Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction

A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmania infantum, Leishmania braziliensis, Leishmania guyanensis and Leishmania amazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC₅₀ = 99.8–26.8 μM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [K_b = 1.14 × 10⁵ M⁻¹ (6) and 3.26 × 10⁵ M⁻¹ (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV–visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [K_b = 2.5 × 10⁴ M⁻¹ (6) and 1.9 × 10⁴ M⁻¹ (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.     

Two DNA binding modes of a zinc-metronidazole and biological evaluation as a potent anti-cancer agent

A complex of metronidazole (MTZ) with zinc ion was synthesized and characterized by UV-Vis, Fourier transform infrared (FT-IR), ¹H-NMR, X-ray crystallography and thermal gravimetric-differential thermal analysis (TG-DTA). The cytotoxicity effect of the synthesized complex investigated over SKNMC, A549, MCF-7, and MCDK cell lines and the results have shown that it has high cytotoxic potential over cancer cell lines. In order to clarify the mechanism of cell cytotoxicity, the oxidative stress and binding of the complex to the calf thymus-DNA studied by evaluating the intrinsic binding constant and defining thermodynamic parameters of complex over the DNA accompanying with in silico molecular modeling method. For this purpose, the complex optimized at the B3LYP/LANL2DZ level and docked over the DNA structure. The results revealed that the metronidazole-zinc complex interacted with DNA via hydrogen binding and electrostatic interaction to the minor groove region and phosphate backbone of DNA, respectively.     

Microbial and xanthine dehydrogenase inhibitory activity of some flavones

Xanthine dehydrogenase (XDH) is responsible for the pathological condition called Gout. In the present study different flavones synthesized from chalcone were evaluated in vitro for their inhibitory activity. Inhibitory activity of flavones on XDH was determined in terms of inhibition of uric acid synthesis from Xanthine. The enzymatic activity was found maximum at pH 7.5 and temperature 40°C. The flavones 6-chloro-2-[3-(4–hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F₁) and 6-chloro-7methyl-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F₂),were noncompetitive and competitive inhibitor with Ki values 1.1 and 0.22 respectively. The flavones (F₁), (F₂), 6-chloro-2-[3-(4-chloro-phenyl)-1phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F₃), 8-bromo-6-chloro-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F₄), 2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F₅) and 6-methyl-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F₆) were also screened for their antimicrobial activity, measured in terms of zone of inhibition. A broad spectrum antifungal activity was obtained against Trichoderma viridae, Candida albicans, Microsporum cannis, Penicillium chrysogenum and Fusarium moniliformae. In case of Aspergillus niger and Aspergillus flavous only spore formation was affected, while antibacterial activity was observed against Staphylococcus aureus, Bacillus subtilis and Serratia marsecens only. The flavones were further analyzed for quantitative structural activity relationship study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were revealed by PALLAS software in terms of their molecular weight. Log P values were also studied. The result showed both the F₁ and F₂ flavones as antigout and therefore supports the development of novel drugs for the treatment of gout.     

Effects of Ca2+ on the activity and stability of methanol dehydrogenase

The effects of exogenously added Ca²⁺ on the enzymatic activity and structural stability of methanol dehydrogenase were studied for various Ca²⁺ concentrations. Methanol dehydrogenase activity increased significantly with increasing concentration of Ca²⁺, approaching saturation at 200 mM Ca²⁺. The effect of Ca²⁺ on the activation of MDH was time dependent and Ca²⁺ specific and was due to binding of the metal ions to the enzyme. Addition of increasing concentration of Ca²⁺ caused a decrease of the intrinsic tryptophan fluorescence intensity in a concentration-dependent manner to a minimum at 200 mM, but with no change in the fluorescence emission maximum wavelength or the CD spectra. The results revealed that the activation of methanol dehydrogenase by Ca²⁺ occurred concurrently with the conformational change. In addition, exogenously bound Ca²⁺ destabilized MDH. The potential biological significance of these results is discussed.     

Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles

Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained.     

Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity

Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.     

Membrane damage as first and DNA as the secondary target for anti‐candidal activity of antimicrobial peptide P7 derived from cell‐penetrating peptide ppTG20 against Candida albicans

P7, a peptide analogue derived from cell‐penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti‐Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l ‐phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin‐treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC‐P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Carbon monoxide dehydrogenase in mycobacteria possesses a nitric oxide dehydrogenase activity

CO dehydrogenase (CO-DH) catalyzes the oxidation of CO to CO(2) in carboxydobacteria. Cell-free extracts prepared from several mycobacteria, including Mycobacterium tuberculosis H37Ra, showed NO dehydrogenase (NO-DH) activity in a reaction mixture containing sodium nitroprusside (SNP) as the source of NO. The association of the NO-DH activity with CO-DH was revealed by activity staining and confirmed by enzyme assay with purified CO-DH from Mycobacterium sp. strain JC1, a carboxydotrophic mycobacterium. SNP stimulated the production of CO-DH with a coincidental increase in NO-DH activity in the bacterium, further supporting this association and implying the existence of a possible SNP-induced CO-DH gene expression. The addition of purified CO-DH to cultures of Escherichia coli revealed that the enzyme protected E. coli from SNP-induced killing in a dose-dependant way. The present results indicate that mycobacterial CO-DH also acts as a NO-DH, which may function in the protection of mycobacterial pathogens from nitrosative stress during infection.     

A lipoamide dehydrogenase from Neisseria meningitidis has a lipoyl domain

A protein of molecular weight of 64 kDa (p64k) found in the outer membrane of Neisseria meningitidis shows a high degree of homology with both the lipoyl domain of the acetyltransferase and the entire sequence of the lipoamide dehydrogenase, the E2 and E3 components of the dehydrogenase multienzyme complexes, respectively. The alignment of the p64k with lipoyl domains and lipoamide dehydrogenases from different species is presented. The possible implications of this protein in binding protein-dependent transport are discussed. This is the first lipoamide dehydrogenase reported to have a lipoyl domain. © 1995 Wiley-Liss, Inc.     

Alcohol dehydrogenase activity in diploid and autotetraploid Phlox

The effect of polyploidy on the activity of alcohol dehydrogenase was examined in a series of diploid and synthetic autotetraploid Phlox drummondii. In most cases autotetraploids had about twice as much activity as the corresponding diploids, in two cases autotetraploids had about 1.5 times more activity, and in one wild seed pair the activity of the tetraploid was somewhat lower.This study was supported in part by NSF grants (DEB 76-19914, DAL; 4392, AMT, BMS 75-19621, ML).     

Bacteriophage MB78 DNA synthesis is specifically inhibited by the chelating agent ethylene diamine tetraacetic acid

The growth of bacteriophage MB78, a virulent phage of Salmonella typhimurium is extremely sensitive to the chelating agent EDTA. Other chelating agents like EGTA, a specific chelator for Ca²⁺ and orthophenanthroline which chelates Zn²⁺ and Fe²⁺ have no effect. EDTA stops phage MB78 DNA synthesis while synthesis of host DNA and other Salmonella phage DNA are not affected in presence of such low concentrations of EDTA. The present report indicates that some early phage function(s) and most probably the phage DNA synthesis are sensitive to EDTA which is probably due to chelation of Mg²⁺.     

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the β-amyloid precursor protein (βAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with β-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon γ (INFγ). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of β-amyloid, or thiorphan, an inhibitor of neprilysin involved in β-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNγ, which can influence the progression of the AD, significantly reduced the enzyme activity.     

The herpes simplex virus processivity factor, UL42, binds DNA as a monomer

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer in solution. However, UL42 is structurally similar to sliding clamp processivity factors, such as PCNA, which encircle DNA as a multimeric ring. We used chemical crosslinking and electrophoretic mobility-shift assays to investigate whether UL42 oligomerizes upon DNA binding. UL42 did not form intermolecular crosslinks upon treatment with glutaraldehyde in the presence of DNA, whereas proteins that are known to be multimers in solution were successfully crosslinked by this treatment. This result suggests that UL42 does not form multimers on DNA. We next analyzed the composition of UL42:DNA complexes using electrophoretic mobility-shift assays. UL42 was mixed with a maltose-binding protein-UL42 fusion protein before being added to DNA. The patterns of electrophoretic mobility of the resultant protein:DNA complexes were those predicted if each isoform of UL42 binds to DNA as a monomer. From this result and the failure of UL42 to form crosslinks, we infer that UL42 binds DNA as a monomer.     

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α₂β₂ hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Synthesis,antitumor activity and DNA binding features of benzothiazolyl and benzimidazolyl substituted isoindolines

In this paper novel isoindolines substituted with cyano and amidino benzimidazoles and benzothiazoles were synthesized as new potential anti-cancer agents. The new structures were evaluated for antiproliferative activity, cell cycle changes, cell death, as well as DNA binding and topoisomerase inhibition properties on selected compounds. Results showed that all tested compounds exerted antitumor activity, especially amidinobenzothiazole and amidinobenzimidazole substituted isoindolin-1-ones and benzimidazole substituted 1-iminoisoindoline that showed antiproliferative effect in the submicromolar range. Moreover, the DNA-binding properties of selected compounds were evaluated by biophysical and biochemical approaches including thermal denaturation studies, circular dichroism spectra analyses and topoisomerase I/II inhibition assays and results identified some of them as strong DNA ligands, harboring or not additional topoisomerase II inhibition and able to locate in the nucleus as determined by fluorescence microscopy. In conclusion, we evidenced novel cyano- and amidino-substituted isoindolines coupled with benzimidazoles and benzothiazoles as topoisomerase inhibitors and/or DNA binding compounds with potent antitumor activities.