scholarly journals Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor

2019 ◽  
Vol 20 (7) ◽  
pp. 1802
Author(s):  
Stefania Pucciarelli ◽  
Silvia Vincenzetti ◽  
Massimo Ricciutelli ◽  
Oumarou Camille Simon ◽  
Anna Teresa Ramadori ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Dissociation Constants ◽  
Gold Compound ◽  
Inhibitory Effects ◽  
Binding Properties ◽  
Enzyme Active Site ◽  
E Coli ◽  
Kd Values ◽  
Gold Compounds

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan’s emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh3)2AuCl in the presence of NADPH to 21.4 ± 3.85 µM for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = −5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

High-resolution gold labeling

1993 ◽  
Vol 51 ◽  
pp. 330-331
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya ◽  
Kyra Carbone ◽  
Martha Simon ◽  
Beth Lin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Polypeptide Chain ◽  
External Surface ◽  
Gold Cluster ◽  
Macromolecular Complex ◽  
Gold Compound ◽  
Central Cavity ◽  
Chain Folding ◽  
E Coli ◽  
Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Synergistic Antimicrobial Activities of Combinations of Vanillin and Essential Oils of Cinnamon Bark, Cinnamon Leaves and Cloves

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1406
Author(s):  
Rita Cava-Roda ◽  
Amaury Taboada-Rodríguez ◽  
Antonio López-Gómez ◽  
Ginés Benito Martínez-Hernández ◽  
Fulgencio Marín-Iniesta
Keyword(s):  
Antimicrobial Activity ◽  
Essential Oils ◽  
Food Packaging ◽  
Antimicrobial Effect ◽  
Antimicrobial Activities ◽  
Inhibitory Effects ◽  
Cinnamon Bark ◽  
E Coli ◽  
Pure Compounds ◽  
Main Component

Plant bioactive compounds have antimicrobial and antioxidant activities that allow them to be used as a substitute for synthetic chemical additives in both food and food packaging. To improve its sensory and bactericidal effects, its use in the form of effective combinations has emerged as an interesting possibility in the food industry. In this study, the antimicrobial activities of essential oils (EOs) of cinnamon bark, cinnamon leaves, and clove and the pure compounds vanillin, eugenol, and cinnamaldehyde were investigated individually and in combination against Listeria monocytogenes and Escherichia coli O157:H7. The possible interactions of combinations of pure compounds and EOs were performed by the two-dimensional checkerboard assay and isobologram methods. Vanillin exhibited the lowest antimicrobial activity (MIC of 3002 ppm against L. monocytogenes and 2795 ppm against E. coli O157:H7), while clove and cinnamon bark EOs exhibited the highest antimicrobial activity (402–404 against L. monocytogenes and 778–721 against E. coli O157:H7). For L. monocytogenes, pure compound eugenol, the main component of cinnamon leaves and clove, showed lower antimicrobial activity than EOs, which was attributed to the influence of the minor components of the EOs. The same was observed with cinnamaldehyde, the main component of cinnamon bark EO. The combinations of vanillin/clove EO and vanillin/cinnamon bark EO showed the most synergistic antimicrobial effect. The combination of the EOs of cinnamon bark/clove and cinnamon bark/cinnamon leaves showed additive effect against L. monocytogenes but indifferent effect against E. coli O157:H7. For L. monocytogenes, the best inhibitory effects were achieved by cinnamon bark EO (85 ppm)/vanillin (910 ppm) and clove EO (121 ppm)/vanillin (691 ppm) combinations. For E. coli, the inhibitory effects of clove EO (104 ppm)/vanillin (1006 ppm) and cinnamon leaves EO (118 ppm)/vanillin (979 ppm) combinations were noteworthy. Some of the tested combinations increased the antimicrobial effect and would allow the effective doses to be reduced, thereby offering possible new applications for food and active food packaging.

scholarly journals The Protein Interactome of Glycolysis in Escherichia coli

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 16
Author(s):  
Shomeek Chowdhury ◽  
Stephen Hepper ◽  
Mudassir K. Lodi ◽  
Milton H. Saier ◽  
Peter Uetz
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Allosteric Regulation ◽  
Glycolytic Enzymes ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Interacting Protein ◽  
E Coli ◽  
Protein Interactome ◽  
The Impact

Glycolysis is regulated by numerous mechanisms including allosteric regulation, post-translational modification or protein-protein interactions (PPI). While glycolytic enzymes have been found to interact with hundreds of proteins, the impact of only some of these PPIs on glycolysis is well understood. Here we investigate which of these interactions may affect glycolysis in E. coli and possibly across numerous other bacteria, based on the stoichiometry of interacting protein pairs (from proteomic studies) and their conservation across bacteria. We present a list of 339 protein-protein interactions involving glycolytic enzymes but predict that ~70% of glycolytic interactors are not present in adequate amounts to have a significant impact on glycolysis. Finally, we identify a conserved but uncharacterized subset of interactions that are likely to affect glycolysis and deserve further study.

scholarly journals Interaction of Temporin-L Analogues with the E. coli FtsZ Protein

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 704
Author(s):  
Angela Di Somma ◽  
Carolina Canè ◽  
Antonio Moretta ◽  
Angela Duilio
Keyword(s):  
Protein Interactions ◽  
Bacterial Infections ◽  
Antibacterial Properties ◽  
Bacterial Cell Division ◽  
Structural Determinants ◽  
E Coli ◽  
Functional Studies ◽  
Division Process ◽  
Z Ring ◽  
Peptide Complexes

The research of new therapeutic agents to fight bacterial infections has recently focused on the investigation of antimicrobial peptides (AMPs), the most common weapon that all organisms produce to prevent invasion by external pathogens. Among AMPs, the amphibian Temporins constitute a well-known family with high antibacterial properties against Gram-positive and Gram-negative bacteria. In particular, Temporin-L was shown to affect bacterial cell division by inhibiting FtsZ, a tubulin-like protein involved in the crucial step of Z-ring formation at the beginning of the division process. As FtsZ represents a leading target for new antibacterial compounds, in this paper we investigated in detail the interaction of Temporin L with Escherichia coli FtsZ and designed two TL analogues in an attempt to increase peptide-protein interactions and to better understand the structural determinants leading to FtsZ inhibition. The results demonstrated that the TL analogues improved their binding to FtsZ, originating stable protein-peptide complexes. Functional studies showed that both peptides were endowed with a high capability of inhibiting both the enzymatic and polymerization activities of the protein. Moreover, the TL analogues were able to inhibit bacterial growth at low micromolar concentrations. These observations may open up the way to the development of novel peptide or peptidomimetic drugs tailored to bind FtsZ, hampering a crucial process of bacterial life that might be proposed for future pharmaceutical applications.

scholarly journals In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 13
Author(s):  
Elena Forte ◽  
Sergey A. Siletsky ◽  
Vitaliy B. Borisov
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Reductase Activity ◽  
Ammonia Concentration ◽  
High Concentration ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cytochrome Bo3 ◽  
Oxygen Reductase

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Screening of Commercial and Pecan Shell–Extracted Liquid Smoke Agents as Natural Antimicrobials against Foodborne Pathogens

2012 ◽  
Vol 75 (6) ◽  
pp. 1148-1152 ◽  
Author(s):  
ELLEN J. VAN LOO ◽  
D. BABU ◽  
PHILIP G. CRANDALL ◽  
STEVEN C. RICKE
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Salmonella Enteritidis ◽  
Antioxidant Properties ◽  
Inhibitory Effects ◽  
Natural Antimicrobials ◽  
E Coli ◽  
Liquid Smoke ◽  
Water Based ◽  
Smoke Sample

Liquid smoke extracts have traditionally been used as flavoring agents, are known to possess antioxidant properties, and serve as natural alternatives to conventional antimicrobials. The antimicrobial efficacies of commercial liquid smoke samples may vary depending on their source and composition and the methods used to extract and concentrate the smoke. We investigated the MICs of eight commercial liquid smoke samples against Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli. The commercial liquid smoke samples purchased were supplied by the manufacturer as water-based or concentrated extracts of smoke from different wood sources. The MICs of the commercial smokes to inhibit the growth of foodborne pathogens ranged from 0.5 to 6.0% for E. coli, 0.5 to 8.0% for Salmonella, and 0.38 to 6% for S. aureus. The MIC for each liquid smoke sample was similar in its effect on both E. coli and Salmonella. Solvent-extracted antimicrobials prepared using pecan shells displayed significant differences between their inhibitory concentrations depending on the type of solvent used for extraction. The results indicated that the liquid smoke samples tested in this study could serve as effective natural antimicrobials and that their inhibitory effects depended more on the solvents used for extraction than the wood source.

scholarly journals Auranofin-Mediated NRF2 Induction Attenuates Interleukin 1 Beta Expression in Alveolar Macrophages

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 632
Author(s):  
Stephanie B. Wall ◽  
Rui Li ◽  
Brittany Butler ◽  
Ashley R. Burg ◽  
Hubert M. Tse ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Inflammatory Responses ◽  
Interleukin 1 ◽  
Direct Interaction ◽  
Synthesis Methods ◽  
Related Factor ◽  
Gold Compound ◽  
Nuclear Factor ◽  
Glutathione Synthesis ◽  
Gold Compounds

Background: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1β expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis. Methods: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2. Results: Expression of IL-1β, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1β expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1β. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II. Conclusions: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1β synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1β directly inhibits transcription of the IL1β gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation.

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