Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships

There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit     

Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.     

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.     

Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [¹⁴C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [¹⁴C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase.     

Chloroplast Dedifferentiation in Mechanically Isolated Asparagus Cells during Culture Initiation

Mechanically isolated asparagus (Asparagus officinalis) mesophyll cells dedifferentiate and divide when cultured in the dark in a medium containing sucrose. A strong correlation was observed between the onset of cell division and a loss of photosynthetic capacity. For the first 8 to 9 d of culture, there was no change in chloroplast size or morphology. However, following this period, the chloroplasts divided to form smaller proplastid-like structures. The gross chlorophyll content of the cell population did not change, suggesting that the loss of photosynthetic potential was not by senescence. Northern analysis showed that mRNA of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase was undetectable within 1 d postisolation, which was quicker than in dark-treated plants. The mRNA of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase decreased to low levels within 2 d of cell isolation. Both the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase protein showed a gradual reduction in abundance, falling to basal levels by days 6 to 7, which coincided with the onset of rapid cell division. A similar trend was observed with chloroplast rRNA molecules, which decreased to basal levels by day 6 in culture.     

Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli

A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes. A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R. sphaeroides form II RuBPC/O genes. Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis.     

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.     

Intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach studied by electron paramagnetic resonance spectroscopy

Two enzyme-metal-bound intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) have been studied by electron paramagnetic resonance (EPR) spectroscopy. Their rates of approach to a stationary state are different and their relative amounts at steady state are dependent on the concentration of ribulose 1,5-bisphosphate. It is therefore proposed that enzyme-metal-coordinated ribulose 1,5-bisphosphate and an enzyme-metal-coordinated enediolate anion of it, where bound ribulose 1,5-bisphosphate appears first, constitute the two EPR-detectable intermediates, respectively.     

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.     

Characterization of antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides showed no cross-reactivity towards the form I enzyme as evidenced by a lack of immunopreciptation. In addition, this antiserum failed to inhibit form I enzymatic activity.     

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.     

The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase

The relative specificities of the carboxylase and oxygenase reactions catalyzed by the recombinant large subunit core (L8) of Synechococcus ribulose 1,5-bisphosphate carboxylase have been determined. The L8 core still retained the ability to catalyze both reactions but at a much reduced turnover rate, about 0.6% of the holoenzyme. The fate of ribulose 1,5-bisphosphate during carboxylation and oxygenation by L8 was compared with the Synechococcus holoenzyme (reconstituted from L8 and recombinant small subunits), the carboxylase from Rhodospirullum rubrum, and that of spinach. The absence of small subunits had no significant effect on the partitioning of the bisphosphate substrate between the two reactions. Thus the course of the two competing reactions is a characteristic of the structural elements that compose the L-subunits, whereas the S-subunits exert their effect on factors common to both reactions such as the specificity of the bisphosphate substrate.     

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.     

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.     

Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides.

A heterologous phosphoribulokinase (PRK) gene probe was used to analyze two recombinant plasmids isolated from a Rhodopseudomonas (Rhodobacter) sphaeroides gene library. These plasmids were previously shown to carry the genes for form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O). Southern blot hybridization analysis indicated that there were two PRK genes linked to the RuBPC/O coding sequences. Restriction mapping showed the arrangement of the duplicate sets of PRK and RuBPC/O to be distinct. Subcloning of the hybridizing PRK sequences downstream of the lac promoter of pUC8 allowed expression of the two PRK enzymes in Escherichia coli. Analysis of the purified proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed polypeptides with molecular weights of 32,000 and 34,000 corresponding to the form I and form II PRKs, respectively. Preliminary experiments on sensitivity to NADH regulation suggested that the two PRK enzymes differ in catalytic properties.     

Isolation and some properties of ribulose-1, 5-bisphosphate carboxylase-oxygenase from red kidney bean primary leaves

Purification of ribulose-1,5-bisphosphate carboxylase from primary leaves of Phaseolus vulgaris var. Red Kidney with ammonium sulfate precipitation, ion exchange chromatography, and gel filtration resulted in the complete loss of detectable oxygenase activity and the retention of a low velocity and a high K(m) form of both the carboxylase and oxygenase. The polyethylene glycol-6000-purified ribulose-1, 5-bisphosphate oxygenase displayed a broad pH optimum (7.9-9.4) and a high K(m) for O(2) and ribulose 1,5-bisphosphate (0.90 mm and 0.25 mm, respectively). Initiation of the oxygenase reaction with protein rather than ribulose 1,5-bisphosphate resulted in reduced activity. The enzymes prepared by the two purification procedures were electrophoretically different.Etiolated primary leaf tissue exhibited low rates of both carboxylase and oxygenase. Similar developmental kinetic activity was observed for both reactions during greening. Photosynthetic (14)CO(2) fixation was inhibited 95% by 100% N(2) gas during the first 24 hours of greening, but the inhibition was rapidly overcome by 48 to 72 hours of light exposure.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Three partial reactions of ribulose-bisphosphate carboxylase require both large and small subunits

Three partial reactions of ribulose-bisphosphate carboxylase/oxygenase were measured in the presence and absence of small subunits using the enzyme from the cyanobacterium, Synechococcus ACMM 323, whose small subunits may be reversibly dissociated from its octameric, large-subunit core. These partial reactions were: the exchange of the proton at C-3 of the substrate, ribulose 1,5-bisphosphate, with the medium which is indicative of C-2, C-3 enolization; the hydrolysis of the 6-carbon reaction intermediate, 3-keto-2-carboxy-D-arabinitol 1,5-bisphosphate, to two molecules of 3-phosphoglycerate; and the decarboxylation of the 6-carbon intermediate, which is catalyzed only by the deactivated, divalent metal-ion-free carboxylase. None of these partial reactions was catalyzed by the small-subunit-depleted, large-subunit octamer to an extent greater than that expected from the residual small subunit content (about 3%), implying that small subunits are required for all three reactions. Clearly, the small subunit's influence is not restricted to any single stage of the catalytic sequence. Under conditions where it was possible to demonstrate tight binding of the reaction-intermediate analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, to the large-subunit octamer, no binding of the 6-carbon intermediate could be detected. We suggest that either the tight-binding form of the 6-carbon intermediate is the hydrated gem-diol, not the ketone, or the large subunits by themselves intrinsically possess a trace of catalytic activity which discharges any bound intermediate before it can be measured.