Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ALDOLASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 559 records · Page 31Linked to original sources
PubMed1973

Aldolase catalysis: single base-mediated proton activation.

The enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, catalyzes several reactions, the natural ones being (i) the exchange of hydrogen atoms of the methyl groups of pyruvate with protons of the solvent (C-H synthesis) and (ii) the reversible condensation of pyruvate with D-glyceraldehyde-3-phosphate (C-C synthesis). Previous work has provided chemical evidence for the occurrence of a protein-bound carboxylate group adjacent to the Schiff's base-forming lysine in the active site geometry. This carboxylate could provide the basic group postulated to participate in proton activation catalyzed by aldolases. With the use of three-dimensional models, it is shown that simple rotation about a carbon-carbon bond of the side chain will allow the base to assume the two positions necessary for proton activation in either the C-H synthesis or the C-C synthesis catalyzed by KDPG aldolase. This single base hypothesis provides a model wherein all reagents can approach a single face of the active site and is consistent with the stereochemistry thought to occur in the aldolase reaction.

Aldehyde-Lyases↗
PubMed2003

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

Acetyl Coenzyme A↗
PubMed1992

Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene.

Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C. J. Vlahos and E. E. Dekker, J. Biol. Chem. 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers. The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product. Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E. coli DH5 alpha F'IQ. When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein. All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized. A strain of E. coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene.

Amino Acid Sequence↗
PubMed2002

Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase (Tk-Fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100 degrees C. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and K(m) values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.

Journal Article↗
PubMed1995

D-glucosaminate aldolase activity of D-glucosaminate dehydratase from Pseudomonas fluorescens and its requirement for Mn2+ ion.

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-D-gluconate and ammonia: alpha,beta-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3-4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn2+ + pyridoxal 5'-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn2+ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn2+, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn2+ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.

Aldehyde-Lyases↗
PubMed1989

Effect of threonine and glycine concentrations on threonine aldolase activity of yogurt microorganisms during growth in a modified milk prepared by ultrafiltration.

To evaluate the combined effects of threonine and glycine concentrations during growth on threonine aldolase activity (EC 2.1.2.1) of yogurt microorganisms, Streptococcus thermophilus and Lactobacillus bulgaricus, a modified milk growth medium was prepared using UF to deplete the free amino acid level. Threonine and glycine were added according to a 2x2x2 factorial design at 5 or 195 microg.ml(-1) along with a standard amino acid mixture. Acetaldehyde production and threonine aldolase activity were evaluated utilizing headspace gas chromatography. Results showed that threonine and glycine concentrations did not affect growth or titratable acidity. The high concentration of threonine in combination with low glycine in the growth medium resulted in increased acetaldehyde synthesis by both microorganisms. Conversely, high glycine with low threonine decreased acetaldehyde synthesis. High threonine and low glycine increased threonine aldolase activity of cell-free extracts from S. thermophilus and L. bulgaricus, whereas high glycine and low threonine reduced threonine aldolase activity of both microorganisms.

Acetaldehyde↗
PubMed1985

Evidence for an essential arginine residue in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. Modification with 1,2-cyclohexanedione.

Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.

Amino Acids↗
PubMed2000

Stereochemistry of nonnatural aldol reactions catalyzed by DHAP aldolases.

A coupled enzymatic assay was developed for quantitative determination of the stereoisomeric products formed in aldol reactions catalyzed by dihydroxyacetone phosphate (DHAP)-dependent aldolases. Three of the four stereoisomers could be determined directly; the fourth one was calculated. This procedure is based on the reversibility of the aldol reaction and requires no derivatization or work-up of the product samples, only removal or inactivation of the biocatalyst. In comparison with other methods the enzymatic assay is highly accurate and fast. Determination of isomer formation with 10 different acceptor substrates applying this procedure gave unprecedented insight in the stereochemistry of fructose-1,6-bisphosphate aldolase from Staphylococcus carnosus and l-rhamnulose-1-phosphate aldolase from E. coli.

Aldehyde-Lyases↗
PubMed1981

Two cases of red cell aldolase deficiency associated with hereditary hemolytic anemia in a Japanese family.

Two cases of red cell aldolase deficiency associated with congenital nonspherocytic hemolytic anemia are reported. The proband is a fourteen-month-old Japanese boy. Consanguineous marriage was not proven but probable in this family, as the parents were born in the same small island. The proband had moderate to mild anemia aggravated by upper respiratory infections, 1 cm hepatomegaly and 2.5 cm splenomegaly, but was unremarkable in other respects and has thus far not shown mental or growth retardation. He did not have dysmorphic features. The red cell aldolase activity was 6% of the normal mean. The enzyme was unstable with respect to heat, and Km for fructose 1,6-diphosphate (F-1,6-DP) was high. The parents and other heterozygotes showed intermediate activity between that of the proband and that of normal subjects. Red cell F-1,6-DP concentration in this case was remarkably increased. Red cell glucose consumption, and lactate formation, as well as hexose monophosphate shunt activity, were decreased as compared with a comparable reticulocyte-rich hereditary spherocytosis patient. Hexose monophosphate dehydrogenase by a high concentration of F-1,6-DP in his red cells. As a result of family study, another homozygous aldolase deficiency case associated with hemolytic anemia was found. He is 13 years old and a nephew of the proband's paternal grandmother. His hemolytic anemia also is moderate to mild and aggravated by upper respiratory infections. He does not seem to have mental or growth retardation, nor does he possess dysmorphic features.

Adolescent↗
PubMed1996

Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

Amino Acid Sequence↗
PubMed1996

Thermal-induced unfolding domains in aldolase identified by amide hydrogen exchange and mass spectrometry.

Amide hydrogen exchange has been measured in short segments of intact rabbit muscle aldolase at temperatures of 14-50 degrees C by the protein fragmentation/mass spectrometry method (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). Deuterium levels in some segments did not change over the temperature range of the measurements, whereas deuterium levels in other segments increased rapidly with temperature. These results demonstrate that the equilibrium constant for local unfolding, Kunf, of some segments increases with temperature in the low temperature range (14-30 degrees C) of this study. Aldolase begins to lose activity at temperatures above 40 degrees C. In the 40-50 degrees C temperature range, Kunf is greater than 10(-4) in some regions and less than 10(-6) in other regions. This wide range of regional stability in the temperature range where aldolase begins to denature is interpreted in terms of cooperative unfolding/folding domains. Regions of highest stability were located along the hydrophobic subunit binding surface. It is proposed that hydrogen exchange might be used to identify unfolding domains in multidomain proteins whose thermodynamic properties have been determined by differential scanning calorimetry.

Amides↗
PubMed1995

Properties of fructose-1,6-bisphosphate aldolase from Escherichia coli: an NMR analysis.

A class II Zn(2+)-dependent fructose-1,6-bisphosphate (FBP)- aldolase was purified from an overproducer strain of Escherichia coli and characterized by standard biochemical techniques and 13C NMR spectroscopy. The principal finding of these studies was identification, by 13C NMR spectroscopy, of an enzyme-bound reaction intermediate, the enediol(ate) form of dihydroxyacetone phosphate (DHAP). Formation of this intermediate requires the presence of Zn2+ and is pH dependent, with increasing amounts of this tautomer appearing at alkaline pH's. This pH dependence closely parallels the pH activity profile of the enzyme, suggesting an involvement of the enediol-DHAP form in the reaction pathway. In addition to these results the following observations were made on this enzyme: (a) E. coli FBP aldolase binds and utilizes only the carbonyl forms of FBP and DHAP; (b) the function of Zn2+ in this metalloaldolase appears to be polarization of the C = O bond of DHAP; (c) activity of this enzyme is unaffected by glycolytic intermediates or nucleotide phosphates such as ATP. Although these studies provide some information about the catalytic mechanism of E. coli FBP aldolase, they do not provide an explanation for the apparent regulation of this enzyme reported in previous in vivo NMR studies. While the possibility that the enzyme is allosterically regulated cannot be excluded at this time, an interesting possibility suggested by this and other studies is that in E. coli glycolytic substrates may be channeled through a multienzyme complex.

Binding Sites↗
PubMed1995

Analysis of the in vitro translation product of a novel-type Drosophila melanogaster aldolase mRNA in which two carboxyl-terminal exons remain unspliced.

Drosophila melanogaster generates three different types of aldolase mRNAs from a single gene by selective usage of the triplicate exons 4 (4 alpha, 4 beta, and 4 gamma), which encode three different isozymes having respective carboxyl termini. We have found the presence of a novel-type mRNA (named alpha beta) in which two final exons, 4 alpha and 4 beta, were retained unspliced. Herein, a cDNA clone containing the alpha beta sequence was inserted into pINIII and expressed in an Escherichia coli system. The product, which exhibited aldolase activity, was found to be isozyme alpha from the primary structure and the enzymological properties, with the 4 alpha sequence alone being present as the carboxyl terminus. In tissues of D. melanogaster, the production of mRNA encoding exon 4 alpha is known to be restrained to a low level. This may be understood by the fact that the aldolase gene of this species does not have a typical poly(A) signal at the 3' end in exon 4 alpha. Instead, the transcript-encoding exons, 4 alpha and 4 beta, might be produced when AATATA, which resides downstream of the coding frame in exon 4 beta, is recognized as a poly(A) signal during RNA processing.

Alternative Splicing↗
PubMed1995

Characterization of recombinant human aldolase B and purification by metal chelate chromatography.

Recombinant human aldolase B and the native enzyme purified from human liver were found to be identical in size, charge, structure, Km constants for fructose-1,6-bis(phosphate) and fructose-1-phosphate, and the activity ratio of the two substrates. Thus recombinant aldolase B is a valid model for the native enzyme and can be used to study mutations that cause hereditary fructose intolerance or others designed in the active site. Addition of six histidine residues to the amino-terminus of the recombinant enzyme did not alter its structural or functional characteristics and allowed for purification by immobilized metal affinity chromatography. This purification protocol does not require a stable or active enzyme and will facilitate the study of mutant aldolase B enzymes that would otherwise be difficult to purify.

Base Sequence↗
PubMed1994

Growth-arrested dependence of aldolase A L-type mRNA expression in rodent cell lines.

Two ubiquitous (L- and F-type) and one muscle-specific (M-type) mRNA species are generated by the human aldolase A gene. Despite the high degree of sequence similarities in the promoter region between human and rodents, no L-type mRNA expression has yet been found in the latter. Here we demonstrate that L-type aldolase A mRNA is expressed during the differentiation of mouse myogenic C2.7 and rat oligodendrocyte precursor CEINGE C13 cells. The L-type mRNA expression is increased during differentiation and is associated with cell-growth arrest caused by nocodazole treatment or serum deprivation in C2.7 and CEINGE C13 cells, respectively. The L-type aldolase A mRNA is correctly processed at the L1-L2 junction.

Animals↗
PubMed1999

The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity.

The structure of a class II fructose-1,6-bisphosphate aldolase in complex with the substrate analogue and inhibitor phosphoglycolohydroxamate (PGH) has been determined using X-ray diffraction terms to a resolution of 2.0 A (1 A=0.1 nm). The crystals are trigonal, space group P3121 with a=b=78.24 A, c=289.69 A. The asymmetric unit is a homodimer of (alpha/beta)8 barrels and the model has refined to give R-work 19.2 %, R-free (based on 5 % of the data) 23.0 %. PGH resembles the ene-diolate transition state of the physiological substrate dihydroxyacetone phosphate. It is well ordered and bound in a deep polar cavity at the C-terminal end of the (alpha/beta)8 barrel, where it chelates the catalytic zinc ion using hydroxyl and enolate oxygen atoms. Trigonal bipyramidal coordination of the zinc ion is completed by three histidine residues. The complex network of hydrogen bonds at the catalytic centre is required to organise the position of key functional groups and metal ion ligands. A well-defined monovalent cation-binding site is observed following significant re-organisation of loop structures. This assists the formation of a phosphate-binding site on one side of the barrel that tethers PGH in the catalytic site. The positions of functional groups of substrate and putative interactions with key amino acid residues are identified. Knowledge of the complex structure complements the results of spectroscopic and site-directed mutagenesis studies, and contributes to our understanding of the mechanism and substrate specificity of this family of enzymes. A reaction mechanism distinct from that proposed for other class II aldolases is discussed. The results suggest that the class II aldolases should be sub-divided into two groups on the basis of both distinct folds and mechanism.

Binding Sites↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1995

Enzyme-enzyme interaction in the chloroplast: glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase.

Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.

Amino Acid Sequence↗