Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective

Marine and terrestrial photosynthetic and chemoautotrophic microorganisms assimilate considerable amounts of carbon dioxide. Like green plastids, the predominant means by which this process occurs is via the Calvin-Benson-Bassham reductive pentose phosphate pathway, where ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a paramount role. Recent findings indicate that this enzyme is subject to diverse means of control, including specific and elaborate means to guarantee its high rate and extent of synthesis. In addition, powerful and specific means to regulate Rubisco activity is a characteristic feature of many microbial systems. In many respects, the diverse properties of microbial Rubisco enzymes suggest interesting strategies to elucidate the molecular basis of CO₂/O₂ specificity, the holy grail of Rubisco biochemistry. These systems thus provide, as the title suggests, different perspectives to this fundamental problem. These include vast possibilities for imaginative biological selection using metabolically versatile organisms with well-defined genetic transfer capabilities to solve important issues of Rubisco specificity and molecular control. This review considers the major issues of Rubisco biochemistry and regulation in photosynthetic microoganisms including proteobacteria, cyanobacteria, marine nongreen algae, as well as other interesting prokaryotic and eukaryotic microbial systems recently shown to possess this enzyme.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

Activation-dependent changes in microenvironment of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase

The spinach ribulose 1,5-bisphosphate carboxylase/oxygenase was labelled with o-phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o-phthalaldehyde, namely 1-(hydroxyethylthio)-2-beta hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH less than 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to greater than 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.     

Covalent modification of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodomicrobium vannielii

The most abundant phosphorus-containing polypeptide in the purple non-sulphur bacterium Rhodomic-robium vannielii has been identified by a combination of immunoprecipitation and sucrose density gradient centrifugation as the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. The covalent modification of the large subunit involves the phosphorylation of one or more tyrosine residues and appears to occur prior to assembly of the large subunit into the mature enzyme. In addition, the phosphorylated form of the large subunit was found to exist in at least two distinct protein complexes of Mr 410,000 and 440,000.     

The dimerization of folded monomers of ribulose 1,5-bisphosphate carboxylase/oxygenase

Spontaneous refolding and reconstitution processes of dimeric ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum have been investigated using size-exclusion high performance liquid chromatography (HPLC), spectroscopic, and activity measurements. When the unfolded Rubisco in guanidine hydrochloride is diluted at 4 degrees C, a folding intermediate (Rubisco-I) is rapidly formed, which remains in an unstable monomeric state and gradually develops into folded monomer (Rubisco-M) at 4 degrees C but undergoes irreversible aggregation at 25 degrees C. Refolding of Rubisco-I to Rubisco-M is a very slow process, taking about 20 h for 70% conversion at 4 degrees C. Rubisco-M is stable at 4 degrees C and is capable of forming an active dimer spontaneously when incubated at a temperature higher than 10 degrees C. The dynamic dimerization process has been measured in a temperature range of 4-35 degrees C by HPLC, and the results demonstrate that the dimerization is strongly facilitated by the temperature. It is found that dithiothreitol is essential for the spontaneous reconstitution of Rubisco.     

Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z

An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.     

Partial reduction in ribulose 1,5-bisphosphate carboxylase/oxygenase activity by carboxypeptidase A

Treatment with carboxypeptidase A of ribulose bisphosphate carboxylase/oxygenase (rubisco) from spinach and Chlamydomonas, but not tobacco, reduced activity by 60-70%. Further studies with the spinach enzyme indicated that only one amino acid from each of the large (valine) and small (tyrosine) subunits was removed and the loss of activity was correlated with modification of the large subunit. The modified enzyme also had a two-fold greater Km for RuBP but CO2/O2 specificity was only 5% lower and may not be significantly different. The relative rates of release of valine and tyrosine also depended on the presence or absence of RuBP or CO2 plus Mg during treatment. The results indicate that the C-terminal amino acid in the large subunit of spinach, which is not located near the active site region, plays a previously unrecognized role in determining the catalytic activity of the enzyme.     

Inhibition of ribulose 1,5-bisphosphate carboxylase/oxygenase by 2-carboxyarabinitol-1-phosphate

In some plants, 2-carboxy-d-arabinitol 1-phosphate (CA 1P) is tightly bound to catalytic sites of ribulose, 1,5-bisphosphate carboxylase/oxygenase (rubisco). This inhibitor's tight binding property results from its close resemblance to the transition state intermediate of the carboxylase reaction. Amounts of CA 1P present in leaves varies with light level, giving CA 1P characteristics of a diurnal modulator of rubisco activity. Recently, a specific phosphatase was found that degrades CA 1P, providing a mechanism to account for its disappearance in the light. The route of synthesis of CA 1P is not known, but could involve the branched chain sugar, hamamelose. There appear to be two means for diurnal regulation of the number of catalytic sites on rubisco: carbamylation mediated by the enzyme, rubisco activase, and binding of CA 1P. While strong evidence exists for the involvement of rubisco activase in rubisco regulation, the significance of CA 1P in rubisco regulation is enigmatic, given the lack of general occurrence of CA 1P in plant species. Alternatively, CA 1P may have a role in preventing the binding of metabolites to rubisco during the night and the noncatalytic binding of ribulose bisphosphate in the light.     

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     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Phylogeny and evolution of the ribulose 1,5-bisphosphate carboxylase/oxygenase genes in prokaryotes

The review considers the phylogeny and evolution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is the key enzyme of the autotrophic Calvin-Benson cycle and the most abundant protein on Earth. RuBisCO occurs in several structural and functional forms, including fully functional forms I, II, and III, which catalyze carboxylation/oxygenation of ribulose 1,5-bisphosphate, and RuBisCO-like form IV, which lacks carboxylating activity. The genomic localization, operon structure, and copy number of the RuBisCO genes vary among different autotrophic organisms. The RuBisCO gene phylogeny substantially differs from the phylogeny of other conserved genes, including the 16S rRNA gene. The difference is due to duplication/deletion and horizontal gene transfer events that were common in the evolution of autotrophic organisms.     

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase

Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 A resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 A Calpha-Calpha) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 A Calpha-Calpha). Structural analysis of the mutant enzymes identified regions (residues 50-72 of the small subunit and residues 161-164 and 259-264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.     

Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides.

Form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides is inactivated upon the addition of organic acids to photolithoautotrophically grown cultures. Activity recovers after the dissipation of the organic acid from the culture. The inactivation process depends on both the concentration of the organic compound and the nitrogen status of the cells. The inactivated RubisCO has been purified and was shown to exhibit mobility on both nondenaturing and sodium dodecyl sulfate gels different from that of the active enzyme prepared from cells not treated with organic acids. However, the Michaelis constants for ribulose 1,5-bisphosphate and CO2 or O2 were not dramatically altered. Purified inactivated RubisCO could be activated in vitro by increasing the temperature or the levels of Mg(II), and this activation was accompanied by changes in the electrophoretic mobility of the protein. When foreign bacterial RubisCO genes were expressed in an R. sphaeroides host strain lacking the ability to synthesize endogenous RubisCO, only slight inactivation of RubisCO activity was attained.     

Structural differences in the catalytic subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides.

There are significant differences in the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase isolated from Rhodopseudomonas sphaeroides. Two-dimensional peptide mapping of carboxymethylated large subunits clearly indicates that there are differences in the primary structure of the two proteins. These results are supported by limited proteolysis with three different proteases and by subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data, in conjunction with immunological studies and investigations on the regulation of the two enzymes, support the conclusion that the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase may be different gene products.     

Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions

Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg²⁺ and HCO₃⁻, and then stored for up to 30 days at 4 or −18°C or in liquid N₂. Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg²⁺ and CO₂ and subsequent storage at either 4°C or in liquid N₂ or flushing the samples with N₂ and rapid freezing and storage in liquid N₂ are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved.     

Cloning and expression in Escherichia coli of the form II ribulose 1,5-bisphosphate carboxylase/oxygenase gene from Rhodopseudomonas sphaeroides

The gene encoding the form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) from Rhodopseudomonas (R.) sphaeroides has been identified on a 3-kb EcoRI fragment and cloned into a broad-host-range, high-copy-number plasmid, using the gene from Rhodospirillum (Rs.) rubrum as a hybridization probe. Subclones of the gene from R. sphaeroides in pBR322 and pUC8 show substantial levels of expression and enzymatic activity in whole cells and crude cell extracts of Escherichia coli. This enzymatic activity has been shown to be similar in many respects to that of the protein purified from R. sphaeroides.     

Glutamine synthetase in ribulose 1,5-bisphosphate carboxylase/oxygenase deficient tobacco mutants in cell suspension culture

In two tobacco mutants lacking ribulose, 1,5-bisphosphate carboxylase/oxygenase the amount of glutamine synthetase and its activity were determined and compared with the wild type green cells. It was shown that in these two mutants glutamine synthetase protein content was six times lower than in the wild type. This situation was comparable to that found in etiolated cells where ribulose 1,5-bisphosphate carboxylase/oxygenase was absent. These observations suggest that a common regulatory mechanism might control the dual light dependent biosynthesis of both enzymes. The results have also implications concerning the efficiency of the reassimilation of ammonia by chloroplastic glutamine synthetase during the photorespiratory process.