Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

Active oxygen (AO) species generated in plants under stress conditions trigger degradation of Rubisco (EC 4.1.1.39). To find out whether AO species activate proteases or make the protein susceptible to proteolysis, purified and ¹⁴C-labelled Rubisco protein was incubated with stromal preparations obtained from barley (Hordeum vulgare L.) leaves. The protein was degraded into distinct fragments only after a treatment with AO. This result shows that AO-treated Rubisco has been modified to become a substrate for stromal protease(s) and dismisses the possibility of protease activation. Upon degradation, distinct fragments accumulated with time. The fragmentation pattern was indistinguishable from that obtained with intact chloroplasts subjected to oxidative conditions (cf. M. Desimone et al., 1996, Plant Physiol 111: 789–796). Degradation required ATP-hydrolysis, since AMP, ADP or non-hydrolysable ATP-analogs did not support proteolysis. The ClpP-deficient stromal preparations degraded AO-modified Rubisco, making the involvement of the ClpC/P protease unlikely. Received: 1 September 1997 / Accepted 15 November 1997     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the enzyme assimilating CO2 in biology. Despite serious efforts, using many different methods, a detailed understanding of activity and regulation in Rubisco still eludes us. New results in X-ray crystallography may provide a structural framework on which to base experimental approaches for more detailed analyses of the function of Rubisco at the molecular level. This article gives a critical review of the field and summarizes recent results from structural studies of Rubisco.     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.     

Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation

When photoheterotrophic Euglena gracilis Z Pringsheim was subjected to nitrogen (N)-deprivation, the abundant photosynthetic enzyme ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was rapidly and selectively degraded. The breakdown began after a 4-h lag period and continued for a further 8 h at a steady rate. After 12 h of starvation, when the amount of Rubisco was reduced to 40%, the proteolysis of this enzyme slowed down while degradation of other proteins started at a similar pace. This resulted in a decline of culture growth, chloroplast disassembly — as witnessed by chlorophyll (Chl) loss — and cell bleaching. Experiments with spectinomycin, an inhibitor of chloroplastic translation, indicated that there was an absolute increase in the rate of Rubisco degradation in the N-deprived culture as compared with control conditions, where no significant carboxylase breakdown was detected. Oxidative aggregation of Rubisco (as detected by non-reductive electrophoresis) and association of the enzyme to membranes increased with time of N-starvation. Fluorescent labeling of oxidized cysteine (Cys) residues with monobromobimane indicated a progressive oxidation of Cys throughout the first hours of N-deprivation. It is concluded that Rubisco acts as an N store in Euglena, being first oxidized, and then degraded, during N-starvation. The mobilization of Rubisco allows sustained cell growth and division, at almost the same rate as the control (non-starved) culture, during 12 h of N-deprivation. Afterwards, breakdown is extended to other photosynthetic structures and the whole chloroplast is dismantled while cell growth is greatly reduced.Abbreviations Chl chlorophyll - Cys cysteine - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate We thank Drs. Pablo Vera and Ismael Rodrigo (Univ. Politécnica, Valencia, Spain) for advice and facilities in raising and collecting the anti-Rubisco serum. This work was supported by grants PB87-0353 and PB92-0821 of DGICYT and by a fellowship of the Spanish Ministerio de Educación y Ciencia (awarded to C.G.-F.).     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

Thermal properties and oxygenase activity of ribulose-1,5-bisphosphate carboxylase from the thermophilic purple bacterium, Chromatium tepidum

Abstract Ribulose-1,5-biphosphate carboxylase (RuBPCase) partially purified from the thermophilic purple bacterium Chromatium tepidum displayed maximum carboxylase activity at 50°C, while enzyme from a related mesophilic species, Chromatium vinosum , was completely inactive at 50°C. RuBPCase from C. tepidum showed ribulose-1,5- bisphosphate-dependent oxygenase activity, and, in addition, O₂ was found to partially destroy carboxylase activity. It is concluded that thermophilic purple bacteria produce heat-stable RuBPCase and that all RuBPCases, even those from an obligate anaerobe such as C. tepidum , have associated oxygenase activity.     

Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts

In intact chloroplasts isolated from mature pea leaves (Pisum sativum L.), the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was rapidly fragmented into several products upon illumination in the presence of 1 mM dithiothreitol (DTT). Very similar effects on LSU stability could be observed when illuminated chloroplasts were poisoned with cyanide which, like DTT, inhibits important plastid antioxidant enzymes, or when a light-dependent hydroxyl radical-producing system was added to the incubation medium. Moreover, DTT-stimulated light degradation of LSU was markedly delayed in the presence of scavengers of active oxygen species (AOS). It is therefore suggested that light degradation of LSU in the presence of DTT is mainly due to inhibition of the chloroplast antioxidant defense system and the subsequent accumulation of AOS in intact organelles. When chloroplasts were isolated from nonsenescent or senescent leaves, LSU remained very stable upon incubation without DTT, indicating that the antioxidant system was still functional in the isolated chloroplasts during leaf ageing. Our data support the notion that AOS might be important for the degradation of Rubisco in vivo under oxidative stress.     

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)^–1·s^–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)^–1·^–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m^–2·s^–1·bar^–1) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 bar (18.3 M). From measurements of the photocompensation point (_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.Abbreviations and Symbols A CO₂-assimilation rate - g_w conductance for CO₂ transfer from the substomatal cavities to the sites of carboxylation - K_c, K_o Michaelis-Menten constants for carboxylation, oxygenation of Rubisco - k_cat V_cmax/[active site] - O partial pressure of O₂ at the site of carboxylation - p_c partial pressure of CO₂ at the site of carboxylation - p_i intercellular CO₂ partial pressure - R_d day respiration (non-photorespiratory CO₂ evolution) - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - S_c/o relative specificity factor for Rubisco - SSu small subunit of Rubisco - V_cmax, V_omax maximum rates of Rubisco carboxylation, oxygenation - _* partial pressure of CO₂ in the chloroplast at which photorespiratory CO₂ evolution equals the rate of carboxylation     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Cloning and expression of the genes for ribulose-1,5-bisphosphate carboxylase from Spirulina platensis

The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase have been cloned from the filamentous cyanobacterium Spirulina platensis. The two genes, located very closely on a 4.6 kbp DNA fragment, appear to be expressed although to a different extent in minicells of Escherichia coli. The amount of large subunit produced in the bacterial host represents at least 10% of the total protein.