Structure-based Reevaluation of the Mechanism of Class I Fructose-1,6-bisphosphate Aldolase

The enzymatic reaction carried out by class I fructose-1,6-bisphosphate aldolase is known in great detail in terms of reaction intermediates, but the precise role of individual amino acids in the active site is poorly understood. Therefore, on the basis of the crystallographic structure of the complex between aldolase and dihydroxyacetone phosphate a molecular modelling study was undertaken to predict the Michaelis complex with fructose-1,6-bisphosphate and several covalent enzymatic reaction intermediates. This model reveals the unknown 6-phosphate binding site and assigns distinct roles to crucial residues. Asp33 is responsible for aligning the 2-keto function of the substrate correctly for nucleophilic attack by Lys229, and plays a role in carbinolamine formation. Lys146 assists in carbinolamine dehydration and is essential for stabilising the developing negative charge on O4 of fructose-1,6-bisphosphate during hydroxyl proton abstraction by Glu187. Subsequently, Glu187 is also responsible for protonating C1 of the dihydroxyacetone phosphate enamine. In addition, the absolute configuration of the fructose-1,6-bisphosphate carbinol intermediate is shown to be (2S), in agreement with the crystal structure, but opposite from the interpretation in the literature of the stereospecific reduction of the aldolase fructose-1,6-bisphosphate complex with sodium borohydride. It is demonstrated that the outcome of the latter type of experiment critically depends on conformational changes triggered by Schiff base formation. Electronic Supplementary Material available.     

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.     

Crystal structures of LacD from Staphylococcus aureus and LacD.1 from Streptococcus pyogenes: insights into substrate specificity and virulence gene regulation

Staphylococcus aureus LacD, a Class I tagatose-1,6-bisphosphate (TBP) aldolase, shows broadened substrate specificity by catalyzing the cleavage of 1,6-bisphosphate derivatives of d-tagatose, d-fructose, d-sorbose, and d-psicose. LacD.1 and LacD.2 are two closely-related Class I TBP aldolases in Streptococcus pyogenes. Here we have determined the crystal structures of S. aureus LacD and S. pyogenes LacD.1. Monomers of both enzymes are folded into a (β/α)₈ barrel and two monomers associate tightly to form a dimer in the crystals. The structures suggest that the residues E189 and S300 of rabbit muscle Class I fructose-1,6-bisphosphate (FBP) aldolase are important for substrate specificity. When we mutated the corresponding residues of S. aureus LacD, the mutants (L165E, L275S, and L165E/L275S) showed enhanced substrate specificity toward FBP.

Structured summary

lacDbinds to lacD by X-ray crystallography(View interaction)lacD1binds to lacD1 by X-ray crystallography(View interaction)     

Characterization of a Mn-dependent Fructose-1,6-bisphosphate Aldolase in Deinococcus radiodurans

The key enzyme of the glycolytic pathway of Deinococcus radiodurans, fructose-1,6-bisphosphate aldolase, could be induced independently by glucose and Mn. The enzyme exhibited the characteristics of the metal-dependent Class II aldolases. Unlike most Class II aldolases, the deinococcal aldolase preferred Mn, not Zn, as a cofactor. The fbaA gene encoding the deinococcal aldolase was cloned and the protein overproduced in various Escherichia coli expression hosts. However, the overexpressed deinococcal enzyme aggregated and formed inclusion bodies. Dissolving these inclusion bodies by urea and subsequent purification by nickel affinity chromatography, resulted in a protein fraction that exhibited aldolase activity only in the presence of Mn. This active aldolase fraction exhibited masses of about 70 kDa and 35 kDa by gel filtration and by SDS gel electrophoresis, respectively, suggesting that the active aldolase was a dimer.     

Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.     

Antibody inhibition of external aldolase activity in spinach chloroplast preparations

Abstract Antibodies (rabbit) have been prepared against total stroma from isolated spinach (Spinacia oleracea L. cv. Viking II) chloroplasts. These antibodies inhibited most of the aldolase activity present outside the chloroplasts in preparations of intact (80–95%) chloroplasts. They also reduced the amount of labelled fructose-1,6-bisphosphate found in the medium after ¹⁴CO₂ fixation with such preparations. Both intact and broken chloroplasts were strongly agglutinated by the antibodies. The results indicate that the external fructose-1,6-bisphosphate was formed from excreted dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase present outside the chloroplasts. The contamination of organelle preparations with free enzymes or enzymes adsorbed on the outer surface of the organelles is probably a general phenomenon. It is suggested that antibodies can be used as a tool to detect and selectively inhibit such contaminating enzyme activities.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Genes encoding a fructose-1,6-bisphosphate aldolase and a fructose-1,6-bisphosphatase are present within the gene cluster for mimosine degradation in Rhizobium sp. strain TAL1145

Rhizobium sp. strain TAL1145 can catabolize mimosine, a toxic amino acid produced by the tree-legume leucaena. The mid and pyd genes involved in mimosine degradation in TAL1145 are located in two clusters within a 25-kb region in the chromosome, which was cloned in plasmid pUHR263. A 5.5-kb EcoRI fragment, located between the mid and pyd genes in pUHR263, was characterized by sequencing and transposon-insertion mutagenesis and six open reading frames (ORF) were identified. Based on high homologies with other known proteins and conserved signature domains, ORF1 and ORF2 were identified as fba and fbp genes, encoding fructose-1,6-bisphosphate aldolase (FBA) and fructose-1,6-bisphosphatase (FBP), respectively. The fba mutant showed a slightly reduced growth rate compared to TAL1145 while the fbp mutant did not show any growth defects. Both mutants could catabolize mimosine and formed normal nitrogen-fixing nodules on leucaena, suggesting that these genes are not involved in mimosine degradation and symbiosis.     

Salt mediated changes in some enzymes of carbohydrate metabolism in halotolerantCladosporium sphaerospermum

Cladosporium sphaerospermum, isolated from salt pans was halotolerant. When grown in the presence of salt, the activities of invertase, isocitrate lyase, fructose-1,6 diphosphate aldolase and malate dehydrogenase were found to be increased and that of amylase decreased. Both, enzyme activation as well as an increase inde novo synthesis of enzymes were found to be some of the mechanisms of salt mediated changes. This may be one of the adaptive mechanisms, in halotolerantCladosporium sphaerospermum.     

Glycolytic gene expression in amphibious Acorus calamus L. under natural conditions

Acorus calamus L is an amphibious plant, which is exposed to periods of flooding and consequently hypoxic conditions as a part of its natural life cycle. Previous experiments under laboratory conditions have shown that the plant can survive for two months in the complete absence of oxygen, and that during this period the expression of genes encoding the glycolytic enzymes fructose-1,6-bisphosphate aldolase (ALD), pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is induced in leaves and rhizomes (Bucher and Kuhlemeier, 1993). Here we studied the expression of ALD and ADH through two years in the natural habitat of A. calamus. Under natural conditions roots and rhizomes were always submerged but newly grown leaves emerged in spring; in autumn the leaves senesced and the whole plant was submerged again. High Ald and Adh mRNA levels in leaf and rhizome were found only in winter when the leaves were entirely submerged. Upon leaf emergence in spring the mRNA levels rapidly declined. Under controlled experimental conditions expression of Ald and Adh was not induced by low temperature. The combination of laboratory and field experiments supports the hypothesis that oxygen deprivation rather than low temperature is a major regulator of glycolytic gene expression in A. calamus. The possible role of other environmental factors is also discussed.Abbreviations ADH alcohol dehydrogenase - Adh gene encoding ADH - ALD cytoplasmic fructose-1,6-bisphosphate aldolase - Ald gene encoding ALD - PDC pyruvate decarboxylase - Pdc gene encoding PDC     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

High level expression in Escherichia coli,purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO₂ and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl--d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg²⁺ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg²⁺ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg²⁺ as cofactor and the inhibition by Ca²⁺. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.Abbreviation DTT dithiothreitol - PCR polymerase chain reaction - EDTA (ethylenedinitrilo)tetraacetic     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.