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Applied Microbiology and Biotechnology Dec 2018Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic... (Review)
Review
Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.
Topics: Antineoplastic Agents; Azurin; Bacteriocins; Cations; Cell Membrane; Humans; Nisin; Pediocins; Protein Engineering; Pyocins
PubMed: 30338356
DOI: 10.1007/s00253-018-9420-8 -
Inorganic Chemistry May 2016Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed...
Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.
Topics: Ascorbic Acid; Azurin; Binding Sites; Catalysis; Copper; Electrochemical Techniques; Kinetics; Mutagenesis, Site-Directed; Nitrite Reductases; Oxidation-Reduction; Protein Engineering; Sodium Nitrite
PubMed: 27055058
DOI: 10.1021/acs.inorgchem.5b03006 -
Inorganic Chemistry May 2021Blue copper proteins continue to challenge experiment and theory with their electronic structure and spectroscopic properties that respond sensitively to the...
Blue copper proteins continue to challenge experiment and theory with their electronic structure and spectroscopic properties that respond sensitively to the coordination environment of the copper ion. In this work, we report state-of-the art electronic structure studies for geometric and spectroscopic properties of the archetypal "Type I" copper protein azurin in its Cu(II) state. A hybrid quantum mechanics/molecular mechanics (QM/MM) approach is used, employing both density functional theory (DFT) and coupled cluster with singles, doubles, and perturbative triples (CCSD(T)) methods for the QM region, the latter method making use of the domain-based local pair natural orbital (DLPNO) approach. Models of increasing QM size are employed to investigate the convergence of critical geometric parameters. It is shown that convergence is slow and that a large QM region is critical for reproducing the short experimental Cu-SCys112 distance. The study of structural convergence is followed by investigation of spectroscopic parameters using both DFT and DLPNO-CC methods and comparing these to the experimental spectrum using simulations. The results allow us to examine for the first time the distribution of spin densities and hyperfine coupling constants at the coupled cluster level, leading us to revisit the experimental assignment of the S hyperfine splitting. The wavefunction-based approach to obtain spin-dependent properties of open-shell systems demonstrated here for the case of azurin is transferable and applicable to a large array of bioinorganic systems.
Topics: Azurin; Density Functional Theory; Models, Molecular; Protein Conformation; X-Ray Absorption Spectroscopy
PubMed: 33939922
DOI: 10.1021/acs.inorgchem.1c00640 -
Journal of Biological Inorganic... Feb 2019The Cu center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a...
The Cu center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the Cu site (the TtCu protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0-9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCu increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the Cu site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCu through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.
Topics: Bacterial Proteins; Copper; Diethyl Pyrocarbonate; Electron Transport Complex IV; Histidine; Models, Molecular; Oxidation-Reduction; Thermus thermophilus
PubMed: 30523412
DOI: 10.1007/s00775-018-1632-y -
Frontiers in Bioscience (Landmark... Nov 2022Azurin, a bacterial cupredoxin firstly isolated from the bacterium , is considered a potential alternative therapeutic tool against different types of cancer.
BACKGROUND
Azurin, a bacterial cupredoxin firstly isolated from the bacterium , is considered a potential alternative therapeutic tool against different types of cancer.
AIMS
In this work we have explored the relationship possibly existing between azurin and colorectal cancer (CRC), in light of the evidence that microbial imbalance can lead to CRC progression.
METHODOLOGY/RESULTS
To this aim, the presence of azurin coding gene in the DNA extracted from saliva, stool, and biopsy samples of 10 CRC patients and 10 healthy controls was evaluated by real-time PCR using primers specifically designed to target the azurin coding gene from different bacterial groups. The correlation of the previously obtained microbiota data with real-time PCR results evidenced a "preferential" enrichment of seven bacterial groups in some samples than in others, even though no statistical significance was detected between controls and CRC. The subset of azurin gene-harbouring bacterial groups was representative of the entire community.
CONCLUSIONS
Despite the lack of statistical significance between healthy and diseased patients, HTS data analysis highlighted a kind of "preferential" enrichment of seven bacterial groups harbouring the azurin gene in some samples than in others.
Topics: Humans; Azurin; Genes, Bacterial; Pseudomonas aeruginosa; Microbiota
PubMed: 36472111
DOI: 10.31083/j.fbl2711305 -
Biochemical and Biophysical Research... Feb 2022EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a...
EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a peroxidase, respectively. A Gram-negative bacterium, Sphingomonas sp. strain A1, encodes EfeU, EfeO, EfeB together with alginate-binding protein Algp7, a truncated EfeO-like protein (EfeO), in the genome. The typical EfeO (EfeO) consists of N-terminal cupredoxin and C-terminal M75 peptidase domains. Here, we detail the structure and function of bacterial EfeB and EfeO. Crystal structures of strain A1 EfeB and Escherichia coli EfeO were determined at 2.30 Å and 1.85 Å resolutions, respectively. A molecule of heme involved in oxidase activity was bound to the C-terminal Dyp peroxidase domain of EfeB. Two domains of EfeO were connected by a short loop, and a zinc ion was bound to four residues, Glu156, Glu159, Asp173, and Glu255, in the C-terminal M75 peptidase domain. These residues formed tetrahedron geometry suitable for metal binding and are well conserved among various EfeO proteins including Algp7 (EfeO), although the metal-binding site (HxxE) is proposed in the C-terminal M75 peptidase domain. This is the first report on structure of a typical EfeO with two domains, postulating a novel metal-binding motif "ExxE-//-D-//-E" in the EfeO C-terminal M75 peptidase domain.
Topics: Amino Acid Motifs; Azurin; Bacterial Proteins; Binding Sites; Biological Transport; Cation Transport Proteins; Crystallography, X-Ray; Escherichia coli Proteins; Heme; Iron; Metals; Molecular Conformation; Oxidoreductases; Protein Binding; Protein Conformation; Protein Domains; Protein Structure, Secondary; Sphingomonas
PubMed: 35081501
DOI: 10.1016/j.bbrc.2022.01.055 -
Journal of the American Society For... May 2020Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational...
Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40-50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.
Topics: Amino Acid Sequence; Azurin; Binding Sites; Calcium; Calmodulin; Lanthanoid Series Elements; Metals; Micrococcal Nuclease; Models, Molecular; Spectrometry, Mass, Electrospray Ionization
PubMed: 32275426
DOI: 10.1021/jasms.0c00066 -
The Journal of Physical Chemistry. B Jan 2023Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference...
Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and ° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases ° and reveal properties of the protein microenvironment surrounding tryptophan.
Topics: Azurin; Tryptophan; Oxidation-Reduction; Hydrogen; Water
PubMed: 36542812
DOI: 10.1021/acs.jpcb.2c06677 -
Pharmaceutics Jun 2023Azurin is a natural protein produced by that exhibits potential anti-tumor, anti-HIV, and anti-parasitic properties. The current study aimed to investigate the...
Azurin is a natural protein produced by that exhibits potential anti-tumor, anti-HIV, and anti-parasitic properties. The current study aimed to investigate the potential of azurin protein against breast cancer using both in silico and in vitro analyses. The amino acid sequence of Azurin was used to predict its secondary and tertiary structures, along with its physicochemical properties, using online software. The resulting structure was validated and confirmed using Ramachandran plots and ERRAT2. The mature azurin protein comprises 128 amino acids, and the top-ranked structure obtained from I-TASSER was shown to have a molecular weight of 14 kDa and a quality factor of 100% by ERRAT2, with 87.4% of residues in the favored region of the Ramachandran plot. Docking and simulation studies of azurin protein were conducted using HDOCK and Desmond servers, respectively. The resulting analysis revealed that Azurin docked against p53 and EphB2 receptors demonstrated maximum binding affinity, indicating its potential to cause apoptosis. The recombinant azurin gene was successfully cloned and expressed in a BL21 (DE3) strain using a pET20b expression vector under the control of the pelB ladder, followed by IPTG induction. The azurin protein was purified to high levels using affinity chromatography, yielding 70 mg/L. In vitro cytotoxicity assay was performed using MCF-7 cells, revealing the significant cytotoxicity of the azurin protein to be 105 µg/mL. These findings highlight the potential of azurin protein as an anticancer drug candidate.
PubMed: 37514012
DOI: 10.3390/pharmaceutics15071825 -
The Journal of Physical Chemistry. B Feb 2019We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCu, where two adjacent tryptophan residues (W124 and W122) are inserted...
We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCu, where two adjacent tryptophan residues (W124 and W122) are inserted between the Cu center and a Re photosensitizer coordinated to a H126 imidazole (Re = Re(H126)(CO)(dmp), dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) Re(H126)(CO)(dmp)WWCu product. The Re126FWCu mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCu} and {Re126FWCu} are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCu} contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO)(dmp)//(W122)' unit; then (Cu)' is oxidized intramolecularly (60-90 ns) by (W122)', forming Re(H126)(CO)(dmp)WWCu//(Cu)'. The photocycle is closed by ∼1.6 μs Re(H126)(CO)(dmp) → //(Cu)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCu. Importantly, dimerization makes Re126FWCu photoreactive and, as in the case of {Re126WWCu}, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of Re(H126)(CO)(dmp)//Cu. Although two adjacent W124 and W122 indoles dramatically enhance Cu → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCu} does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCu} or {Re126FWCu} also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO)(dmp)//(W122)' charge-separated state decays to the ground state by ∼50 ns Re(H126)(CO)(dmp) → //(W122)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.
Topics: Azurin; Copper; Electron Transport; Electrons; Imidazoles; Light; Models, Molecular; Mutagenesis; Oxidation-Reduction; Protein Structure, Tertiary; Pseudomonas aeruginosa; Quantum Theory; Tryptophan; Water
PubMed: 30673250
DOI: 10.1021/acs.jpcb.8b11982