Cyanobacterial Metallothionein Research Articles

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA

Among all approaches used for the semisynthesis of natural or chemically modified products, enzyme-assisted ligation is among the most promising and dynamically developing approaches. Applying an efficient C247A mutant of Oldenlandia affinis plant ligase OaAEP1 and solid-phase peptide synthesis chemistry, we present the chemoenzymatic synthesis of a complete sequence of the cysteine-rich and metal-binding cyanobacterial metallothionein Synechococcus metallothionein A (SmtA). Zn(II) and Cd(II) binding to the newly synthesized SmtA showed identical properties to the protein expressed in Escherichia coli. The presented approach is the first example of the use of OaAEP1 mutant for total protein synthesis of metallothionein, which occurs in mild conditions preventing cysteine thiol oxidation. The recognition motif of the applied enzyme could naturally occur in the protein structure or be synthetically or genetically incorporated in some loops or secondary structure elements. Therefore, we envision that this strategy can be used for efficiently obtaining SmtA and for a wide range of proteins and their derivatives.

A distinct molecular signature on anhydrobiotic cyanobacterial metallothioneins

Anhydrobiosis refers to a state of suspended animation in which some organisms enter when exposed to extreme desiccation, ensuring them an outstanding tolerance to several physical stresses due to molecular and cellular adaptations. Metallothioneins (MTs) are short cysteine-rich metal-chelating proteins that work as a cellular protection element in metal ion-rich conditions. Here we aimed to investigate possible molecular signatures in primary and tertiary structures in anhydrobiotic cyanobacterial MTs. Anhydrobiotic and non-anhydrobiotic cyanobacterial MT amino acid sequences were retrieved from NCBI database and aligned in Clustal Omega server. Additionally, the amino acid compositions of these sequences were determined by GeneRunner. Further, we carried out homology-modeling via SWISS-MODEL, structural superposition in UCSF Chimera 1.4 Matchmaker tool and ligand-binding site prediction via COFACTOR. In silico analyses revealed specific divergences in amino acid positions between MT groups, evidencing positive and negative selections, however without affecting final protein structures. Some of these changes on polypeptide sequence potentially enhance protein stabilization during desiccation, whereas others possibly act as additional metal-ion coordinating residues. Analyses on the molecular adaptations on anhydrobiotic cyanobacterial MTs help shed light on their molecular functions and biological roles, as well as may have applications on the development of desiccation- and metal-tolerant organisms.

Cry Protein Crystal-Immobilized Metallothioneins for Bioremediation of Heavy Metals from Water

Cry proteins have been the subject of intense research due to their ability to form crystals naturally in Bacillus thuringiensis (Bt). In this research we developed a new strategy that allows for the removal of cadmium and chromium from wastewater by using one Cry protein, Cry3Aa, as a framework to immobilize tandem repeats of the cyanobacterial metallothionein SmtA from Synechococcus elongatus (strain PCC 7942). SmtA is a low molecular weight cysteine-rich protein known to bind heavy metals. A series of Cry3Aa-SmtA constructs were produced by the fusion of one, three, or six tandem repeats of SmtA to Cry3Aa. Overexpression of these constructs in Bt resulted in the production of pure Cry3Aa-SmtA fusion crystals that exhibited similar size, crystallinity, and morphology to that of native Cry3Aa protein crystals. All three Cry3Aa-SmtA constructs exhibited efficient binding to cadmium and chromium, with the binding capacity correlated with increasing SmtA copy number. These results suggest the potential use of Cry3Aa-SmtA crystals as a novel biodegradable and cost-effective approach to the removal of toxic heavy metals from the environment.

NmtA, a novel metallothionein of Anabaena sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress

Metallothioneins (MTs) are low molecular weight, sulfhydryl-containing, cysteine-rich, metal-binding proteins. Eukaryotes have multiple metallothionein genes; however, there is dearth of reports on prokaryotic metallothioneins. Bacterial MTs with SmtA from Synechococcus PCC 7942 as prototype have been studied in the context of cadmium detoxification. In this study, a smtA related ORF, namely nmtA, was identified in the heterocystous, nitrogen-fixing cyanobacterium, Anabaena PCC 7120. A recombinant N-terminal histidine-tagged Anabaena NmtA protein was overexpressed in Escherichia coli and purified. The protein was identified by peptide mass fingerprinting using MALDI-TOF Mass Spectrometry as putative metallothionein of Anabaena PCC 7120 with a calculated mass of ∼6.1 kDa. While the native metallated NmtA exhibited resistance against proteolysis, metal free apo-NmtA resulting from acid and dithiothreitol (DTT) treatment could be digested by proteinase K revealing a metal dependent proteolytic protection of NmtA. Expression of nmtA in Anabaena PCC 7120 was induced evidently by cadmium, zinc and copper but not by uranium or hydrogen peroxide. Recombinant Anabaena PCC 7120 overexpressing NmtA protein revealed superior cadmium tolerance but showed limited influence against oxidative stress tolerance as compared with the strain carrying vector alone. In contrast, a mutant of Synechococcus PCC 7942 deficient in MT locus was found to be highly susceptible to H2O2 indicating a likely involvement of cyanobacterial MT in protection against oxidative damage. Overall, the study improved our understanding of metal tolerance mechanisms in Anabaena PCC 7120 by demonstrating a key role of NmtA in cadmium tolerance.

Equilibrium Isotherm, Kinetic Modeling, Optimization, and Characterization Studies of Cadmium Adsorption by Surface-Engineered Escherichia coli

Background:Amongst the methods that remove heavy metals from environment, biosorption approaches have received increased attention because of their environmentally friendly and cost-effective feature, as well as their superior performances.Methods:In the present study, we investigated the ability of a surface-engineered Escherichia coli, carrying the cyanobacterial metallothionein on the cell surface, in the removal of Ca (II) from solution under different experimental conditions. The biosorption process was optimized using central composite design. In parallel, the kinetics of metal biosorption was studied, and the rate constants of different kinetic models were calculated.Results:Cadmium biosorption is followed by the second-order kinetics. Freundlich and Langmuir equations were used to analyze sorption data; characteristic parameters were determined for each adsorption isotherm. The biosorption process was optimized using the central composite design. The optimal cadmium sorption capacity (284.69 nmol/mg biomass) was obtained at 40°C (pH 8) and a biomass dosage of 10 mg. The influence of two elutants, EDTA and CaCl2, was also assessed on metal recovery. Approximately, 68.58% and 56.54% of the adsorbed cadmium were removed by EDTA and CaCl2 during desorption, respectively. The Fourier transform infrared spectrophotometer (FTIR) analysis indicated that carboxyl, amino, phosphoryl, thiol, and hydroxyl are the main chemical groups involved in the cadmium bioadsorption process.Conclusion:Results from this study implied that chemical adsorption on the heterogeneous surface of E. coli E and optimization of adsorption parameters provides a highly efficient bioadsorbent.

Poly-γ-glutamic acid coupled Pseudomonas putida cells surface-displaying metallothioneins: composited copper(ii) biosorption and inducible flocculation in aqueous solution

Schematic illustration of the preparation, biosorption, and induced flocculation of the biocomposite MB546-PGA.

Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium.

Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)(n)Zn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. (1)H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu(+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+) and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

Improvement of Cd2+ uptake ability of SmtA protein by Lys/Cys mutation; experimental and theoretical studies

The improved Cd2+ surface affinity characteristics of a mutated cyanobacterial metallothionein SmtA (K45C) were investigated via experimental and theoretical methods. Molecular dynamics simulations were carried out using a model of Cd2+ and other ions enclosed in a fully hydrated simulation box with the wild-type or mutated SmtA protein. The theoretical results suggested that mutated SmtA was more powerful in absorption of Cd2+ than the wild-type protein. Then, the mutated smtA gene (from Synechococcus PCC 7942) was synthesized by simplified gene synthesis method and expressed on isopropyl-beta-d-thiogalactopyranoside induction. The protein expression was investigated by SDS-PAGE and verified by Western blotting. Finally, cadmium uptake ratio of mutant protein toward wild type was analyzed by atomic absorption. This study is the first example of cytoplasmic expression of a mutant protein. Experimental results also verified that the mutation intensifies uptake of Cd2+ ions.

Genetically encoded FRET-based nanosensor for in vivo monitoring of zinc concentration in physiological environment of living cell

Heavy metals are ubiquitous in nature, thus constituting a serious environmental problem in many countries. Due to their toxicity, there is an obvious need to determine them at trace levels. Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. To analyze the uptake and metabolism of zinc in Escherichia coli, a high affinity FRET (Fluorescence Resonance Energy Transfer)–based nanosensor was developed using the cyanobacterial metallothionein SmtA flanked by CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein). The chimeric sensor protein was expressed in E. coli BL21 (DE3) and purified by affinity chromatography. Following binding of zinc, FRET ratio increased with increasing the Zn2+ concentration. Affinity mutants were constructed with binding constants ranging from 50μM to 1mM. In vitro analysis with bacterially purified sensor showed that it is specific for Zn2+ and responds only weakly to Cd2+ with no response for other biological metal ions. Moreover, the sensor activity in vitro was stable with extreme changes in pH. In vivo analysis of nanosensor response in E. coli showed that free Zn2+ accumulate in the bacterial cells and is slowly metabolized.

Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium

Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20.

V- type proton ATP-ase to target cancer cells with the aid of Cyanobacterial metallothionein

ATP-ases are a group of enzymes that utilizes ATP hydrolysis, and the subsequent release of energy, to achieve a cellular function. The cellular functions involving ATP-ases are plentiful and diverse including initiation of DNA replication, DNA repair and remodeling, protein folding and chaperoning, protein degradation, intracellular transport, and ion transport. A large number of these enzymes represent attractive drug targets, and drugs targeting ATP-ases, such as proton pump inhibitors. Two families of molecular chaperones, heat shock protein 90 and heat shock protein 70, possess N-terminal nucleotide binding domains (NBD) and require ATP-ase activity for their functions. NBD is charged and highly polar in nature and there is no crystal structure yet published. These two families of ATP-ases represent significant therapeutic targets for the treatment of cancer. The ATP-ase activity of Hsp90, in interaction to Cyanobacterial mettalothionein is essential for targeting cancer affected cells and can be used highly as a drug targeting molecule. Inhibition of ATP-ase activity at nucleotide binding site of the Hsp90 leads to prevent tumor growth. Till now, only two antibiotics (ansamycin and geldanamycin) were discovered for inhibiting ATP-ase enzymes. This article discuss about Vtype proton ATP-ases, interaction with Cyanobacterial mettalothionein for targeting cancer spoiled cells in Cancer treatment.

Surface Display of Bacterial Metallothioneins and a Chitin Binding Domain on Escherichia coli Increase Cadmium Adsorption and Cell Immobilization

To increase the level of adsorption of cadmium ions to the surface of Escherichia coli, we fused cyanobacterial metallothioneins, SmtA (from Synechococcus elongatus PCC 3601) and MtnA (from Synechococcus vulcanus) to the E. coli cell surface using a Lpp'-OmpA-based display system. E. coli strains expressing Lpp'-OmpA-SmtA-linker-ChBD (chitin-binding domain from Bacillus pumillus SG2 chitinase S; chiS) and Lpp'-OmpA-MtnA-linker-ChBD on their surface adsorbed more cadmium compared to the E. coli cells expressing only the Lpp'-OmpA-linker-ChBD hybrid. These constructs also were bound to chitin through their chitin-binding domain, allowing them to be immobilized on a chitin matrix. We assessed surface presentation of Lpp'-OmpA-SmtA-linker-ChBD, Lpp'-OmpA-MtnA-linker-ChBD, and Lpp'-OmpA-linker-ChBD using immunostaining. The Lpp'-OmpA-SmtA-linker-ChBD chimera adsorbed metal and was bound to chitin with the highest efficiency compared to the other chimeras, suggesting that it is an effective bioadsorbent. This is the first example of coupling metal adsorption with cell immobilization using a whole-cell bioadsorbent.

Reactivity of polynuclear zinc‐thiolate sites

AbstractThe proton affinities of a series of mono‐ and polynuclear zinc‐thiolate compounds, mimicking zing finger protein and metallothioneins (MTs) sites, have been investigated by density functional theory calculations. This allows to evaluate the intrinsic nucleophilicity of synthetic and natural zinc‐bound thiolates and to compare their relative reactivities. The site specificity of the experimental thiolate alkylation for the Zn4Cys11 clusters in MTs is well reproduced, as well as the relative reactivities of ZnHis2Cys2, ZnHisCys3, and ZnCys4 zinc finger sites. Our results also show that terminal thiolates, bound to only one Lewis acid, are more reactive than bridging thiolates. Synthetic inorganic clusters and the Zn4Cys9His2 cluster found in a cyanobacterial MT are less reactive than Zn4Cys11 and Zn3Cys9 clusters in MTs. These results allow discussing the influence of the protein backbone and residues on the reactivity of these natural clusters. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Zinc‐Handling in Cyanobacteria: An Update

Zinc is a constituent of all six classes of enzymes, plays important roles in gene regulation, and is thought to be essential for most organisms. Despite initial discoveries of cyanobacterial metallothioneins, zinc efflux pumps and uptake systems, and zinc sensors, our knowledge of the zinc requirements, uptake, and detoxification mechanisms of cyanobacteria is still limited. Although cyanobacteria occupy extremely diverse habitats, most available data pertains to freshwater species, and almost no studies of zinc-handling mechanisms have been conducted in marine species. The current report highlights what is known about zinc homeostasis in cyanobacteria, and presents an analysis of the 40 sequenced cyanobacterial genomes.

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.

Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry

Metals bound to proteins play key roles in structure stabilization, catalysis, and metal transport in cells, but metals may also be toxic. As a consequence, cells have developed mechanisms to control metal concentrations through binding to proteins. We have used a hyphenated strategy linking gel electrophoresis with laser ablation-inductively coupled plasma-mass spectrometry in order to detect, map, and quantify metal-binding proteins synthesized in Escherichia coli under zinc- and cadmium-stress conditions. We report the development of a powerful analytical method suitable for detection and characterization of metalloproteins in complex, unfractionated bacterial cell extracts. The approach was validated by using an E. coli strain overexpressing the cyanobacterial metallothionein protein SmtA. We observed induction of SmtA synthesis by zinc and binding of both zinc and cadmium cations by this protein. A profile of zinc- and cadmium-binding proteins was obtained from E. coli cytoplasmic fractions. Analysis of induction patterns and metal contents demonstrated the presence of proteins with high metal content which, on further study, should lead to the identification of novel metal-binding proteins.

Inert site in a protein zinc cluster: isotope exchange by high resolution mass spectrometry.

Using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, we show that one Zn2+ ion in the Zn4 cluster of cyanobacterial metallothionein is inert to exchange with 67Zn2+. We suggest that this is zinc in site A, which together with the surrounding alpha and beta secondary structure forms a zinc-finger fold.

SmtB−DNA and Protein−Protein Interactions in the Formation of the Cyanobacterial Metallothionein Repression Complex: Zn2+ Does Not Dissociate the Protein−DNA Complex in Vitro

SmtB−DNA and Protein−Protein Interactions in the Formation of the Cyanobacterial Metallothionein Repression Complex: Zn²⁺ Does Not Dissociate the Protein−DNA Complex in Vitro

SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro.

The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.

Microbial metallothioneins

Bacterial metallothioneins bind, sequester and buffer excess intracellular zinc. At present, the vast majority of the available experimental data relate to cyanobacterial metallothionein, SmtA, from Synechococcus PCC 7942. SmtA is required for normal resistance to zinc and smtA-mediated zinc resistance has been used as a selectable marker. The imidazole groups of histidine residues, in addition to the thiol groups of cysteine residues, co-ordinate zinc in bacterial metallothioneins. The structure of bacterial metallothionein must facilitate some discrimination between 'adventitious' and 'adventageous' zinc-binding sites such that under excess zinc conditions metal is predominantly scavenged from the former. It remains unclear whether or not bacterial metallothionein also acts as a zinc store that supplies zinc-requiring proteins or if under some conditions it deactivates a subset of proteins via zinc removal. Expression of smtA is induced in response to elevated concentrations of zinc via the action of SmtB. SmtB has some sequence similarity to the arsenic responsive repressor ArsR and genes encoding related proteins are present in many bacterial genomes. Metal perception by SmtB differs from ArsR. The latter contains a characteristic Cys-Val-Cys motif associated with a DNA-binding helix-turn-helix (the ArsR motif), while the former contains metal-binding motifs associated with a carboxyl-terminal alpha-helix that forms the interface between SmtB dimers (the SmtB motif). Some SmtB-ArsR family proteins, including the zinc sensor ZiaR from the cyanobacterium Synechocystis PCC 6803, have the metal-sensory motifs of both SmtB and ArsR. The mechanisms of action, and the features that allow discrimination between different metal ions by these sensors, are discussed.