The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase.
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ABSTRACT:
SUBMITTER: Freemont PS
PROVIDER: S-EPMC1148774 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.
| S-EPMC2144250 | biostudies-literature
Project description:Fructose-1,6-bisphosphate aldolase (aldolase) is an essential enzyme in glycolysis and gluconeogenesis. In addition to this primary function, aldolase is also known to bind to a variety of other proteins, a property that may allow it to perform 'moonlighting' roles in the cell. Although monomeric and dimeric aldolases possess full catalytic activity, the enzyme occurs as an unusually stable tetramer, suggesting a possible link between the oligomeric state and these noncatalytic cellular roles. Here, the first high-resolution X-ray crystal structure of rabbit muscle D128V aldolase, a dimeric form of aldolase mimicking the clinically important D128G mutation in humans associated with hemolytic anemia, is presented. The structure of the dimer was determined to 1.7 angstroms resolution with the product DHAP bound in the active site. The turnover of substrate to produce the product ligand demonstrates the retention of catalytic activity by the dimeric aldolase. The D128V mutation causes aldolase to lose intermolecular contacts with the neighboring subunit at one of the two interfaces of the tetramer. The tertiary structure of the dimer does not significantly differ from the structure of half of the tetramer. Analytical ultracentrifugation confirms the occurrence of the enzyme as a dimer in solution. The highly stable structure of aldolase with an independent active site is consistent with a model in which aldolase has evolved as a multimeric scaffold to perform other noncatalytic functions.
| S-EPMC2631105 | biostudies-literature
Project description:Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.
| S-EPMC3169401 | biostudies-literature
Project description:The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].
| S-EPMC1146794 | biostudies-other
Project description:The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0?Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.
| S-EPMC4157416 | biostudies-literature
Project description:Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO(4), 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml(-1) and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C222(1), with unit-cell parameters a=121.46, b=135.82, c=61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C222(1) and the unit-cell parameters were a=121.96, b=137.61, c=62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.
| S-EPMC3169402 | biostudies-literature
Project description:Fructose 1,6-bisphosphate aldolase is a ubiquitous cytosolic enzyme that catalyzes the fourth step of glycolysis. Aldolases are classified into three groups: Class-I, Class-IA, and Class-II; all classes share similar structural features but low amino acid identity. Apart from their conserved role in carbohydrate metabolism, aldolases have been reported to perform numerous non-enzymatic functions. Here we review the myriad "moonlighting" functions of this classical enzyme, many of which are centered on its ability to bind to an array of partner proteins that impact cellular scaffolding, signaling, transcription, and motility. In addition to the cytosolic location, aldolase has been found the extracellular surface of several pathogenic bacteria, fungi, protozoans, and metazoans. In the extracellular space, the enzyme has been reported to perform virulence-enhancing moonlighting functions e.g., plasminogen binding, host cell adhesion, and immunomodulation. Aldolase's importance has made it both a drug target and vaccine candidate. In this review, we note the several inhibitors that have been synthesized with high specificity for the aldolases of pathogens and cancer cells and have been shown to inhibit classical enzyme activity and moonlighting functions. We also review the many trials in which recombinant aldolases have been used as vaccine targets against a wide variety of pathogenic organisms including bacteria, fungi, and metazoan parasites. Most of such trials generated significant protection from challenge infection, correlated with antigen-specific cellular and humoral immune responses. We argue that refinement of aldolase antigen preparations and expansion of immunization trials should be encouraged to promote the advancement of promising, protective aldolase vaccines.
| S-EPMC8385298 | biostudies-literature
Project description:The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
| S-EPMC5544942 | biostudies-literature
Project description:Glycolysis is one of the important ways by which Echinococcus multilocularis acquires energy. Fructose-1, 6-bisphosphate aldolase (FBA) plays an important role in this process, but it is not fully characterized in E. multilocularis yet. The results of genome-wide analysis showed that the Echinococcus species contained four fba genes (FBA1-4), all of which had the domain of FBA I and multiple conserved active sites. EmFBA1 was mainly located in the germinal layer and the posterior of the protoscolex. The enzyme activity of EmFBA1 was 67.42 U/mg with Km and Vmax of 1.75 mM and 0.5 mmol/min, respectively. EmFBA1 was only susceptible to Fe3+ but not to the other four ions (Na+, Ca2+, K+, Mg2+), and its enzyme activity was remarkably lost in the presence of 0.5 mM Fe3+. The current study reveals the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in E. multilocularis.
| S-EPMC8781991 | biostudies-literature
Project description:Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6-bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu-plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas subterminal lysine residues were not involved.
| S-EPMC4831477 | biostudies-literature