (Ruuska et al, 1998;Whitney and Andrews, 2001b), or used to measure K c 21%O 2 at 25°C, pH 8.0 using 14 CO 2 fixation assays as described in Whitney and Sharwood (2007). The K c assays were initiated by adding the activated soluble protein extract into septum capped scintillation vials containing CO 2 -free air equilibrated assay buffer (100 mM Hepps-NaOH, 15 mM MgCl 2 , 0.6 mM ribulose-P 2 , 0.1 mg mL 21 carbonic anhydrase) containing 0 to 44 mM 14 CO 2 .…”

“…The K c assays were initiated by adding the activated soluble protein extract into septum capped scintillation vials containing CO 2 -free air equilibrated assay buffer (100 mM Hepps-NaOH, 15 mM MgCl 2 , 0.6 mM ribulose-P 2 , 0.1 mg mL 21 carbonic anhydrase) containing 0 to 44 mM 14 CO 2 . The assays were stopped after 1 min with 0.5 volumes of 25% (v/v) formic acid and processed for scintillation counting (Whitney and Sharwood, 2007 …”

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

“…When hybrid enzymes composed of Synechococcus large subunits and the small subunits of other higher ⍀ Rubisco enzymes from prokaryotes, algae, and plants were expressed in vitro or in E. coli, they had the same or slightly lower ⍀ values relative to that of Synechococcus Rubisco (23)(24)(25)(26)(27)(28). It has been difficult to study the contribution of small subunits to the function of eukaryotic Rubisco enzymes because eukaryotic subunits cannot assemble into functional Rubisco when expressed in E. coli (29,30), and plant small subunits are coded by a family of rbcS genes in the nucleus that cannot be eliminated and replaced with foreign copies. However, pea small subunits were introduced into Arabidopsis thaliana to form a heterologous Rubisco enzyme that had a small decrease in carboxylation activity nearly equal to the percentage of foreign small subunits in the holoenzyme (31).…”