Activity and properties of fructose bisphosphatase of turbellaria <Emphasis Type="Italic">Phagocata sibirica</Emphasis>

Activity and properties of fructose bisphosphatase (FBPase) were studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. Dependence of the FBPase reaction rate on the substrate concentration was studied. For realization of the enzyme activity, the high affinity to substrate and the presence of bivalent cations (Mg²⁺ or Mn²⁺) are necessary. There was studied the effect of various effectors as well as of monovalent (Na⁺, K⁺, Li⁺, and NH⁺) and bivalent (Zn²⁺ and Cu²⁺) cations.     

Wiedereinführung vonBryopsis lyngbyei (Bryopsidales,Chlorophyta) als selbständige Art

Zusammenfassung 1. Die bisher in der Algenflora Helgolands alsBryopsis plumosa (Huds.) C. Ag. geführte Art wurde alsBryopsis lyngbyei Hornemann erkannt.2. Der Umriß der dicht zweizeilig gefiederten Art ist einer Vogelfeder ähnlich.Bryopsis lyngbyei ist diözisch; ihr Lebenszyklus schließt ein fadenförmiges Sporophytenstadium ein, das nur stephanokonte Zoosporen erzeugt.3. Die negativen Kreuzungsversuche mit Herkünften vonBryopsis plumosa europäischer Küstenabschnitte erweisen die Selbständigkeit vonBryopsis lyngbyei.

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Reestablishment ofBryopsis lyngbyei (Bryopsidales, Chlorophyta) as separate species

This study investigates the taxonomic status of a Helgoland species hitherto regarded asBryopsis plumosa (Huds.)Ag. The habitus of the plant concerned agrees well with that of the original specimens ofBryopsis lyngbyei Hornemann in the Botanical Museum of Copenhagen. Pinnules arise distichously from the axis; the life cycle of the dioecious species includes a filamentous sporophytic stage which produces only stephanokontic zoids. Crosses with samples ofBryopsis plumosa from European coasts were unsuccessful; thusBryopsis lyngbyei is evidently a separate species.

     

Exogenous expression of the wheat chloroplastic fructose-1,6-bisphosphatase gene enhances photosynthesis in the transgenic cyanobacterium, <Emphasis Type="BoldItalic">Anabaena</Emphasis> PCC7120

The paper reports a study on the genetic regulation of photosynthesis by introducing the gene encoding wheat chloroplastic fructose-1,6-bisphosphatase (FBPase) into the cyanobacterium Anabaena PCC7120. The gene was RT-PCR amplified from wheat and modified by replacement of the 5′-terminal encoding sequence with optimal and A/T-rich codons to promote prokaryotic expression. The resultant FBPase gene was ligated downstream of the strong promoter, PpsbA of expression vector pRL-439, then inserted into of shuttle vector pDC-08. The resulting shuttle expression vector (pDC-fbp) was transferred into the filamentous, heterocystour cyanobacterium, Anabaena PCC7120, by the tri-parental conjugation transfer method. Protein expression of FBPase in the transgenic Anabaena was 126.5% higher than in wild type cells, and the enzyme activity of transgenic cells was 1.41-fold higher than that of wild type cells. Under atmospheric conditions of 360 μmol mol⁻¹ CO₂, Anabaena cells overexpressing the FBPase gene further showed increases in net photosynthesis (117.2%) and true photosynthesis (122.5%) as compared to wild type cells. In addition, transgenic Anabaena grew faster and contained more Chl a than did wild type cells. Together, these results indicate that introduction of the wheat chloroplastic FBPase gene into Anabaena increase photosynthesis and cell growth; furthermore, these trends were more evident under stress condition (higher CO₂ concentration). This is the first report of enhanced photosynthesis in cyanobacteria expressing genes from higher plants.     

Cloning,structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)⁺ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg²⁺ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme. Accession number for pea chloroplast FBPase coding sequence: X68826 in the European Molecular Biology Laboratory (EMBL)     

Central Cavity of Fructose-1,6-bisphosphatase and the Evolution of AMP/Fructose 2,6-bisphosphate Synergism in Eukaryotic Organisms

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P₂) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P₂ synergism in pFBPase is absent in eFBPase. Fru-2,6-P₂ induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P₂ synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser⁴⁵ → His substantially fills the central cavity of pFBPase, and the triple mutation Ser⁴⁵ → His, Thr⁴⁶ → Arg, and Leu¹⁸⁶ → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P₂ on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P₂ exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P₂ complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P₂ synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P₂ synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.     

Fructose bisphosphatase of cestodes of <Emphasis Type="Italic">Bothriocephalus scorpii</Emphasis>

There have been studied the activity and properties of fructose bisphosphatase (FBPase) of Bothriocephalus scorpii parasitizing in pyloric appendages of the goby Myoxocephalus brandti. All subcellular fractions of B. scorpii (12 g/cytosol, 105 g/cytosol, mitochondria, and microsomes) have the FBPase activity. The enzyme has a high affinity to substrate and needs the presence of bivalent cations (Mg²⁺ or Mn²⁺). AMP inhibits the enzyme significantly. Action of various effectors has been studied. The monovalent (Na⁺, K⁺, Li⁺, NH₄ ⁺) and bivalent cations (Zn²⁺ and Cu²⁺) inhibit the enzyme activity.     

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.     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C₃ (spinach) and C₄ (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000-dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.

Kalanchoë FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoë ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.

Kalanchoë FBPase could be partially activated by Mg²⁺ at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca²⁺, especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoë SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg²⁺ concentration. However, like FBPase, increased Mg²⁺ concentrations enhanced the thioredoxin-linked activation of this enzyme.

In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoë leaves which required reduced thioredoxin m for activity.

These results indicate that Kalanchoë FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C₃ and C₄ plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the functional segregation of glucan synthesis and degradation.

     

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1

To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t _1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg²⁺, while Li⁺ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.     

The Mechanism of Calcium-Induced Inhibition of Muscle Fructose 1,6-bisphosphatase and Destabilization of Glyconeogenic Complex

The mechanism by which calcium inhibits the activity of muscle fructose 1,6-bisphosphatase (FBPase) and destabilizes its interaction with aldolase, regulating glycogen synthesis from non-carbohydrates in skeletal muscle is poorly understood. In the current paper, we demonstrate evidence that Ca²⁺ affects conformation of the catalytic loop 52–72 of muscle FBPase and inhibits its activity by competing with activatory divalent cations, e.g. Mg²⁺ and Zn²⁺. We also propose the molecular mechanism of Ca²⁺-induced destabilization of the aldolase–FBPase interaction, showing that aldolase associates with FBPase in its active form, i.e. with loop 52–72 in the engaged conformation, while Ca²⁺ stabilizes the disengaged-like form of the loop.     

Chlorophyll fluorescence quenching and violaxanthin deepoxidation of FBPase antisense plants at low light intensities and low temperatures

Genetically modified potato (Solanum tuberosum L. cv. Desiree) and tobacco (Nicotiana tabacum cv. Samsun N.N.) plants were used to analyze the effects exerted by the chloroplastic (cp) fructose- 1,6-bisphosphatase (FBPase) on the regulation of light energy discrimination at the level of photosystem II. The cp-FBPase activity was progressively inhibited by an mRNA antisense to this FBPase. The chlorophyll fluorescence quenching parameters of these transgenic plants were compared to those of wild-type and transgenic plants that were acclimated to low temperatures. In particular various lines of the transgenic potato and tobacco plants were exposed to a temperature treatment of 10 and 20°C for 10 days. Light intensities were kept low to reduce photoinhibition so that we could analyze exclusively the effects of a modification in the carbon fixation cycle on the chlorophyll fluorescence quenching parameters. The photon flux densities (PFDs) employed at the level of the middle leaves of all plants were set to two different values of 10 μmol m^?2 s^?1 and 50 μmol m^?2 s^?1. Subsequent to this 10-day acclimation the chlorophyll-fluorescence parameters of all plants were measured. Photoinhibition as expressed by the F_y/F_m ratio was minor in plants subjected to a PFD of 10 μmol m^?2 s^?1. Higher photon fluence rates of 50 μmol m^?2 s^?1 at temperatures of 10°C gave rise to a significant reduction in the F_y/F_m ratios obtained from the transgenic plants which were characterized by a restriction in cp-FBPase capacity to 20% of normal activity. Furthermore, a progressive inhibition of the cp-FBPase activity induced an amplified nonphotochemical quenching of chlorophyll fluorescence with in the genetically manipulated species (except at 10°C and 50 μmol m^?2 s^?1). The increase in nonphotochemical quenching depended upon light and temperature. Photochemical quenching of light quanta within the antisense plants declined relative to that in the wild type. To further characterize the mechanisms producing higher levels of nonphotochemical fluorescence quenching. we analyzed several of the xanthophyll cycle pigments. The deepoxidation state of the xanthophyll cycle pigments in potato plants increased with attenuating FBPase activities under all conditions. For tobacco plants, this elevation of the deepoxidation state was only observed at a PFD of 50 μmol m^?2 s^?1.     

Purification,kinetic studies,and homology model of Escherichia coli fructose-1,6-bisphosphatase

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Molecular modeling studies on phosphonic acid-containing thiazole derivatives: design for fructose-1,6-bisphosphatase inhibitors

Presently, an in silico modeling was carried out on a series of 63 phosphonic acid-containing thiazole derivatives as fructose-1,6-bisphosphatase (FBPase) inhibitors using CoMFA/CoMSIA and molecular docking methods. The CoMFA and CoMSIA models using 51 molecules in the training set gave r _cv² values of 0.675 and 0.619, r ² values of 0.985 and 0.979, respectively. The systemic external validation indicated that our CoMFA and CoMSIA models possessed high predictive powers with r ₀² values of 0.995 and 0.994, r _m(test)² values of 0.887 and 0.860, respectively. The 3D contour maps of the CoMFA and CoMSIA provided smooth and interpretable explanation of the structure-activity relationship for the inhibitors. Molecular docking studies revealed that a phosphonic group was essential for binding to the AMP binding site, and some key features were also identified. The analyses of the 3D contour plots and molecular docking results permitted interesting conclusions about the effects of different substituent groups at different positions of the common scaffold, which might guide the design of novel FBPase inhibitors with higher activity and bioavailability. A set of 60 new analogues were designed by utilizing the results revealed in the present study, and were predicted with significantly improved potencies in the developed models. The findings can be quite useful to aid the designing of new fructose-1,6-biphophatase inhibitors with improved biological response.     

Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Fructose-1,6-bisphosphatase from a cold-hardy insect: Control of cryoprotectant glycerol catabolism

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol^?1. K_m values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2,6P₂), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P₂ increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I₅₀ values for AMP and F2,6P₂ increase at 22°C (compared with 5°C), high glycerol levels override F2,6P₂ inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley-Liss, Inc.     

Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysC^fbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the P_EFTUfbp mutant. As revealed by ¹³C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysC^fbr P_EFTUfbp and 46% for C. glutamicum lysC^fbr. The flux for NADPH supply was 180% in the P_EFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique