Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase: a step toward carbon dioxide fixation bioprocess

Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.     

Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress

The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as reflected by the kinetics of proteolysis with subtilisin. The response of the DM strain to saline stress revealed that the absence of Cys449 and Cys459 intensifies Rubisco degradation and the covalent disulfide and non-disulfide polymerization of the enzyme in vivo. Saline stress also induced Rubisco translocation to a membrane (M) fraction that contained only covalently polymerized enzyme. Rubisco mobilization to this M fraction was enhanced also in the DM strain. Altogether, these results indicate that Cys449 and Cys459 participate in the modulation of the conformational changes promoted by oxidative modifications retarding processes related to the catabolism of the enzyme in vivo.     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants

Transgenic manipulation of the photosynthetic CO2-fixing enzyme, ribulose bisphosphate carboxylase/oxygenase (Rubisco) in higher plants provides a very specific means of testing theories about photosynthesis and its regulation. It also encourages prospects for radically improving the efficiencies with which photosynthesis and plants use the basic resources of light, water, and nutrients. Manipulation was once limited to variation of the leaf's total content of Rubisco by transforming the nucleus with antisense genes directed at the small subunit. More recently, technology for transforming the small genome of the plastid of tobacco has enabled much more precise manipulation and replacement of the plastome-encoded large subunit. Engineered changes in Rubisco's properties in vivo are reflected as profound changes in the photosynthetic gas-exchange properties of the leaves and the growth requirements of the plants. Unpredictable expression of plastid transgenes and assembly requirements of some foreign Rubiscos that are not satisfied in higher-plant plastids provide challenges for future research.     

Control of photosynthetic carbon dioxide fixation by the boundary layer, stomata and ribulose 1,5-biphosphate carboxylase/oxygenase

Abstract Coefficients describing the sensitivity of the rate of photosynthetic carbon dioxide fixation to small changes in the stomatal conductance and boundary layer conductance are derived. These sensitivity or ‘control’ coefficients, together with those for the carboxylase and oxygenase activities of ribulose 1,5-bisphosphate carboxylase/oxygenase, are calculated from standard gas exchange data and apply under conditions where leaf temperature and water vapour concentration at the leaf surface remain largely constant. It is shown that the magnitude of the control coefficients depends on conditions such as photon flux density, ambient CO₂ concentration and relative humidity at the leaf surface. The extension of this analysis to encompass the sensitivity of the photosynthetic fluxes to changes in enzyme concentrations and kinetic properties is also discussed.     

Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase

Pierce, J. 1988. Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase. - Physiol. Plant. 72: 690–698.
The idea of enhancing plant productivity by minimizing the apparently wasteful process of photorespiration has been an enduring one. Since the relative fluxes of carbon through the competing pathways of photosynthesis and photorespiration are determined by the kinetic properties of a single enzyme, ribulose bisphosphate carboxylase/oxygenase, it has been conjectured that genetic modification of this protein could provide more productive plants. Recent advances in techniques for studying ribulose bisphosphate carboxylase/oxygenase hold promise for determining whether such modifications will prove possible.     

Formation of the tight-binding inhibitor, 3-ketoarabinitol-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase is O2-dependent

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) not only catalyzes carboxylation and oxygenation of ribulose-1,5-bisphosphate (RuBP), but it can also act either as an epimerase or isomerase converting RuBP into xylulose-1,5-bisphosphate (XuBP) or 3-ketoarabinitol-1,5-bisphosphate (KABP), respectively, a process called misfire. XuBP is formed as a result of misprotonation at C3 of the RuBP-enediol. It is released from Rubisco active sites and accumulates in the reaction mixture. Increasing the amounts of CO₂ or O₂ decreases XuBP production. However, KABP synthesis, which has been proposed to be only a product due to C2 misprotonation of the RuBP-endiol, is dependent upon the presence of O₂. KABP remains tightly bound to Rubisco active sites after its formation, causing the loss of Rubisco activity (fallover). The results suggest that the non-stabilized form of the peroxy-intermediate in the oxygenase reaction can be converted in a backreaction to KABP and molecular oxygen. The stabilization of the peroxy-intermediate due to the presence of Mn²⁺ instead of Mg²⁺ eliminates the formation of KABP.     

Regulation of the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in a cyanobacterium, Synechococcus PCC7942

When cyanobacterium cells are grown under extremely low CO₂ concentration, the number of carboxysomes, structures containing ribulose-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), is known to increase. This suggests that Rubisco helps to regulate photosynthesis in cyanobacteria. However, no studies have been done on the changes of Rubisco content and activity in response to the extracellular CO₂ concentration, and no information is available on its effect on photosynthesis. To elucidate the relationship between the expression responses of Rubisco and extracellular CO₂, wild-type cells (Synechococcus PCC7942) and carboxysome-lacking cells were grown under various CO₂ concentrations, and Rubisco activity was determined. In both strains, Rubisco activity increased when the cells were grown under a CO₂ concentration around, or less than, K _1/2(CO₂) of photosynthesis. In carboxysome-lacking cells, Rubisco activity increased five to six times at most, and a simultaneous increase in the rate of photosynthesis was observed. These results suggest that stimulation of expression of Rubisco occurs to compensate for the decrease in the rate of photosynthesis under CO₂-limited conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protection and enhancement of ribulose 1,5 bisphosphate carboxylase activity by exogenous proteins

When assayedin vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1,5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement.     

Inactivation of the monocistronic rca gene in Anabaena variabilis suggests a physiological ribulose bisphosphate carboxylase/oxygenase activase-like function in heterocystous cyanobacteria

There was no discernible effect after incubating recombinant Anabaena Rubisco and carboxyarabinitol 1-phosphate with the product of the Anabaena rca gene. Since the unactivated cyanobacterial Rubisco is not readily inhibited by ribulose 1,5-bisphosphate and fallover is not observed, a genetic basis for the function of the Rubisco activase-like gene (rca) was sought. The monocistronic rca gene was inactivated in vivo and resulting mutant strains of A. variabilis were found to be incapable of synthesizing immunologically detected RCA protein. The requirement for the product of the rca gene in the light was further examined by measuring Rubisco activity in permeabilized whole cells of wild-type and rca mutant strains at different light intensities. In a 1% CO₂-air atmosphere, inactivation of rca reduced the ability of A. variabilis to elevate Rubisco activity under high light (73 mol quanta m^–2 s^–1), but had little effect under low light (8 mol m^–2 s^–1). For air-grown cultures, differences in the rates exhibited by the wild-type and rca mutant to fully activate Rubisco during a whole-cell assay were enhanced by increases in light intensity. The significance of the rca mutation was underlined by effects on growth as, unlike the wild-type, growth rates did not increase after cells transferred from low to high light intensities. Higher exogenous CO₂ concentrations (1%) were required to sustain a normal growth rate for the A. variabilis rca mutant. When grown in air levels of CO₂, the rca mutant not only needed longer times to double in cell density but also exhibited greatly diminished Rubisco activity compared with the wild-type strain. Despite the unusual properties of cyanobacterial Rubisco, these results suggest a physiological role for the product of the rca gene in maximizing the activity of Rubisco in heterocystous cyanobacteria.     

Light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the genus Phaseolus

Modulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in low light and darkness was measured in A) 25 genotypes from the four cultivated species of Phaseolus (P. vulgaris, P. acutifolius, P. lunatus and P. coccineus), B) 8 non-cultivated Phaseolus species, and C) the related species Macroptileum atropurpureum. The activity ratio of Rubisco (the ratio of initial and total Rubisco activities, which reflects Rubisco carbamylation), and the molar activity of fully-activated Rubisco (which primarily reflects the inhibition of Rubisco activity by carboxyarabinitol 1-phosphate, CA1P) were assayed in leaves from the cultivated species sampled at midday in full sunlight, in low light at dusk (60 to 100 mol photons m^-2s^-1), and after at least 4 h in darkness. Dark inhibition of Rubisco molar activity was compared in both cultivated and non-cultivated species. In all cultivated genotypes, a significant reduction of the activity ratio of Rubisco was measured in leaves sampled at low light; however, the molar activity of fully activated Rubisco was not greatly reduced in these low light samples. In darkened leaves, molar activities substantially declined in most Phaseolus species with 11 of 13 exhibiting greater than 60% reduction. In P. vulgaris, the reduction of molar activity was extensive (greater than 69%) in all genotypes studied, which included wild progenitors as well as ancient and advanced cultivars. These results indicate that at low light late in the day, modulation of Rubisco activity is primarily through changes in carbamylation state, with CA1P playing a more limited role. By contrast in the dark, binding of CA1P dominates the modulation of Rubisco activity in Phaseolus in a pattern that appears to be conserved within a species, but can vary significantly between species within a genus. The degree of CA1P inhibition in Phaseolus was associated with phylogenetic affinities within the genus, as the species with extensive dark-inhibition of Rubisco activity tended to be more closely related to each other than to species with reduced inhibition of Rubisco activity.Abbreviations CA1P carboxyarabinitol 1-phosphate - CABP carboxyarabinitol bisphosphate - PFD photon flux density between 400 and 700 nm - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Measurements of the CO2/O2 specificity of ribulose 1, 5-bisphosphate carboxylase/oxygenase by 31P- and 1H-NMR

High resolution NMR spectroscopy has been demonstrated to be capable of measuring the CO2/O2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase. 31P-NMR provides a simple method for quantitatively determining the ratio of the products, 3-phosphoglycerate and phosphoglycolate. Both the specificity factor and degree of the reaction can be measured during the reaction without the need for complete consumption of ribulose bisphosphate or removal of it from the reaction mixture. Inorganic phosphate can also be detected and this may be used for monitoring the phosphatase activity and pH changes. Measurement by highly sensitive 1H-NMR is most time-efficient and is particularly suitable for the Rubisco with a high specificity factor. By optimizing the experimental conditions, it is possible to follow the simultaneous reactions in situ. The NMR method has been applied to three Rubisco enzymes with different values of specificity factor. Both 31P- and 1H-NMR gave similar results, agreeing with those previously reported by other methods.     

Activation and inhibition of spinach ribulose 1,5-bisphosphate carboxylase by 2-phosphoglycolate

Ribulose 1,5-bisphosphate carboxylase when activated by preincubation with 1 mM bicarbonate and 10 mM magnesium chloride can be further activated ca 20–500% by incubating with 2.5 mM phosphoglycolate depending upon the pH of the preincubation medium. The activation effects were seen only under specific preincubation conditions. The activation by phosphoglycolate was a slow reaction requiring ca 15 min for maximal effect. Even though magnesium was essential for phosphoglycolate activation, concentrations higher than 15 mM progressively inhibited the activation of the enzyme by phosphoglycolate. When added directly to the reaction mixture, phosphoglycolate was a potent inhibitor of the carboxylase activity. Even under preincubating conditions, phosphoglycolate showed slight inhibitory effect at 0.1 mM and activation was observed at concentrations higher than 0.5 mM. The K_A value for phosphoglycolate was 2.8 mM.     

Functional metagenomic selection of ribulose 1, 5‐bisphosphate carboxylase/oxygenase from uncultivated bacteria

Ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO₂‐dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO‐encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO₂/O₂ specificity typical of form II enzymes. X‐ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO₂‐fixing enzymes not previously characterized.     

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

A rapid method to determine the CO₂/O₂ specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO₂ and O₂ fixation at varying CO₂/O₂ ratios to determine the relative rates of each reaction. CO₂ fixation is measured by the incorporation of the moles of¹⁴CO₂ into 3-phosphoglycerate, while O₂ fixation is determined by subtraction of the moles of CO₂ fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.     

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type