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Journal of the American Chemical Society Mar 2022Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials,...
Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials, and active-site reorganization energies. Although the minimal frustration of the folding landscape enables this tuning, residual frustration in the vicinity of the metallocofactor can allow conformational fluctuations required for protein function. We show here that the constrained copper site in wild-type azurin is governed by an intricate pattern of minimally frustrated local and distant interactions that together enable rapid electron flow to and from the protein. In contrast, sluggish electron transfer reactions (unfavorable reorganization energies) of active-site azurin variants are attributable to increased frustration near to as well as distant from the copper site, along with an exaggerated oxidation-state dependence of both minimally and highly frustrated interaction patterns.
Topics: Azurin; Copper; Electron Transport; Electrons; Pseudomonas aeruginosa
PubMed: 35171591
DOI: 10.1021/jacs.1c13454 -
International Microbiology : the... Dec 2019Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming...
Molecular screening and genetic diversity analysis of anticancer Azurin-encoding and Azurin-like genes in human gut microbiome deduced through cultivation-dependent and cultivation-independent studies.
Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level.
Topics: Adolescent; Adult; Azurin; Child; Culture Media; Feces; Female; Gastrointestinal Microbiome; Genetic Variation; Humans; Male; Phylogeny; Pseudomonas aeruginosa; Young Adult
PubMed: 30895406
DOI: 10.1007/s10123-019-00070-8 -
Journal of Chemical Theory and... Jun 2021Biomolecules with metal ion(s) (e.g., metalloproteins) play many important biological roles. However, accurate structural determination of metalloproteins, particularly...
Biomolecules with metal ion(s) (e.g., metalloproteins) play many important biological roles. However, accurate structural determination of metalloproteins, particularly those containing transition metal ion(s), is challenging due to their complicated electronic structure, complex bonding of metal ions, and high number of conformations in biomolecules. Quantum refinement, which was proposed to combine crystallographic data with computational chemistry methods by several groups, can improve the local structures of some proteins. In this study, a quantum refinement method combining several multiscale computational schemes with experimental (X-ray diffraction) information was developed for metalloproteins. Various quantum refinement approaches using different ONIOM (our own -layered integrated molecular orbital and molecular mechanics) combinations of quantum mechanics (QM), semiempirical (SE), and molecular mechanics (MM) methods were conducted to assess the performance and reliability on the refined local structure in two metalloproteins. The structures for two (Cu- or Zn-containing) metalloproteins were refined by combining two-layer ONIOM2(QM1/QM2) and ONIOM2(QM/MM) and three-layer ONIOM3(QM1/QM2/MM) schemes with experimental data. The accuracy of the quantum-refined metal binding sites was also examined and compared in these multiscale quantum refinement calculations. ONIOM3(QM/SE/MM) schemes were found to give good results with lower computational costs and were proposed to be a good choice for the multiscale computational scheme for quantum refinement calculations of metal binding site(s) in metalloproteins with high efficiency. Additionally, a two-center ONIOM approach was employed to speed up the quantum refinement calculations for the Zn metalloprotein with two remote active sites/ligands. Moreover, a recent quantum-embedding wavefunction-in-density functional theory (WF-in-DFT) method was also adopted as the high-level method in unprecedented ONIOM2(CCSD-in-B3LYP/MM) and ONIOM3(CCSD-in-B3LYP/SE/MM) calculations, which can be regarded as novel pseudo-three- and pseudo-four-layer ONIOM methods, respectively, to refine the key Zn binding site at the coupled-cluster singles and doubles (CCSD) level. These refined results indicate that multiscale quantum refinement schemes can be used to improve the structural accuracy obtained for local metal binding site(s) in metalloproteins with high efficiency.
Topics: Azurin; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Histone Acetyltransferases; Ligands; Metalloproteins; Metals; Molecular Dynamics Simulation; Quantum Theory
PubMed: 34032440
DOI: 10.1021/acs.jctc.1c00148 -
Quarterly Reviews of Biophysics Nov 2015Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Employing photosensitizer-modified... (Review)
Review
Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Employing photosensitizer-modified iron and copper proteins, we have shown that single-step electron tunneling can occur on nanosecond to microsecond timescales at distances between 15 and 20 Å. We also have shown that charge transport can occur over even longer distances by hole hopping (multistep tunneling) through intervening tyrosines and tryptophans. In this perspective, we advance the hypothesis that such hole hopping through Tyr/Trp chains could protect oxygenase, dioxygenase, and peroxidase enzymes from oxidative damage. In support of this view, by examining the structures of P450 (CYP102A) and 2OG-Fe (TauD) enzymes, we have identified candidate Tyr/Trp chains that could transfer holes from uncoupled high-potential intermediates to reductants in contact with protein surface sites.
Topics: Azurin; Bacillus megaterium; Bacterial Proteins; Catalytic Domain; Copper; Cytochrome P-450 Enzyme System; Electron Transport; Electrons; Escherichia coli; Iron; Molecular Conformation; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidative Stress; Oxygen; Oxygenases; Surface Properties; Tryptophan; Tyrosine
PubMed: 26537399
DOI: 10.1017/S0033583515000062 -
Preparative Biochemistry & Biotechnology 2019In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade using the Nisin Controlled Gene Expression (NICE) System. In addition, the...
In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for and were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade strain. As a result, the recombinant strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.
Topics: Anti-Bacterial Agents; Azurin; Bacillus cereus; Cell Line; Cloning, Molecular; Escherichia coli; Gene Amplification; Humans; Lactococcus lactis; Plasmids; Pseudomonas aeruginosa; Recombinant Proteins; Transformation, Genetic
PubMed: 31156029
DOI: 10.1080/10826068.2019.1621894 -
3 Biotech Jul 2019In the present study, the simultaneous application of gene of and - antigen on the induction of immune responses against breast cancer tumors was investigated in...
In the present study, the simultaneous application of gene of and - antigen on the induction of immune responses against breast cancer tumors was investigated in BALB/c mice. The pBudCE4.1-azurin-MAM-A recombinant vector was generated and prepared at a large scale. This recombinant vector alone or combined with chitosan nanoparticles was infused into the hip muscle of animals. Animals were divided into the "prevention" and "therapy" categories. The animals of prevention category were first, immunized by a recombinant vector and then exposed to chemical cancer inducers; while the animals in the therapy category were first treated with chemical compounds and then infused by a recombinant plasmid. The tumor tissues, infusion sites, and blood specimens were collected and examined by serological, molecular, and histological tests. The breast tumor incidence in the infused animals by recombinant plasmid alone or combined with nanoparticles (in both prevention and therapy categories) compared with infused mice by empty pBudCE4.1 vector was significantly decreased (< 0.05). These results were supported by histological studies using H&E staining. The ELISA and q-real-time PCR techniques showed the range of IFN-γ, IL-12, IL-4, and IL-17A cytokines in the infused mice by recombinant vector alone or combined with nanoparticles compared to the healthy mice and infused animals by intact pBudCE4.1 were significantly increased (< 0.05). Accordingly, the expression of the tumor markers , , and were significantly decreased in treated mice either by the sole recombinant vector or combined with nanoparticles (< 0.05). These findings indicated that pBudCE4.1-azurin-MAM-A recombinant vector plays an essential role against the formation and expansion of breast tumors in the animal model. In addition, this recombinant vector is safe and has the proper ability to stimulate the immune system. In addition, the chitosan nanoparticle represents a promising adjuvant for DNA vaccine delivery, which improves the immune system stimulation and boosts the vaccine performance.
PubMed: 31245235
DOI: 10.1007/s13205-019-1804-7 -
The Journal of Physical Chemistry. B Apr 2022We investigate the events characterizing the steps of the unfolding pathway of blue copper metalloprotein azurin using replica exchange molecular dynamics (REMD). Our...
We investigate the events characterizing the steps of the unfolding pathway of blue copper metalloprotein azurin using replica exchange molecular dynamics (REMD). Our studies show that the unfolding of azurin begins with the melting of α-helix and β-sheets II and V. This is followed by the melting of other β-sheets and the exposure of hydrophobic protein core to the solvent, resulting in disruptions of its tertiary structure. Free energy surfaces constructed at different temperatures portray different basins that signify the stability of different melted structures in the unfolding process. The contact maps at different temperatures reveal that the strong hydrophobic interaction within the core of the protein is the vital force that renders high stability to this protein. Analysis of the individual β-sheets by looking into their amino acid sequence shows that β-sheets with charged side chains on the surface melt fast compared to others. The β-barrel of azurin is able to dynamically rearrange, and it helps the protein to preserve its hydrophobic core, holding back the native topology from melting fast. B-factor analysis shows that residues of β-sheets III, IV, and VII deviate less from their initial structure at the transition temperature.
Topics: Azurin; Copper; Hydrophobic and Hydrophilic Interactions; Metalloproteins; Molecular Dynamics Simulation; Protein Folding
PubMed: 35324174
DOI: 10.1021/acs.jpcb.2c00622 -
The Journal of Physical Chemistry... Dec 2023We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or...
We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or lower current values compared to fully coherent transport. This depends on the structural details of the junctions as well as the alignment of the protein orbitals. Moreover, we show how the asymmetry of the curves can be affected by the position of the tip in the junction and that, under specific conditions, such a hopping mechanism is consistent with a fairly low temperature dependence of the current. Finally, we show that increasing the number of hopping sites leads to higher hopping currents. Our findings, from fully quantum calculations, provide deep insight to help guide the interpretation of experimental curves on highly complex systems.
PubMed: 38059566
DOI: 10.1021/acs.jpclett.3c02702 -
Biomolecules Apr 2023Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological...
Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological systems. Still, Cu/ is an essential micronutrient in many organisms, while no known biological process requires silver. In human cells, copper regulation and trafficking is strictly controlled by complex systems including many cytosolic copper chaperones, whereas some bacteria exploit the so-called "blue copper" proteins. Therefore, evaluating the controlling factors of the competition between these two metal cations is of enormous interest. By employing the tools of computational chemistry, we aim to delineate the extent to which Ag might be able to compete with the endogenous copper in its Type I (T1Cu) proteins, and where and if, alternatively, it is handled uniquely. The effect of the surrounding media (dielectric constant) and the type, number, and composition of amino acid residues are taken into account when modelling the reactions in the present study. The obtained results clearly indicate the susceptibility of the T1Cu proteins to a silver attack due to the favorable composition and geometry of the metal-binding centers, along with the similarity between the Ag/Cu-containing structures. Furthermore, by exploring intriguing questions of both metals' coordination chemistry, an important background for understanding the metabolism and biotransformation of silver in organisms is provided.
Topics: Humans; Copper; Silver
PubMed: 37189429
DOI: 10.3390/biom13040681 -
Accounts of Chemical Research May 2023"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes...
"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (Ni) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the Ni center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced Ni center. The elusive EPR-active S = 1/2 Ni-CH species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological Ni species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.
Topics: Azurin; Acetyl Coenzyme A; Nickel; Carbon Monoxide; Metalloproteins; Electron Spin Resonance Spectroscopy
PubMed: 37042748
DOI: 10.1021/acs.accounts.2c00824