The mechanism of Rubisco activase: Insights from studies of the properties and structure of the enzyme.

Rubisco, the primary carboxylating enzyme in photosynthesis, must be activated to catalyze CO2 fixation. The concept of an 'activase', a specific protein for activating Rubisco, was first introduced in 1985 based largely on biochemical and genetic studies of a high CO2-requiring mutant of Arabidopsis (Salvucci et al. (1985) Photosynth Res 7: 193-201). Over the past ten years, details about the occurrence, structure, and properties of Rubisco activase have been elucidated. However, the mechanism of action of Rubisco activase remains elusive. This review discusses the need for and function of Rubisco activase and summarizes information about the properties and structure of Rubisco activase. The information is evaluated in the context of the mechanism of Rubisco activase.

The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.).

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.

Localization of light-inducible and tissue-specific regions of the spinach ribulose bisphosphate carboxylase/oxygenase (rubisco) activase promoter in transgenic tobacco plants.

Deletions in the spinach rubisco activase (Rca) promoter in transgenic tobacco were analyzed to define the regions necessary for conferring light-inducible and tissue-specific expression. Transgenic plants were constructed with Bal 31 deletions of the Rca promoter fused to the coding region of the bacterial reporter gene beta-glucuronidase (GUS). Analysis of the Rca deletion mutants localized the region conferring normal expression downstream from -294 relative to the Rca transcription start site. A second set of transgenic plants containing the cauliflower mosaic virus (CaMV) 35S enhancer fused to the 3' end of the Rca/GUS constructs demonstrated the presence of a light-responsive element between -150 and -78 active in leaves. Regions 10 bp long within the light-responsive region, which included putative G box and GT elements, were removed by recombinant polymerase chain reaction. Deletion of the G box element resulted in a loss of gene expression in the leaves of transgenic tobacco, while deletion of the GT motif caused a 10-100-fold increase in expression in roots. However, site-directed mutagenesis of the GT motif resulted in expression patterns identical to the normal promoter. These experiments demonstrated that light-inducible and tissue-specific expression of the Rca promoter involves multiple cis elements proximal to the transcription start site, and that interactions between these elements are essential for regulating expression.

Molecular basis of the ribulose-1,5-bisphosphate carboxylase/oxygenase activase mutation in Arabidopsis thaliana is a guanine-to-adenine transition at the 5'-splice junction of intron 3.

Analysis of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene and gene products from Arabidopsis thaliana wild-type plants and the Rubisco activase-deficient mutant strain showed that the rca mutation caused GT to be changed to AT at the 5'-splice junction of intron 3 in the six-intron pre-mRNA. Northern blot analysis, genomic and cDNA sequencing, and primer extension analysis indicated that the mutation causes inefficient and incomplete splicing of the pre-mRNA, resulting in the accumulation of three aberrant mRNAs. One mutant mRNA was identical with wild-type mRNA except that it included intron 3, a second mRNA comprised intron 3 and exons 4 through 7, and the third mRNA contained exons 1 through 3. The G-to-A transition is consistent with the known mechanism of mutagenesis by ethyl methanesulfonate, the mutagen used to create the Rubisco activase-deficient strain.

Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and rubisco activase.

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chlamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanacee species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid changes that have arisen by evolutionary divergence in one of these enzymes through spontaneous mutation or selection pressure have led to compensatory changes in the other enzyme.

Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis.

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase.

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K(m) value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K(m) values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V(max) of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.

Expression of the two isoforms of spinach ribulose 1,5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain.

The two isoforms of ribulose 1,2-bisphosphate carboxylase activase (Rbu-P2 carboxylase) from spinach (Spinacea oleracea L.) were individually purified from Escherichia coli transformed with expression vectors for the appropriate cDNAs. Both isoforms catalyzed activation of Rbu-P2 carboxylase (ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) and ATP hydrolysis. The kinetics of the two isoforms with respect to ATP concentration were different, in that the 45-kDa polypeptide exhibited a sigmoidal response while a rectangular response was observed with the 41-kDa isoform. These observations suggest that the additional domain at the C terminus of the 45-kDa isoform modulates the ATP regulation of activity. Lysine 169, at the putative ATP-binding site of the 41-kDa form of Rbu-P2 carboxylase activase, was changed to arginine, isoleucine, and threonine by directed mutagenesis. These mutations abolished Rbu-P2 carboxylase activase and ATPase activities, as well as the capability of the protein to bind ATP. These results confirm that lysine 169 is an essential residue.

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide.

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase.

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO(2) (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.

Chlamydomonas reinhardtii Phosphoribulokinase : Sequence, Purification, and Kinetics.

The sequence and kinetic properties of phosphoribulokinase purified from Chlamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chlamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chlamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. The transit peptide cleavage site was determined by Edman analysis of the mature protein. The precursor protein consists of a 31 amino acid transit peptide and a 344 amino acid mature polypeptide. The mature polypeptide has a calculated molecular weight of 38.5 kilodaltons and a pl of 5.75. The V(max) of the purified enzyme was 465 micromoles per minute per milligram, with apparent K(m) values of 62 micromolar ATP and 56 micromolar ribulose 5-phosphate. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Southern blot analysis of Chlamydomonas genomic DNA indicated the presence of a single phosphoribulokinase gene. Comparison of the mature proteins from Chlamydomonas and spinach revealed 86 amino acid differences in primary structure (25% of the total) without a major difference in kinetic properties. The transit peptides of the spinach and Chlamydomonas proteins possessed little sequence homology.

A novel role for light in the activation of ribulosebisphosphate carboxylase/oxygenase.

Light stimulated the activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) in a buffered lysed chloroplast system in the presence of saturating concentrations of ATP. This indicates a role for light in the rubisco activase activation system in addition to the previously identified requirement for the synthesis of ATP. Rubisco activation was nearly as great at low irradiance (10 micromoles of photons per square meter per second) as at high irradiance (1000 micromoles of photons per square meter per second). Light stimulation of activation occurred at both low bicarbonate (equivalent to air levels of CO(2)) and high bicarbonate (10 mm) concentrations. Light activation was inhibited by DCMU and glyoxylate. Methyl viologen did not inhibit light activation, and dithiothreitol did not stimulate activation in the dark, indicating that the ferredoxin/thioredoxin system was not involved. Following a transition of the lysed chloroplasts from light to dark, the light-dependent increase in activation ceased immediately. The experiments were conducted with chloroplasts from spinach (Spinacea oleracea L.), a species which was previously shown not to contain the endogenous inhibitor of rubisco, 2-carboxyarabinitol 1-phosphate. Assays of total rubisco activity in the light and dark confirmed the absence of such a tight binding inhibitor of activity. The observations reported here cannot be explained by current hypotheses of the role of light in rubisco activation and demonstrate that in addition to providing ATP needed for rubisco activase activity, at least one other light-dependent reaction is required for regulating the activation state of rubisco in vivo.

Glyoxylate inhibition of ribulosebisphosphate carboxylase/oxygenase activation in intact, lysed, and reconstituted chloroplasts.

At bicarbonate concentrations equivalent to air levels of CO2, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.

Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO(2).

Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO(2) for growth, and a unique cotton cell line that grows at ambient CO(2) (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C(3) mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of (14)C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO(2) fixation occurred primarily by the C(3) pathway. Photorespiration occurred at 330 microliters per liter CO(2), 21% O(2) as indicated by the synthesis of high levels of (14)C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO(2) fixation. Short-term CO(2) fixation in the presence and absence of carbonic anhydrase showed CO(2), not HCO(3) (-), to be the main source of inorganic carbon taken up by the low CO(2)-requiring cotton cells. The cells did not have a CO(2)-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO(2)-requiring cotton cells, present in the high CO(2)-requiring soybean cell lines, and absent in other high CO(2) cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO(2) concentrations.

Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis.

Sequence analysis of ribulosebisphosphate carboxylase/oxygenase (rubisco) activase cDNA and genomic clones isolated from spinach and Arabidopsis thaliana indicates that the two polypeptides of rubisco activase arise from alternative splicing of a common pre-mRNA. In spinach, two 5' splice sites are used in processing a single 137-nucleotide intron near the 3' end of the primary transcript. This intron was either removed completely or, alternatively, the first 22 nucleotides of the intervening sequence were retained in the mature rubisco activase mRNA. The 22-nucleotide auxiliary exon contains an in-frame ochre termination codon and leads to the synthesis of a 41-kilodalton polypeptide. Removal of the entire 137-nucleotide intervening sequence results in the synthesis of a larger 45-kilodalton polypeptide. Thus, alternative splicing of the spinach rubisco activase mRNA results in the synthesis of two polypeptides that are identical except for 37 additional amino acids at the C terminus of the 45-kilodalton polypeptide. This conclusion was confirmed by Cleveland peptide mapping and by N-terminal and C-terminal amino acid sequence analyses of both purified polypeptides. This method of producing the two rubisco activase polypeptides may be an evolutionarily conserved feature in higher plants because a nearly identical process occurs in the production of the two rubisco activase polypeptides in Arabidopsis. In Arabidopsis, an alternatively spliced intron resides at precisely the same position as the alternatively spliced intron in spinach and results in the synthesis of 44-kilodalton and 47-kilodalton rubisco activase polypeptides. In contrast to spinach, however, the retained portion of the intervening sequence does not contain an in-frame termination codon. Rather, a shift in reading frame leads to termination of translation of the smaller polypeptide within the coding region of the larger polypeptide.

Catalysis of Ribulosebisphosphate Carboxylase/Oxygenase Activation by the Product of a Rubisco Activase cDNA Clone Expressed in Escherichia coli.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase activity was obtained from a partially purified extract of Escherichia coli transformed with a 1.6-kilobase spinach (Spinacia oleracea L.) cDNA clone. This activity was ATP-dependent. Catalysis of rubisco activation by spinach and cloned rubisco activase was accompanied by the same extent of carboxyarabinitol bisphosphate-trapped (14)CO(2) as occurred in spontaneous activation, indicating that rubisco carbamylation is one facet of the rubisco activase reaction. The CO(2) concentration required for one-half maximal rubisco activase activity was about 8 micromolar CO(2). These observations are consistent with the postulated role of rubisco activase in regulating rubisco activity in vivo.

Structure and expression of spinach leaf cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase.

Ribulosebisphosphate carboxylase/oxygenase activase is a recently discovered enzyme that catalyzes the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase ["rubisco"; ribulose-bisphosphate carboxylase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in vivo. Clones of rubisco activase cDNA were isolated immunologically from spinach (Spinacea oleracea L.) and Arabidopsis thaliana libraries. Sequence analysis of the spinach and Arabidopsis cDNAs identified consensus nucleotide binding sites, consistent with an ATP requirement for rubisco activase activity. A derived amino acid sequence common to chloroplast transit peptides was also identified. After synthesis of rubisco activase in vitro, the transit peptide was cleaved and the protein was transported into isolated chloroplasts. Analysis of spinach and Arabidopsis nuclear DNA by hybridization indicated a single rubisco activase gene in each species. Leaves of spinach and Arabidopsis wild type contained a single 1.9-kilobase rubisco activase mRNA. In an Arabidopsis mutant lacking rubisco activase protein, mRNA species of 1.7 and 2.1 kilobases were observed under conditions of high-stringency hybridization with a wild-type cDNA probe. This observation indicates that the lesion in the mutant arises from an error in mRNA processing.