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.     

Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH₄)₂SO₄ was stable when frozen as small beads in liquid nitrogen and stored at −80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO₂ and Mg²⁺ activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.     

Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase

Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using ¹⁴CO₂ and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H₂O₂ formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.     

Is ribulose bisphosphate carboxylase present in guard cell chloroplasts

Evidence for and against the presence of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in guard cell chloroplasts is presented. Methods to investigate this question, including immunocytochemistry, are compared.     

Interactions of hydrogen peroxide with ribulose bisphosphate carboxylase oxygenase

Hydrogen peroxide inhibited both carboxylase and oxygenase activities of purified, and fully activated, spinach ribulose-1,5-bisphosphate (RuP2) carboxylase-oxygenase. Inhibition of the carboxylase reaction was mixed competitive with respect to CO2 (Ki = 1.2 mM) and uncompetitive with respect to RuP2. For the oxygenase reaction, H2O2 was a competitive inhibitor with respect to O2 (Ki = 2.1 mM) and an uncompetitive inhibitor with respect to RuP2. H2O2 did not alter the stoichiometry between CO2 and RuP2 in the carboxylase reaction, indicating that H2O2 was not itself a substrate for the enzyme. RuP2 decreased the rate of deactivation of the enzyme which occurred at limiting CO2 concentrations. H2O2 greatly enhanced this stabilizing effect of RuP2 but had no effect on the rate of deactivation in the absence of RuP2. The inhibitory and stabilizing effects of H2O2 varied similarly with H2O2 concentration. These instantaneous, reversible effects of H2O2 were readily distinguishable from an irreversible inhibitory effect which occurred quite slowly, and in the absence of RuP2. These observations are discussed in relation to the enzyme's catalytic mechanism and its activation-deactivation transformations.     

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.     

The effects of bivalent cations on ribulose bisphosphate carboxylase/oxygenase.

The half-saturation constants for binding of the bivalent cations (Mg2+, Ni2+, Co2+, Fe2+ and Mn2+) to ribulose bisphosphate carboxylase/oxygenase from Glycine max and Rhodospirillum rubrum were measured. The values obtained were dependent on the enzyme and the cation present, but were the same for both oxygenase and carboxylase activities. Ribulose bisphosphate rather than its cation complex was the true substrate. The kinetic parameters Vmax.(CO2), Vmax.(O2), Km(CO2), Km(O2), and K1(O2) were determined for both enzymes and each cation activator. The evolutionary and mechanistic implications of these data are discussed.     

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.     

Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported beta-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts

The large subunit of ribulose bisphosphate carboxylase from Anacystis nidulans 6301, and the β subunit of chloroplast ATP synthase from maize, were fused to the transit peptide of the small subunit of ribulose bisphosphate carboxylase from soybean. These proteins were assayed for post-translational import into isolated pea chloroplasts. Both proteins were imported into chloroplasts. Imported large subunits were associated with two distinct macromolecular structures. The smaller of these structures was a hybrid ribulose bisphosphate carboxylase holoenzyme, and the larger was the binding protein oligomer. Time-course experiments following import of the large subunit revealed that the amount of large subunit associated with the binding protein oligomer decreased over time, and that the amount of large subunit present in the assembled holoenzyme increased. We also observed that imported small subunits of ribulose bisphosphate carboxylase, although predominantly present in the holoenzyme, were also found associated with the binding protein oligomer. In contrast, the imported β subunit of chloroplast ATP synthase did not assemble into a thylakoid-bound coupling factor complex.     

Carbon dioxide assimilation by leaves, isolated chloroplasts, and ribulose bisphosphate carboxylase from spinach

The relationship between rate of photosynthesis and CO(2) concentration has been reinvestigated using isolated spinach (Spinacia oleracea) chloroplasts. The apparently low CO(2) concentration required for half-maximal photosynthesis is shown to result partly from a ceiling imposed by electron transport. In double reciprocal plots of rate against CO(2) concentration, this ceiling results in departures from linearity at high CO(2) concentrations. If these rate limitations are disregarded in extrapolation the "true" CO(2) concentration required for half maximal carboxylation by intact chloroplasts is approximately 46 mum (CO(2)).When assayed under comparable conditions, ribulose bisphosphate carboxylase from these chloroplasts also shows an apparent Km (CO(2)) of approximately 46 mum, suggesting that its characteristics are not modified by extraction. An improved assay for ribulose bisphosphate carboxylase yielded rates of carboxylation considerably higher than those previously reported, the highest maximal velocities recorded approaching 1000 mumoles CO(2) fixed mg(-1) chlorophyll hr(-1) at 20 C. With such Km and V(max), values the carboxylase would be able to achieve, at concentrations of CO(2) less than atmospheric, rates of CO(2) fixation equal to those displayed by the parent tissue or by the average plant under favorable conditions in its natural environment.     

Synthesis and assembly of large subunits into ribulose bisphosphate carboxylase/oxygenase in chloroplast extracts

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [32P]-pBR322 at moderately high stringency. Moreover, nick-translated [32-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the 32P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Cooperative binding of carboxyarabinitol bisphosphate to the regulatory sites of ribulose bisphosphate carboxylase/oxygenase from spinach.

Ribulose bisphosphate carboxylase (RuBisCO) binds carboxyarabinitol bisphosphate (CABP) on its regulatory sites [Yokota, A. (1991) J. Biochem. 110, 246-252]. The characteristics of the equilibrium binding of CABP to the sites were examined by the gel-filtration method. Since RuBisCO binds CABP on the substrate sites with a dissociation constant of less than 10 pM, CABP bound exclusively to the substrate sites at less than 5 microM. Plotting the number of CABP bound to the sites other than the substrate sites against the concentration of CABP gave a typical "bumpy" curve; the binding number in the intermediate plateau at 20 to 40 microM CABP was 3.7 to 4.4 mol per mol of RuBisCO and that at the saturating concentration of CABP was 7.6 to 7.8 mol per mol of RuBisCO. The Hill plot of their relationship gave a line which bent strongly at 20 to 40 microM CABP. The best fitting of the data to the equations derived from the binding model constructed according to the reported model [Teipel, J. & Koshland, D.E., Jr. (1969) Biochemistry 8, 4656-4663] showed that the binding of CABP to the regulatory sites proceeded with positive cooperativity both before and after the plateau. The dissociation constant decreased from 31 to 14 microM by the factor of 1/1.3 in the former group and 490 to 0.7 microM by the factor of 1/8.9 in the latter with increasing binding number of CABP.     

Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum

Crystalline tobacco (Nicotiana tabacum L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was prepared using a procedure which protected the enzyme from hydrolysis by endogenous proteases. Leaves were extracted in a buffered medium containing casein, leupeptin, and high concentrations of MgSO4 and NaHCO3. After filtration through ion-exchange resin to remove contaminants, the enzyme was concentrated by precipitation with polyethylene glycol and crystal formation was induced by low-salt dialysis. The crystalline enzyme had a measured specific activity of 1.7 mumol CO2 mg protein-1 min-1, and about 93% of the enzyme could be activated with Mg2+ and CO2. Crystalline enzyme prepared in the absence of casein exhibited an activity which was only one-third of this rate and only about 70% of the enzyme could be activated with Mg2+ and CO2. Casein-extracted enzyme was resolved into distinct bands corresponding to the large (55,000) and small (14,000) subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of enzyme prepared according to the latter procedure was found to be composed of five different polypeptides of slightly decreasing molecular weight. Only about one-third of the large subunits were of the 55,000 molecular weight type. No differences between the two preparations were observed in the Km (CO2) and apparent Km (ribulose bisphosphate).     

Cloning,expression and directed mutagenesis of the genes for ribulose bisphosphate carboxylase/oxygenase

The dominant natural form of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of large (L) 55-kDa and small (S) 15-kDa subunits. This enzyme (as the L₈S₈ form) is widely distributed among oxygenic photosynthetic species and among chemosynthetic bacteria. Another form lacking small subunits is found as an L₂ dimer in Rhodospirillum rubrum or an L oligomer of uncertain aggregation state from Rhodopseudomonas spharoides. The present article reviews two basically different approaches in cloning the R. rubrum gene for RuBisCO. One results in high level expression of this gene product fused with a limited aminoterminal stretch of -galactosidase and the other results in expression of wild-type enzyme in Escherichia coli. Also reviewed are a number of reports of cloning and assembly of the L₈S₈ enzyme in using E. coli L and S subunit genes from Anacystis nidulans, Anabaena 7120, Chromatium vinosum and Rps. sphaeroides.In vitro oligonucleotide-directed mutagenesis has been applied to the gene for RuBisCO from R. rubrum. In terms of contributing new information to our understanding of the catalytic mechanism for RuBisCO, the most significant replacement has been of lys 166 by a number of neutral amino acids or by arg or his. Results establish that lys 166 is a catalytically essential residue and illustrate the power of directed mutagenesis in understanding structure-function correlates for RuBisCO.Oligonucleotide-directed mutagenesis has also been applied to the first and second conserved regions of the S subunit gene for RuBisCO from A. nidulans. In the latter region, corresponding amino acid changes of trp 55 and trp 58 to phe, singly or together, had little or no effect upon enzyme activity. In contrast, mutagenesis in the first conserved region leading to the following pairs of substitutions: arg10 arg 11 to gly 10 gly11; thr14 phe 15 ser 16 to ala 14 phe 15 ala 16; ser 16 tyr 17 to ala 16 asp 17; or pro 19 pro 20 to ala 19 ala 20, are all deleterious.Advances are anticpated in the introduction and expression of interesting modifications of S (and L) subunit genes in plants. A new method of introducing and expressing foreign genes in isolated etiochloroplasts is identified.Abbreviations RuBisCO ribulose bisphosphate carboxylase/oxygenase - 2-CABP 2-carboxyarabinitol-1,5-bisphosphate - 4-CABP 4-carboxyarabinitol-1,5-bisphosphate     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

Formation of a carboxyarabinitol bisphosphate complex with ribulose bisphosphate carboxylase/oxygenase and theoretical specific activity of the enzyme

¹⁴C-Labeled 2-carboxyarabinitol-1,5-bisphosphate was bound to both nonactivated and CO₂and Mg²⁺ activated forms of ribulose bisphosphate carboxylase/oxygenase. The complex could be precipitated with 20% polyethylene glycol and 20 mm MgCl₂ for quantitation of the moles of the affinity label bound per mole of enzyme. The [¹⁴C]carboxyarabinitol-P₂ bound to the nonactivated enzyme could be exchanged with a 100-fold excess of the unlabeled compound. With the activated enzyme the binding of [¹⁴C]carboxyarabinitol-P₂ was so tight that it did not exchange with the unlabeled compound and a binding stoichiometry of one molecule per active site was assumed. This tight binding was dependent upon pretreatment of the enzyme with both CO₂ and MgCl₂ in the same manner that enzyme activation depended on CO₂ and Mg²⁺ concentrations. Various enzyme preparations from spinach leaves tightly bound [¹⁴C]carboxyarabinitol-P₂ in proportion to their specific activities. By extrapolating to a maximum binding of 8 mol of [¹⁴C]carboxyarabinitol-P₂ per mole of this A₈B₈ enzyme a theoretical specific activity of 2.8 μmol · min^?1 · mg protein^?1 was indicated. Enzyme preparations purified from spinach leaves generally have a specific activity in the range of 1.0 to 2.3.     

Protein-bound ribulose bisphosphate correlates with deactivation of ribulose bisphosphate carboxylase in leaves

Previous reports indicate that ribulose 1,5-bisphosphate (RuBP) binds very tightly to inactive ribulose bisphosphate carboxylase (rubisco) in vitro. Therefore, we decided to investigate whether there was evidence for tight binding of RuBP associated with deactivation of rubisco in vivo. We modified a technique for rapidly separating `free' metabolites from those bound to high molecular compounds. Arabidopsis thaliana plants were illuminated at various irradiances before freezing the leaves in liquid N₂ and assaying rubisco activity and RuBP. The percentage activation of rubisco varied from 37% at low irradiance (45 micromoles quanta per square meter per second) to 100% at high irradiance (800 micromoles quanta per square meter per second). The total amount of RuBP did not vary much with irradiance, but bound RuBP changed from 36% of the total at low irradiance to none at high irradiance. Bound RuBP was significantly correlated with the estimated number of inactive rubisco sites, with a ratio of about 1:1. After a step increase in irradiance, rubisco activation increased and total RuBP increased transiently, but steady levels of both occurred by 10 minutes. The amount of bound RuBP decreased with a similar time course to the estimated decrease in inactive rubisco sites. After a step decrease in irradiance, rubisco deactivated slowly for at least 25 minutes. Bound RuBP increased gradually but did so more slowly than the estimated increase in inactive rubisco sites.     

Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.