Oxyanion Binding Research Articles

Overexpression of a Designed Mutant Oxyanion Binding Protein ModA/WtpA in Acidithiobacillus ferrooxidans for the Low pH Recovery of Molybdenum and Rhenium.

Molybdenum and rhenium are critically important metals for a number of emerging technologies. We identified and characterized a molybdenum/tungsten transport protein (ModA/WtpA) of Acidithiobacillus ferrooxidans and demonstrated the binding of tungstate, molybdate, and chromate. We used computational design to expand the binding capabilities of the protein to include perrhenate. A disulfide bond was engineered into the binding pocket of ModA/WtpA to introduce a more favorable geometric coordination and surface charge distribution for oxyanion binding. The mutant protein experimentally demonstrated a 2-fold higher binding affinity for molybdate and 6-fold higher affinity for perrhenate. The overexpression of the wild-type and mutant ModA/WtpA proteins in A. ferrooxidans cells enhanced the innate tungstate, molybdate, and chromate binding capacities of the cells to up to 2-fold higher. In addition, the engineered cells expressing the mutant protein exhibited enhanced perrhenate binding, showing 5-fold and 2-fold higher binding capacities compared to the wild-type and ModA/WtpA-overexpressing cells, respectively. Furthermore, the engineered cell lines enhanced biocorrosion of stainless steel as well as the recovered valuable metals from an acidic wastewater generated from molybdenite processing. The improved binding efficiency for the oxyanion metals, along with the high selectivity over nontargeted metals under mixed metal environments, highlights the potential value of the engineered strains for practical microbial metal reclamation under low pH conditions.

Mechanistic Insights into the Selectivity for Arsenic over Phosphate Adsorption by Fe3+-Cross-Linked Chitosan Using DFT.

Fe3+-cross-linked chitosan exhibits the potential for selectively adsorbing arsenic (As) over competing species, such as phosphate, for water remediation. However, the effective binding mechanisms, bond nature, and controlling factor(s) of the selectivity are poorly understood. This study employs ab initio calculations to examine the competitive binding of As(V), P(V), and As(III) to neat chitosan and Fe3+-chitosan. Neat chitosan fails to selectively bind As oxyanions, as all three oxyanions bind similarly via weak hydrogen bonds with preferences of P(V) = As(V) > As(III). Conversely, Fe3+-chitosan selectively binds As(V) over As(III) and P(V) with binding energies of -1.9, -1, and -1.8 eV for As(V), As(III), and P(V), respectively. The preferences are due to varying Fe3+-oxyanion donor-acceptor characteristics, forming covalent bonds with distinct strengths (Fe-O bond ICOHP values: - 4.9 eV/bond for As(V), - 4.7 eV/bond for P(V), and -3.5 eV/bond for As(III)). Differences in pKa between As(V)/P(V) and As(III) preclude any preference for As(III) under typical environmental pH conditions. Furthermore, our calculations suggest that the binding selectivity of Fe3+-chitosan exhibits a pH dependence. These findings enhance our understanding of the Fe3+-oxyanion interaction crucial for preferential oxyanion binding using Fe3+-chitosan and provide a lens for further exploration into alternative transition-metal-chitosan combinations and coordination chemistries for applications in selective separations.

Does Charge Influence the Response in Oxyanion Binding Europium(III) Complexes?

AbstractThrough the synthesis of a positively charged europium complex, with a structure derived from the DO2A (1, 4, 7, 10‐tetraazacyclododecane‐1, 7‐diacetic acid), we were able to explore the influence of charge on the interaction between coordinatively unsaturated lanthanide complexes and charged oxyanions. The data suggest that the positive charge of the lanthanide host does not allow better selectivity between the differently and negatively charged oxyanion guests. Although, binding constants seem greatly improved with charge for the α‐hydroxycarboxylates investigated here.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Oxoanion Imprinting Combining Cationic and Urea Binding Groups: A Potent Glyphosate Adsorber.

The use of polymerizable hosts in anion imprinting has led to powerful receptors with high oxyanion affinity and specificity in both aqueous and non-aqueous environments. As demonstrated in previous reports, a carefully tuned combination of orthogonally interacting binding groups, for example, positively charged and neutral hydrogen bonding monomers, allows receptors to be constructed for use in either organic or aqueous environments, in spite of the polymer being prepared in non-competitive solvent systems. We here report on a detailed experimental design of phenylphosphonic and benzoic acid-imprinted polymer libraries prepared using either urea- or thiourea-based host monomers in the presence or absence of cationic comonomers for charge-assisted anion recognition. A comparison of hydrophobic and hydrophilic crosslinking monomers allowed optimum conditions to be identified for oxyanion binding in non-aqueous, fully aqueous, or high-salt media. This showed that recognition improved with the water content for thiourea-based molecularly imprinted polymers (MIPs) based on hydrophobic EGDMA with an opposite behavior shown by the polymers prepared using the more hydrophilic crosslinker PETA. While the affinity of thiourea-based MIPs increased with the water content, the opposite was observed for the oxourea counterparts. Binding to the latter could however be enhanced by raising the pH or by the introduction of cationic amine- or Na+-complexing crown ether-based comonomers. Use of high-salt media as expected suppressed the amine-based charge assistance, whereas it enhanced the effect of the crown ether function. Use of the optimized receptors for removing the ubiquitous pesticide glyphosate from urine finally demonstrated their practical utility.

Allosteric Regulation of 3CL Protease of SARS-CoV-2 and SARS-CoV Observed in the Crystal Structure Ensemble

The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)–disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.

Kinetics of Cation and Oxyanion Adsorption and Desorption on Ferrihydrite: Roles of Ferrihydrite Binding Sites and a Unified Model.

Quantitative understanding the kinetics of toxic ion reactions with various heterogeneous ferrihydrite binding sites is crucial for accurately predicting the dynamic behavior of contaminants in environment. In this study, kinetics of As(V), Cr(VI), Cu(II), and Pb(II) adsorption and desorption on ferrihydrite was studied using a stirred-flow method, which showed that metal adsorption/desorption kinetics was highly dependent on the reaction conditions and varied significantly among four metals. High resolution scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed that all four metals were distributed within the ferrihydrite aggregates homogeneously after adsorption reactions. Based on the equilibrium model CD-MUSIC, we developed a novel unified kinetics model applicable for both cation and oxyanion adsorption and desorption on ferrihydrite, which is able to account for the heterogeneity of ferrihydrite binding sites, different binding properties of cations and oxyanions, and variations of solution chemistry. The model described the kinetic results well. We quantitatively elucidated how the equilibrium properties of the cation and oxyanion binding to various ferrihydrite sites and the formation of various surface complexes controlled the adsorption and desorption kinetics at different reaction conditions and time scales. Our study provided a unified modeling method for the kinetics of ion adsorption/desorption on ferrihydrite.

Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO42−, WO42− and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

An Improved Model of the Trypanosoma brucei CTP Synthetase Glutaminase Domain-Acivicin Complex.

The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1‐Å‐resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure‐based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.

Arsenic oxyanion binding to NOM from dung and aquaculture pond sediments in Bangladesh: Importance of site-specific binding constants

Groundwater contamination by arsenic (As) is a serious public health concern in many different areas worldwide, particularly in the Bengal region. Mobilization and fate of As in natural waters is controlled by a variety of factors including the presence of natural organic matter (NOM). This study experimentally determined conditional distribution coefficients (with KD defined as the ratio between As bound to NOM and truly dissolved As) and apparent stability constants between AsIII oxyanions and NOM from cow dung, chicken dung, and Bangladeshi aquaculture pond sediment prior to and after one year of operation. As-sorption experiments with cow dung as the source of NOM resulted in the highest range for log KD, from 4.7 to 6.3. Pond sediment from Bangladesh after a year of operation for fish production showed greater affinity for binding As oxyanions than fresh sediment prior to fish production. PHREEQCI modeling using constants derived from the experiments along with water chemistry parameters typical for the site supports the dominance of AsIII oxyanion-NOM complexation in this system. Models employing constants from previous studies using purified NOM considerably underestimate observed complexation by environmental NOM; thus, applying site-specific constants to geochemical models will better predict As speciation for the field site.

Dual Guest [(Chloride)3-DMSO] Encapsulated Cation-Sealed Neutral Trimeric Capsular Assembly: Meta-Substituent Directed Halide and Oxyanion Binding Discrepancy of Isomeric Neutral Disubstituted Bis-Urea Receptors

A logically synthesized ortho-phenylenediamine based chloro-methyl disubstituted neutral organic bis-urea receptor L1 with the aid of three symmetry-independent units encapsulates an unusual triangular [(chloride)3-DMSO] guest assembly (complex 1a) via formation of DMSO + host + salt cocrystals within its trimeric paddle-wheel shaped cavity sealed by three n-TBA cations and exhibits diverse anion binding properties with oxyanions along with the chloride complex of its isomeric bromo-methyl disubstituted bis-urea receptor L2. Receptor L2 and L1 both form a similar kind of noncapsular 2:2 host–guest assembly in the presence of excess chloride (complex 2a) and acetate (complex 1b) respectively by noncooperative H-bonding interactions of urea groups which are attributed to the effect of meta-functionalization with respect to the adjacent N–H part of the urea moiety, whereas another planar oxyanion carbonate is doubly encapsulated within the tetrameric capsular cavity of L1 in the solid state (complex 1c). Mor...

A C3v-Symmetric Tripodal Urea Receptor for Anions and Ion Pairs: Formation of Dimeric Capsular Assemblies of the Receptor during Anion and Ion Pair Coordination

A new C(3v)-symmetric urea-based heteroditopic tripodal receptor capable of recognizing both anions and ion pairs was designed, synthesized, and characterized. The protonated receptor forms a sulfate complex which encapsulates a single DMF in the tripodal cavity of the receptor. However, the SO4(2-) anion is located outside the tripodal cavity and is stabilized by N-H···O hydrogen bonds from the urea functions of four receptor cations. With TBAHSO4 the receptor forms a contact ion pair complex, where both the TBA(+) and SO4(2-) groups are pseudoencapsulated in the tripodal cavity of the protonated receptor. Significantly, the receptor forms a charge-separated polymeric ion pair complex with K(+) and HPO4(2-) via formation of a dimeric capsular assembly of the receptor, in which three K(+) encapsulated dimeric capsular assemblies interdigitate to form a precise cavity that further encapsulates HPO4(2-). The receptor also forms an anion complex with CO3(2-) via formation of dimeric capsular self-assembly of the receptor. Solution-state binding studies of the receptor with oxyanions have also been carried out by (1)H NMR titration experiments, which show the oxyanion binding trend HCO3(-) > H2PO4(-) > HSO4(-), whereas no binding with NO3(-) and ClO4(-) anions is observed.

Activation of MgADP-inactivated chloroplast F1-ATPase depends on oxyanion binding to noncatalytic sites

Activation of MgADP-inactivated chloroplast F1-ATPase depends on oxyanion binding to noncatalytic sites

Specific adsorption of tungstate by cell surface display of the newly designed ModE mutant

By cell surface display of ModE protein that is a transcriptional regulator of operons involved in the molybdenum metabolism in Escherichia coli, we have constructed a molybdate-binding yeast (Nishitani et al., Appl Microbiol Biotechnol 86:641-648, 2010). In this study, the binding specificity of the molybdate-binding domain of the ModE protein displayed on yeast cell surface was improved by substituting the amino acids involved in oxyanion binding with other amino acids. Although the displayed S126T, R128E, and T163S mutant proteins adsorbed neither molybdate nor tungstate, the displayed ModE mutant protein (T163Y) abolished only molybdate adsorption, exhibiting the specific adsorption of tungstate. The specificity of the displayed ModE mutant protein (T163Y) for tungstate was increased by approximately 9.31-fold compared to the displayed wild-type ModE protein at pH 5.4. Therefore, the strategy of protein design and its cell surface display is effective for the molecular breeding of bioadsorbents with metal-specific adsorption ability based on a single species of microorganism without isolation from nature.

Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms

Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.

Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases

The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C-C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C-C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C-C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.

Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

BackgroundThe dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.MethodsAlanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined.ResultsKinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole.ConclusionsKinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

The oxyanion hole ofPseudomonas fluorescensmannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites

The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca2+-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

Synthesis and anion-binding properties of new disulfonamide-based receptors

The synthesis of disulfonamide receptor scaffolds for anion binding is reported. Acyclic receptors are found to tightly bind acetate in MeCN- d 3 with dominant 1:1 stoichiometry, a smaller, sequential 1:2 (H+G) association is also found. Constraint of the disulfonamide receptor into macrocycles serves to eliminate the 1:2 binding stoichiometry and X-ray crystal structures of several macrocyclic receptors allow rationalisation of their affinity for acetate binding. l-Valine derived macrocycles maintain tight 1:1 binding of acetate ( K a 1:1 >10 4 M −1) in MeCN- d 3 and display preference for oxyanion binding in more competitive MeCN- d 3/2% H 2O.