Directed mutagenesis shows that the preceding region of the chloroplast fructose-1,6-bisphosphatase regulatory sequence is the thioredoxin docking site.

The alignment of the six higher plant photosynthetic fructose-1,6-bisphosphatases (FBPases) so far sequenced shows a lack of homology in the region which just precedes the cluster engaged in light modulation. Earlier experiments suggested that this region is the docking point in FBPase-thioredoxin (Trx) binding, and could be responsible for the interspecific differences in the enzyme-Trx interaction and Trx ability for FBPase activation. Using a pea chloroplast FBPase-coding cDNA, we have prepared two chimeric clones for FBPase. One of them (pDELFBP) shows a deletion of the 17 amino acids (Leu154 to Glu170) coding sequence, whereas in the second (pPFBPW) the above sequence was substituted by the corresponding one of the wheat enzyme. After Escherichia coli overexpression in pET-3d and later purification, both modified FBPases showed FBPase activity when determined under non-reducing conditions. However, only DELFBP lost the Trx f modulatory effect, indicating the important role played by this fragment in FBPase-Trx interaction and activity. Under these conditions the substituted PFBPW enzyme retains FBPase activity, even though clearly diminished. Superose 12 filtration experiments after preincubating the wild-type and modified FBPases with Trx f, showed the existence of an enzyme-Trx f binding with the wild-type and the substituted PFBPW, but not with the deleted DELFBP protein. Similarly, gradient PAGE under native conditions, followed by Western blot and developing with FBPase and Trx f antibodies, indicated the existence of such a binding between the wild-type and PFBPW, on the one hand, and both Trxs f and m, on the other, although never with the deleted DELFBP enzyme. These results show the central role played by the regulatory site preceding fragment of chloroplast FBPase in its binding with Trx. Computer-aided tridimensional models for the wild-type and modified FBPases are proposed.

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-biophosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceeding 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, this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.

Purification and properties of pea (Pisum sativum L.) thioredoxin f, a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase.

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (Mr) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an Mr of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at - 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP(+)-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa Mr = 6324 M(-1) · cm(-1)). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves.

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg(2+) or Mn(2+), and was free of interfering enzyme activities. Ca(2+) was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg(2+) or Mn(2+) was used as cofactor. In the presence of 5 millimolar Mg(2+), Ca(2+) appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca(2+). However, with Mn(2+) (60 micromolar) as cofactor, Ca(2+) exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca(2+) behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg(2+) or Mn(2+) as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg(2+) as cofactor, but this effect was low with Mn(2+). From these results we suggest that Ca(2+) plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.

Immunogold localization of photosynthetic fructose-1,6-bisphosphatase in pea leaf tissue.

An enriched IgG serum fraction obtained from rabbits immunized against pea chloroplast fructose-1,6-bisphosphatase (FBPase) was used, coupled to colloidal gold (15 nanometer particles) goat anti-rabbit IgG, to analyze by electron microscopy the location of photosynthetic FBPase in pea (Pisum sativum L.) leaf ultrathin sections. In accordance with earlier biochemical studies on distribution of FBPase activity, the enzyme was visualized both in the stromal space and bound to the chloroplast membranes. Some gold particles also appear in the cytoplasm, which can be related to the presence in the cytosol of a high molecular weight precursor of this nuclear coded enzyme.

Light-Induced Nuclear Synthesis of Spinach Chloroplast Fructose-1,6-bisphosphatase.

Etiolated spinach (Spinacia oleracea L. var Winter Giant) seedlings show a residual photosynthetic fructose-1,6-bisphosphatase activity, which sharply rises under illumination. This increase in activity is due to a light-induced de novo synthesis, as it has been demonstrated by enzyme labeling experiments with (2)H(2)O and [(35)S]methionine. The rise of bisphosphatase activity under illumination is strongly inhibited by cycloheximide, but not by the 70S ribosome inhibitor lincocin, which shows the nuclear origin of this chloroplastic enzyme.

Antibiotics from Pseudomonas reptilivora. II. Isolation, purification, and properties.

Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B(1), and B(2). The B(1) and B(2) activities were studied in greater detail than A. The B(1) substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively.