-
Infection and Drug Resistance 2020The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients....
AIM
The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of gene using globally reported sequences was intended.
MATERIALS AND METHODS
Fifty-nine multidrug-resistant isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The gene sequences were compared with the sequence data from other countries. The expression level of gene in isolates with different sequences from different clinical specimens was evaluated by real-time PCR.
RESULTS AND CONCLUSION
About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about genetic variation and significance in pathogenesis.
PubMed: 32765002
DOI: 10.2147/IDR.S248043 -
Communications Biology Jan 2023Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in...
Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.
Topics: Humans; Azurin; Bacterial Proteins; Peptides; Tumor Suppressor Protein p53; Ubiquitination
PubMed: 36650277
DOI: 10.1038/s42003-023-04458-1 -
Metallomics : Integrated Biometal... Mar 2020Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to...
Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to 3D domain swap, but there has been no domain swapping report on a blue copper protein. Here, we found that azurin from Alcaligenes xylosoxidans oligomerizes by the procedure of 2,2,2-trifluoroethanol addition to Cu(i)-azurin at pH 5.0, lyophilization, and dissolution at pH 7.0, whereas it slightly oligomerizes when using Cu(ii)-azurin. The amount of high order oligomers increased with the addition of Cu(ii) ions to the dissolution process of a similar procedure for apoazurin, indicating that Cu(ii) ions enhance azurin oligomerization. The ratio of the absorbance at 460 nm to that at ∼620 nm of the azurin dimer (Abs460/Abs618 = 0.113) was higher than that of the monomer (Abs460/Abs622 = 0.067) and the EPR A‖ value of the dimer (5.85 mT) was slightly smaller than that of the monomer (5.95 mT), indicating a slightly more rhombic copper coordination for the dimer. The redox potential of the azurin dimer was 342 ± 5 mV vs. NHE, which was 50 mV higher than that of the monomer. According to X-ray crystal analysis, the azurin dimer exhibited a domain-swapped structure, where the N-terminal region containing three β-strands was exchanged between protomers. The copper coordination structure was tetrahedrally distorted in the azurin dimer, similar to that in the monomer; however, the Cu-O(Gly45) bond length was longer for the dimer (monomer, 2.46-2.59 Å; dimer, 2.98-3.25 Å). These results open the door for designing oligomers of blue copper proteins by domain swapping.
Topics: Alcaligenes; Azurin; Bacterial Proteins; Copper; Crystallography, X-Ray; Models, Molecular; Protein Conformation; Protein Domains; Protein Multimerization
PubMed: 31956880
DOI: 10.1039/c9mt00255c -
Proteins Oct 2020Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa,...
Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa, with notable occurrences in fungi. The role of these fungal MCOs in lignin degradation sparked an interest due to their potential for application in biofuel production and various other industries. MCOs consist of different protein domains, which led to their classification into two-, three-, and six-domain MCOs. The previously established Laccase and Multicopper Oxidase Engineering Database (https://lcced.biocatnet.de) was updated and now includes 51 058 sequences and 229 structures of MCOs. Sequences and structures of all MCOs were systematically compared. All MCOs consist of cupredoxin-like domains. Two-domain MCOs are formed by the N- and C-terminal domain (domain N and C), while three-domain MCOs have an additional domain (M) in between, homologous to domain C. The six-domain MCOs consist of alternating domains N and C, each three times. Two standard numbering schemes were developed for the copper-binding domains N and C, which facilitated the identification of conserved positions and a comparison to previously reported results from mutagenesis studies. Two sequence motifs for the copper binding sites were identified per domain. Their modularity, depending on the placement of the T1-copper binding site, was demonstrated. Protein sequence networks showed relationships between two- and three-domain MCOs, allowing for family-specific annotation and inference of evolutionary relationships.
Topics: Amino Acid Sequence; Azurin; Binding Sites; Coenzymes; Copper; Data Mining; Databases, Protein; Evolution, Molecular; Fungal Proteins; Fungi; Models, Molecular; Oxidation-Reduction; Oxidoreductases; Oxygen; Protein Binding; Protein Engineering; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Water
PubMed: 32447824
DOI: 10.1002/prot.25952 -
Bioprocess and Biosystems Engineering Mar 2022Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox...
Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.
Topics: Azurin; Bacterial Proteins; Copper; Octoxynol; Polysorbates; Pseudomonas aeruginosa
PubMed: 35039942
DOI: 10.1007/s00449-021-02678-5 -
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 -
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