Julio López Gorgé

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.

Binding features of chloroplast fructose-1,6-bisphosphatase-thioredoxin interaction

It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities.

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.

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Molecular weight transitions of the purified enzyme

Abstract Fructose-1,6-diphosphatase (FDPase) active fractions I and II earlier purified from spinach leaves show similar molecular weights, in the range 92 000–115 000, when calculated by Sephadex filtration and acrylamide gel electrophoresis. Both fractions are stable at acidic and neutral pH, but at pH 8.8 are partly splitted in similar subunits. Acrylamide electrophoresis at different gel concentration (Ferguson plots) show these subunits as monomers of native dimers I and II, with molecular weights between 54 000 and 60 000.

Properties of spinach chloroplast fructose-1,6-diphosphatase

Abstract Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg2+-dependent for activity, with an Optimum Mg2+ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG2+/EDTA ratio is increased to 10; Mn2+ and Co2+ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. Km values are 1·4 × 10−3 M (fraction I) and 1·1 × 10−3 M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).

Isolation and properties of crystalline ferredoxin from lactuca sativa

Abstract Lettuce ferredoxin has been purified to homogeneity, with a yield of 18 mg/kg of denerved leaves. It crystallizes in magnificent needles, often clustered in broom-like sheaves. The absorption spectrum showed maxima at 460, 422, 330 and 274 nm,with a ratio A422/A274, of 0.46. The mM absorption coefficient was 9.74 at 422 nm, and 21.62 at 274 nm. This ferredoxin showed a pI = 4.7 and an E′0 = −425 mV (at pH = 7.7). MWs of 12 400, 11480 and 13000 were obtained by sucrose gradient centrifugation, and on the basis of the amino acid composition and the iron content, respectively, with an average of 12 300. The amino acid analysis showed the existence of one methionine residue per mole, with 105 amino acid residues. There are two iron atoms and two labile sulfide groups per mole; 4 half-cystine residues were found by performic acid oxidation, and 5 cysteine groups when determined by titration with pHMB. The native protein is not fixed on thiol-Sepharose 4B, but it is quantitatively retained after incubation with 8 M urea. Lettuce ferredoxin showed a 62, 58 and 78% effectiveness with the spinach ferredoxin-NADP reductase, nitrite reductase and fructose-1,6-diphosphatase (FDPase), respectively, when compared with the spinach ferredoxin. This different behaviour of both ferredoxins is joined to genetic-structural relationships, and suggests that the role of ferredoxin in FDPase activation is more sophisticated than that of a mere nonspecific reductant.

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Subunit structure

Abstract After chromatography on DEAE-cellulose at pH 5.5 the native photosynthetic fructose-1,6-diphosphatase (FDPase) from spinach leaves is split into a basic subunit , which can be isolated by filtration through a Sephadex-G-100 column; both the native enzyme and the subunit show a similar relative amino acid composition. The behaviour in gel filtration and SDS-polyacrylamide gel electrophoresis identifies this subunit as a protein species with a molecular weight lower than 10 000–12 000, in agreement with the values 6600 and 6573 determined by sucrose gradient centrifugation and from the amino acid composition, respectively. From the molecular weight data found earlier for the native FDPase, the enzyme appears set up by 16 subunits. There are two half-cystine residues per subunit, of which only one is p -hydroxymercuribenzoate-reactive.

Site-directed mutagenesis shows that lysine-299 is essential for activity of pea chloroplast fructose-1,6-bisphosphatase

Abstract Lysine-299 of the pea ( Pisum sativum L. cv. Lincoln) chloroplast fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) was substituted by glycine, glutamic acid and valine by codon mutation of specific nucleotides, and PCR construction using as target a cDNA clone coding for the pea photosynthetic enzyme. The mutagenized Escherichia coli -expressed FBPases showed K m values in the same order of magnitude as that of the wild type-expressed enzyme. On the contrary, the V max of the glutamic acid mutant was fourfold lower than that of the wild type FBPase, whereas the glycine and valine mutants exhibited an intermediate behaviour. These results contrast with those reported for the gluconeogenic FBPase from rat liver, in which a mutation of lysine-274 (the homologous counterpart for lysine-299 of the pea chloroplast enzyme) to alanine produced a sharp increase in the K m value (M.R. El-Magrabi et al., Journal of Biological Chemistry 267 (1992) 6526–6530). We therefore conclude that, as observed with the cytosolic FBPases, lysine-299 of the pea plastidic enzyme plays an essential role in FBPase activity. However, unlike the case with the cytosolic enzyme, this lysine appears more concerned with the normal catalytic process than with substrate binding.

Cloning and molecular features of cytosolic fructose-1,6-bisphosphatase from pea

A cDNA clone encoding for pea (Pisum sativum L.) cytosolic fructose-1,6-bisphosphatase (E.C. 3.1.3.11) has been isolated by reverse transcription-polymerase chain reaction of the total mRNA. The sequence analysis displayed a 341-amino acid protein of about 37300 Da molecular mass, corresponding to the subunit of this homotetrameric enzyme; it showed about 80% homology with the other ten higher plant cytosolic FBPases sequenced so far. The enzyme displayed a strong transcriptional expression in green organs (sessile and petioled leaves, stem, pod and grain), and poor expression in root and senescent basal leaves. It is noteworthy the high FBPase transcriptional expression in pod, which displays up to 4-fold higher content of FBPase-specific mRNA than that of root. The mRNA related to cytosolic FBPase was detected after 24 h continuous illumination of 24-h-dark-grown seedlings; this light-induced transcriptional expression is slower than that of chloroplast FBPase, which appears soon after 2 h light. In both cases the corresponding mRNAs disappeared when the light was turned off. The translational expression was also manifested, both as FBPase protein and activity, after 24 h illumination. This delay in the expression of cytosolic FBPase with respect to that of the plastidic enzyme can be interpreted as an indirect effect induced by a metabolite of the photosynthetic carbon pathway, rather than a direct effect of light on the DNA-expression mechanism. Pea cytosolic FBPase was not activated by dithiothreitol, with or without coupling to thioredoxins f or m. The enzyme showed a half-life of 6 h.

In Vivo Synthesis and Immunological Relationship of Thioredoxin f from Pea and Spinach

Thioredoxins are small proteins (typically 12 kDa m.w.) involved as cofactors in some oxidation-reduction processes, such as ribonucleotide reduction, reduction of sulfur-containing compounds, reductive activation of Calvin cycle enzymes (1,2). In addition, thioredoxin constitute an essential subunit of the DNA polymerase of bacteriophage T7 (3). Even though it has been mainly study in E. coli (4), this protein was isolated from a variety of prokaryotic and eukaryotic organisms. Those from E. coli (4), Corynebacterium nephridii (5), the cyanobacterium Anabaena 7119 (6), Chromatium vinosum (7), Rhodospirillum rubrum (8), bacteriophage T7 (9), and the m-type thioredoxins from spinach chloroplasts (10) have been sequenced. Except for that of bacteriophage T7, all the thioredoxins show considerable homology. The three dimensional structure of E. coli and T7 thioredoxins have been determined (11,12) and, in spite of large differences in amino acid sequences, both show many similarities in their terciary structures.