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At least 973 records · Page 54Linked to original sources
PubMed2005

A binding site for Pur alpha and Pur beta is structurally unstable and is required for replication in vivo from the rat aldolase B origin.

The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Puralpha and Purbeta, i.e., single-stranded DNA-binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Puralpha/beta may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Puralpha/beta proteins to the origin. These observations suggest functional roles of site PPu and Puralpha/beta proteins in replication initiation.

Amino Acid Sequence↗
PubMed2006

Enhancing the synthetic utility of aldolase antibody 38C2.

Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4'-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 degrees C. However, since TPP-treated 38C2 also gave an additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst which gave a single product and could be reused at 40 degrees C five times without loss of activity.

Acetone↗
PubMed2005

Paracoccidioides brasiliensis presents two different cDNAs encoding homologues of the fructose 1,6-biphosphate aldolase: protein isolation, cloning of the cDNAs and genes, structural, phylogenetic, and expression analysis.

A proteomic approach was used to identify a 39 kDa antigen of Paracoccidioides brasiliensis. Amino acid sequences of the N-terminal and of endoproteinase Lys-C digested peptides revealed the protein to be a fructose 1,6-biphosphate aldolase (FBA) Class II of P. brasiliensis. Two cDNA homologues, Pbfba1 and Pbfba2, were cloned and characterized. Pbfba1 encoded a predicted polypeptide of 360 amino acids that was highly homologous in the primary structure to the same enzyme from fungi and bacteria. The other DNA, Pbfba2, encoded a polypeptide predicted to be 363 amino acids. The sequence of Pbfba2 differed significantly from Pbfba1. Phylogenetic and molecular analysis supports the concept of gene duplication for FBAs in P. brasiliensis, constituting a two-member family. Expression analysis demonstrated differential expression for both fbas genes in P. brasiliensis cells.

Amino Acid Sequence↗
PubMed2005

The Krüppel-like zinc-finger protein ZNF224 represses aldolase A gene transcription by interacting with the KAP-1 co-repressor protein.

Transcription factors belonging to the Krüppel-like zinc finger family of proteins participate in the regulation of cell differentiation and development. Although many of these proteins have been identified, little is known about their structure and function. We recently purified ZNF224, a new Krüppel-like zinc finger protein, that contains a Krüppel-associated box (KRAB) domain at the NH2 terminus, and 19 Cys2-His2 zinc-finger domains at the COOH terminus. Using chromatin immunoprecipitation and transient transfection assays, we demonstrate that ZNF224 binds in vivo to the distal promoter of the aldolase A gene and represses its transcription. The results of transient co-transfection experiments show that ZNF224-mediated transcription repression requires the 45-amino acid long KRAB A domain. The ability of KRAB-containing ZNF224 protein to repress transcription depends on specific interaction with the KAP-1 co-repressor molecule. Finally, using selective treatment with the HDAC1 inhibitor trichostatin A, we demonstrate that ZNF224-mediated repression requires histone deacetylases.

Acetylation↗
PubMed2006

High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor.

The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.

Amino Acid Sequence↗
PubMed2005

Purification and characterization of an allosteric fructose-1,6-bisphosphate aldolase from germinating mung beans (Vigna radiata).

Cytosolic fructose-1,6-P(2) (FBP) aldolase (ALD(c)) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 micromol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD(c))-IgG. The enzyme's native M(r) was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg(2+) had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65 degrees C. It demonstrated: (i) a temperature coefficient (Q(10)) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD(c) is bifunctional for FBP and sedoheptulose-1,7-P(2) (K(m) approximately 17 microM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K(i) values=2.2, 3.1 and 7.5mM, respectively). By contrast, the addition of 2mM ATP: (i) reduced V(max) by about 2-fold, (ii) increased K(m)(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h=1.0 and 2.6 in the absence and presence of 2mM ATP, respectively). Potent feedback inhibition of ALD(c) by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.

Allosteric Regulation↗
PubMed1991

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator.

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

Chloramphenicol O-Acetyltransferase↗
PubMed1997

Practical synthesis of 4-hydroxy-3-oxobutylphosphonic acid and its evaluation as a bio-isosteric substrate of DHAP aldolase.

An efficient four step synthesis of the title compound 4-hydroxy-3-oxobutylphosphonate (2) has been developed based on inexpensive 4-ethoxy-1-hydroxybutane-2-one using an Arbusow reaction (59% overall yield). Several dihydroxyacetone-dependent aldolases having different stereospecificities were tested for their acceptance of this phosphonomethyl substrate mimic as the aldol donor. Individual enzymes belonging to both type I (Schiff base formation) and type II (Zn2+ catalysis) mechanistic classes were found to catalyze the stereoselective addition of 2 to simple aldehydes to provide bio-isosteric analogs of sugar 1-phosphates in high yields. The lack of acceptance by specific enzymes is discussed with regard to recent protein X-ray data.

Aldehydes↗
PubMed2001

Bidirectional activity and orientation-dependent specificity of the rat aldolase C promoter in transgenic mice.

We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.

Animals↗
PubMed1997

Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1.

Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FTO2B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5-fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Sp1-dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Sp1 binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FTO2B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Sp1. From these results we conclude that glucose increases the amount of the dephosphorylated form of Sp1 which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.

Animals↗
PubMed2003

A humanized aldolase antibody for selective chemotherapy and adaptor immunotherapy.

Mouse monoclonal antibody 38C2 is the prototype of a new class of catalytic antibodies that were generated by reactive immunization. Through a reactive lysine, 38C2 catalyzes aldol and retro-aldol reactions using the enamine mechanism of natural aldolases. In addition to its remarkable versatility and efficacy in synthetic organic chemistry, 38C2 has been used for the selective activation of prodrugs in vitro and in vivo and thereby emerged as a promising tool for selective chemotherapy. Adding another application with relevance for cancer therapy, designated adaptor immunotherapy, we have recently shown that 38C2 can be chemically programmed to target tumors by formation of a covalent bond of defined stoichiometry with a beta-diketone derivative of an integrin alpha(v)beta(3) targeting RGD peptidomimetic. However, a major limitation for the transition from preclinical to clinical evaluation is the human anti-mouse antibody immune response that mouse 38C2 is likely to elicit in a majority of patients after single administration. Here, we report the humanization of mouse 38C2 based on rational design guided by molecular modeling. In essence, the catalytic center of mouse 38C2, which encompasses a deep hydrophobic pocket with a reactive lysine residue at the bottom, was grafted into a human antibody framework. Humanized 38C2 IgG1 was found to bind to beta-diketone haptens with conserved affinities and revealed strong catalytic activity with identical k(cat) and slightly higher K(M) values compared to the parental mouse antibody. Furthermore, humanized 38C2 IgG1 revealed efficiency in prodrug activation and chemical programming comparable to the parental mouse antibody.

Amino Acid Sequence↗
PubMed2001

Structural and immunological similarities between high molecular weight zinc ion-dependent p-nitrophenylphosphatase and fructose-1,6-bisphosphate aldolase from bovine liver.

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.

4-Nitrophenylphosphatase↗
PubMed1999

Opsin shift in an aldolase antibody.

An antibody-retinal assembly that mimics the opsin shift (OS) of the naturally occurring visual pigments is reported. Both experiments and calculations show that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which exhibits a remarkable opsin red shift (140 nm), represents a useful model system for studying the factors that contribute to the OS.

Antibodies↗
PubMed1994

Cloning, overexpression and isolation of the type II FDP aldolase from E. coli for specificity study and synthetic application.

A stable overexpression E. coli strain containing the plasmid pKEN 2 for the production of the Zn(2+)-dependent FDP aldolase from E. coli has been developed. Approximately 14,000 U of the enzyme (specific activity = 23.3 U/mg) can be obtained from 4-L of growth medium. The enzyme was isolated, purified to homogeneity and used for the studies of stability, substrate specificity and metal ion replacement and dissociation. Crystals of the enzyme have been obtained for structural analysis. This E. coli strain was deposited with the American Type Culture Collection (ATCC #77472).

Base Sequence↗
PubMed1997

Design and synthesis of chiral and racemic phosphonate-based haptens for the induction of aldolase catalytic antibodies.

A novel strategy for the generation of aldolase catalytic antibodies, based on the use of antibody-catalyzed enol ester hydrolysis as a 'trigger' to generate a reactive enolate intermediate, is described. A model system to test this strategy was developed and substrate 8 was synthesized. However, the targeted bifunctional haptens 11 and 33 were synthetically inaccessible, and therefore the alternative phosphonate hapten 39 was prepared. The key step in the synthesis of 39 was the direct generation of an unprotected phosphonate precursor via coupling of the secondary alcohol 37 with CH3P(O)Cl2. The chiral counterpart of hapten 39 was also synthesized from alcohol 46, prepared by Corey's asymmetric reduction method. One polyclonal antibody preparation generated from 39 appeared to catalyze the hydrolysis of the secondary acetate 49, but not the desired aldol cyclization of 8. Possible rationales for this finding are discussed.

Antibodies↗
PubMed1996

Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the alpha/beta barrel within the class I aldolase family.

BACKGROUND: Transaldolase is one of the enzymes in the non-oxidative branch of the pentose phosphate pathway. It transfers a C3 ketol fragment from a ketose donor to an aldose acceptor. Transaldolase, together with transketolase, creates a reversible link between the pentose phosphate pathway and glycolysis. The enzyme is of considerable interest as a catalyst in stereospecific organic synthesis and the aim of this work was to reveal the molecular architecture of transaldolase and provide insights into the structural basis of the enzymatic mechanism. RESULTS: The three-dimensional (3D) structure of recombinant transaldolase B from E. coli was determined at 1.87 A resolution. The enzyme subunit consists of a single eight-stranded alpha/beta-barrel domain. Two subunits form a dimer related by a twofold symmetry axis. The active-site residue Lys132 which forms a Schiff base with the substrate is located at the bottom of the active-site cleft. CONCLUSIONS: The 3D structure of transaldolase is similar to structures of other enzymes in the class I aldolase family. Comparison of these structures suggests that a circular permutation of the protein sequence might have occurred in transaldolase, which nevertheless results in a similar 3D structure. This observation provides evidence for a naturally occurring circular permutation in an alpha/beta-barrel protein. It appears that such genetic permutations occur more frequently during evolution than was previously thought.

Amino Acid Sequence↗