Cross-linking between chloroplast fructose-1,6-bisphosphatase and thioredoxin f

The interaction between chloroplast fructose-1,6-bisphosphatase (FBPase) and thioredoxin (Trx) f , two plant proteins involved in the Benson-Calvin cycle, is mainly of an electrostatic nature [Hermoso et al. (1996) Plant Mol Biol 30: 455–465; Reche et al. (1997) Physiol Plant 101: 463–470; Sahrawy et al. (1997) J Mol Biol 269: 623–630; Hermoso et al. (1999) Physiol Plant 105: 756–762], possibly involving carboxyl groups of the enzyme and amino groups of Trx f . We carried out the covalent stabilization of that ionic complex, for the purpose of studying the interaction between both proteins and the factors that influence it. We have used 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, a reagent able to cross-link carboxyl and amino groups, which allows the formation of covalent bonds between the groups that, in solution, form ionic bonds. A stable functional complex between both proteins was formed. The efficiency in the formation of that complex depends on the redox state of Trx f , ionic strength and pH, showing a strong correlation with the Trx f -dependent enzyme activity. The complex also retains enzyme activity. This suggests that the formation of the covalent complex requires the previous stabilization of a specific functional ionic complex between both proteins, and that in this functional complex carboxyl groups of the enzyme and primary amines of Trx f are involved. This complex is not stable in a tetrameric structure of the enzyme. We could also detect covalent aggregates of FBPase subunits, which indicates the implication of ionic interactions in the stabilization of the tetrameric structure of the enzyme; besides, as molecular filtration experiments and electrophoresis suggest, hydrophobic forces would also be implicated in the enzyme structure.     

Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase

The amino acid sequence of the spinach chloroplast fructose-1,6-bisphosphatase (FBPase) subunit has been determined. Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage. The sequence of spinach chloroplast FBPase shows clear homology (ca. 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose-1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme. The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity. This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines. The recent X-ray crystallographic resolution of pig kidney FBPase (H. Ke, C. M. Thorpe, B. A. Seaton, F. Marcus, and W. N. Lipscomb, 1989, Proc. Natl. Acad. Sci. USA 86, 1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms. It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are conserved in the chloroplast enzyme. The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxin-linked mechanism can be accommodated in the surface of the FBPase molecule.     

Studies on the regulatory properties of chloroplast fructose-1,6-bisphosphatase

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8.The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.     

Studies on the hysteretic properties of chloroplast fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.     

Studies on the regulatory properties of chloroplast fructose-1,6-bisphosphatase.

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8. The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.     

Spinach (Spinacia oleracea) chloroplast sedoheptulose-1,7-bisphosphatase. Activation and deactivation, and immunological relationship to fructose-1,6-bisphosphatase.

In this paper we study activation by dithiothreitol and reduced thioredoxins and deactivation by oxidized thioredoxins f of sedoheptulose-1,7-bisphosphatase. The behaviour of the enzyme when chromatographed on a thioredoxin-Sepharose column is also described. The enzyme is autoxidizable upon removal of reducing agents, and is activated when reduced by any of the thioredoxins. This mechanism may allow the regulation of the Calvin cycle upon light-dark and dark-light transitions. The formation of a stable complex between enzyme and thioredoxin could explain the inhibitory effect of high thioredoxin concentrations. The use of immunological techniques shows that sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase are poorly related immunologically.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisphosphatase resembles other regulatory enzymes involved in photosynthetic CO2 assimilation in its activation by chloroplast metabolites.     

Regulation of the activation of chloroplast fructose-1,6-bisphosphatase: inhibition by spermidine and spermine

The activation of chloroplast fructose-1,6-bisphosphatase by fructose-1,6-bisphosphate, Ca2+, DTT and chloroplast thioredoxin-f is prevented by either spermidine or spermine; on the contrary, other amino compounds do not replace polyamines in this reversible effect. On the other hand, neither spermidine nor spermine modify the catalysis of chloroplast fructose-1,6-bisphosphatase. The effect of spermidine, but not the effect of spermine, is reversed by increasing the concentration of Ca2+ in the activation; higher concentrations of Fructose-1,6-bisphosphate or thioredoxin-f do not restore the control activity. The present results suggest that other regulatory mechanisms may control the activation of fructose-1,6-bisphosphatase in chloroplasts.     

Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat

An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.     

Chloroplast fructose-1,6-bisphosphatase: structure and function

Redox regulation of photosynthetic enzymes has been a preferred research topic in recent years. In this area chloroplast fructose-1,6-bisphosphatase is probably the most extensively studied target enzyme of the CO₂ assimilation pathway. This review analyzes the structure, biosynthesis, phylogeny, action mechanism, regulation and kinetics of fructose-1,6-bisphosphatase in the light of recent findings on structure–function relationship, and from a molecular biology viewpoint. This revised version was published online in June 2006 with corrections to the Cover Date.     

Activation properties of the redox-modulated chloroplast enzymes glyceraldehyde 3-phosphate dehydrogenase and fructose-1,6-bisphosphatase

Rapid changes of enzyme activity are obtained by post-translational modification of cysteine residues of some chloroplast enzymes. Individual fine-tuning is achieved by specific factors acting upon the redox cycle. In order to study the regulatory properties of these enzymes, they are purified from leaves or in a recombinant form from Escherichia coli . The various factors acting upon the enzyme in vivo can be simulated in vitro. However, in these studies, some subtle technical problems can be encountered. Two cases are presented in this article, and an attempt is made to explain some previous, seemingly contradictory results. The Calvin-cycle enzyme glyceraldehyde 3-phosphate dehydrogenase in its less active A8B8 form can be dissociated and thereby activated in vitro simply by diluting out the protein. On the other hand, activation requires the presence of reduced thioredoxin (Td) and an increase in ionic strength when performed at a high protein concentration, as present in vivo. Chloroplast fructose-1,6-bisphosphatase (FBPase) is purified from E. coli as an enzyme similar to that purified from leaves. However, using the standard protocol for lysis of the bacteria leads to a form with some unusual properties as changed isoelectric point, lack of Ca2+/fructose-1,6-bisphosphate (FBP) dependency of reductive activation, and lack of activity at high pH and high Mg2+ concentration. These observations are used in order to better understand the characteristics of the activation/inactivation process.     

Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin

When we compare the primary structures of the six chloroplast fructose-1,6-bisphosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (¹⁴⁹Pro-¹⁶⁷Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase [29], this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Binding and activation of pea chloroplast fructose-1,6-bisphosphatase by homologous thioredoxins m and f

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) binds its putative physiological activator thioredoxin f (Trx f ) at pH 7.9, the pH in the stroma of the illuminated chloroplast. Since Trx m , described as specific in NADP⁺-malate dehydrogenase (NADPMDH) activation, appears in pea (Pisum sativum L.) also to be functional in FBPase modulation, we have here analyzed the effect of pH and the redox status of the chloroplast stroma in the pea FBPase binding of homologous Trx f and m . Both pea Trx were strongly bound by purified FBPase when they were preincubated at pH 7.9 with 2.5 m M dithiothreitol (DTT), but not when the reductant was omitted. As occurs with Trx f the Trx m /FBPase ratio of the complex was 4, but this was only observed with a Trx m /FBPase concentration ratio > 10 in the preincubation mixture. The FBPase-Trx m binding disappeared in the presence of 100 m M NaCl, even with 2.5 m M DTT at pH 7.9, with a concomitant appearance of different aggregation states of the FBPase subunit. A similar FBPase-Trx m complex was detected in the stromal solution when pea chloroplasts were lysed at pH 7.9 in the presence of DTT. No interaction was observed between NADP-MDH and Trx f or m , either in the presence or in the absence of DTT. Pea FBPase showed sigmoidal activation kinetics with pea Trx m , and an S_0.5 of 133 n M versus 6.6 n M with pea Trx f . About 10-fold higher concentration of the former than that of the latter was required for obtaining maximum activity; however, the V_max with Trx f was only 2-fold higher than that with Trx m . We conclude that pea FBPase binds and is activated by the homologous Trx m , even though to a lesser extent than with Trx f . We also deduce that in the light the conditions in the chloroplast stroma are optimal for forming an FBPase-Trx complex.