Enhancement of α- and β-Galactosidase Activity in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> by Different Metal Ions

The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose. α-Galactosidase can digest oligosaccharides like raffinose, while β-galactosidases can hydrolyze lactose. Therefore, selection of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective of this study was to investigate the enhancement of α- and β-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na⁺, K⁺, Fe²⁺, and Mg²⁺ and 1 mM of Mn²⁺ were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest α- and β-galactosidase activity when grown in the medium with added Mn²⁺ ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn²⁺ ions lead to 27, 18% enhancement of α- and β-galactosidase activity over the control group, and therefore, it could be added to the growth culture of CF2-7F to produce enhanced levels of α- and β-galactosidase activity. The addition of Fe²⁺ led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that 0.0274% Mn²⁺ can be used to promote the production for α- and β-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of α- and β-galactosidase activity and have a good potential to be used in the food industry.     

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease

19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K _d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K _d  ≈ 2 mM. Mn(II) paramagnetic broadening in ¹⁹F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999     

Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?

 The interaction of human carbonic anhydrase (hCA) isozymes I and II with cyanamide, a linear molecule isoelectronic with the main physiological substrate of the enzyme, CO₂, was investigated through spectroscopic, kinetic, and X-ray crystallographic studies. We show here that cyanamide is hydrated to urea in the presence of CAs, and that it also acts as a weak non-competitive inhibitor (K _I=61±3 mM and 238±9 mM for hCA II and hCA I, respectively) towards the esterasic activity of these enzymes, as tested with 4-nitrophenyl acetate. Changes in the spectrum of the Co(II)-hCA II derivative observed in the presence of cyanamide suggest that it likely binds the metal ion within the CA active site, adding to the coordination sphere, not substituting the metal-bound solvent molecule. It thereafter undergoes a nucleophilic attack from the metal-bound hydroxide ion, forming urea which remains bound to the metal, as observed in the X-ray crystal structure of hCA II soaked in cyanamide solutions for several hours. The urea molecule is directly coordinated to the active site Zn(II) ion through a protonated nitrogen atom. Several hydrogen bonds involving active site residues Thr199 and Thr200 as well as three water molecules (Wat99, Wat122, and Wat123) further stabilize the urea-hCA II adduct. Kinetic studies in solution further proved that urea acts as a tight binding inhibitor of the two isozymes hCA I and hCA II, with very slow binding kinetics (k _on=2.5×10^–5s^–1M^–1). A mechanism to explain the hydration process of cyanamide by CAs, as well as the tight binding of urea in the active site, is also proposed based on the hypothesis that urea is deprotonated when bound to the enzyme. Cyanamide is thus the first true suicide substrate of this enzyme for which binding has been documented by means of X-ray crystallographic and spectroscopic studies. Received: 26 February 1999 / Accepted: 25 May 1999     

Identification of Phosphate Binding Residues of <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP Synthase

Four positively-charged residues, namely βLys-155, βArg-182, βArg-246, and αArg-376 have been identified as Pi binding residues in Escherichia coli ATP synthase. They form a triangular Pi binding site in catalytic site βE where substrate Pi initially binds for ATP synthesis in oxidative phosphorylation. Positive electrostatic charge in the vicinity of βArg-246 is shown to be one important component of Pi binding.     

Structural Analysis of Divalent Metals Binding to the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Response Regulator Spo0F: The Possibility for <Emphasis Type="Italic">In Vitro</Emphasis> Metalloregulation in the Initiation of Sporulation

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and μESI-MS. NMR studies show that the divalent metals Ca²⁺, Mg²⁺ and Mn²⁺ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu²⁺ show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the β4-α4 loop and the β5/α5 interface, particularly around and including H101. μESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu²⁺, multiple metal-bound species are observed. Subsequent experiments reveal that Mg²⁺ supports phosphotransfer between KinA and Spo0F, while Cu²⁺ fails to support KinA phosphotransfer. Additionally, the presence of Cu²⁺ at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.     

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

 The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted ¹H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo. Received: 8 October 1997 / Accepted: 30 December 1997     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter

OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO₄ are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the μOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO₄ is interpreted in terms of a role of the μOH as a reaction nucleophile.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

The use of free and immobilized <Emphasis Type="Italic">Cunninghamella elegans</Emphasis> for removing cobalt ions from aqueous waste solutions

This paper discusses the possible application to use free and immobilized Cunninghamella elegans for the removal of cobalt from aqueous waste solutions. Results indicated that the maximum uptake occurred at; pH 4.0–5.5 ± 0.2, temperature range between 15 and 50°C and stirring rate 250 rpm. The uptake increased with the increase of metal ion concentration up to 40 ppm. Also, it was found that the best biomass weights used for biosorption were 0.25 and 0.5 g for both free and immobilized biomass. The reuse of control alginate beads, alive and dead immobilized Cunninghamella elegans beads was investigated for five cycles. Results showed that the percent uptake decreased slightly after the first cycle. While, in the case of alginate beads there was increase in the second cycle then returned to the same level of uptake. The uptake of cobalt in the presence of Cr(VI) and Cd(II) at different mixture concentrations 40, 50 and 60 ppm was investigated. The results showed that the uptake amount of Co(II) in the presence of other metal ions was lower than Co(II) alone except for Ca-alginate beads. SEM studies for control alginate beads, alive and dead immobilized Cunninghamella elegans beads were conducted to investigate the beads before and after the accumulation of cobalt ions.     

Structural and functional characterization of <Emphasis Type="Italic">Delphinus delphis</Emphasis> hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC–electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major β (β1 16,022 Da, β2 16,036 Da, β3 16,036 Da, labeled according to their progressive elution times) and two major α globins (α1 15,345 Da, α2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that β2 globin differs from β1 for the substitution Val126 → Leu, while β3 globin differs from β2 for the isobaric substitution Lys65 → Gln. The α2 globin differs from the α1 for the substitution Ser15 → Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC–ESI-MS analysis revealed that the fraction with higher pI (HbI) contained β1, β2 and both the α globins, and the fraction with lower pI (HbII) contained β3 and both the α globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA₀, with HbII showing the more pronounced differences. With respect to HbA₀, either the substitution Proβ5 → Gly or the Proβ5 → Ala is present in all the cetacean β globins sequenced so far, and it has been hypothesized that position 5 of β globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysβ65 → Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA₀.     

Cobalt-substituted derivatives ofCarcinus hemocyanin

Summary Four derivatives ofCarcinus maenas hemocyanin containing Co(II) in the active site have been prepared under different experimental conditions. Two of them contain one Co(II) ion/active site and most probably represent isomeric forms containing Co(II) either in the fast-reacting or in the slow-reacting position within the active site. A third derivative contains two Co(II) ions active site, which reproduces the metal/protein stoichiometry of native hemocyanin. The fourth derivative is a metal hybrid form containing one Cu(I) ion and one Co(II) ion/active site. The derivatives have been characterized by absorption, circular dichroic and fluorescence spectroscopies. The results indicate that in all derivatives the metal is bound with a low coordination number, in agreement with the presence of three histidine residues/copper ion in the native protein. The two alternative metal-binding positions have different structures as shown by the different spectroscopic properties of the bound Co(II) ions. A marked hyperchromic effect on the optical absorption of Co(II) is observed as a result of the presence of a metal ion in the neighbouring metal-binding position in the active site.     

Syntheses and crystal structures of mono-, di- and trinuclear cobalt complexes of a salen type ligand

Three cobalt complexes containing the salen type ligand, bis(salicylidene)-meso-1,2-diphenylethylenediaminato (mdpSal²⁻), are reported. The complexes differ in nuclearity and include the mononuclear, Co(mdpSal) (1), which contains a Co(II) metal center bound to one mdpSal⁻² ligand frame in a square planar geometry. The second complex is the dinuclear [Co(mdpSal)Cl]₂ (2) in which both cobalt ions have been oxidized to the +3 oxidation state. The overall geometry of complex 2 is an edge-sharing bioctahedron with the coordination sphere around each cobalt metal center consisting of one mdpSal⁻² ligand and one Cl⁻ ion. The shared edge between the Co(III) ions contains two bridging phenolate groups, one from each ligand frame. Complex 3 is a linear, mixed valence, trinuclear species, [Co(mdpSal)(OAc)(μ-OAc)]₂Co, with the oxidation states of the metal centers assigned as Co(III)-Co(II)-Co(III). The terminal Co(III) centers are equivalent with the central Co(II) lying on the inversion center of the molecule. Each cobalt ion in 3 adopts an octahedral geometry with the terminal Co(III) ions being bound to one mdpSal²⁻ ligand each. All phenolate groups bridge to the central Co(II). The coordination sphere about each metal center in the trinuclear complex is completed by four acetate groups, two of which bind in a μ-fashion bridging from the terminal Co(III) metal centers to the central Co(II). The complexes have been characterized by X-ray crystallography as well as UV-Vis and IR spectroscopy.     

<Emphasis Type="Italic">π</Emphasis>–<Emphasis Type="Italic">π</Emphasis> interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, π–π interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the π–π interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell π–π interaction. We suggest that the π–π interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability

The effect of metal ions on the activity, the donor substrate specificity, and the stability in organic solvents of Helicobacter pylori α-1,4 fucosyltransferase were studied. The recombinant enzyme was expressed as soluble form in E. coli strain AD494 and purified in a one step affinity chromatography. Its activity was highest in cacodylate buffer at pH 6.5 in the presence of 20 mM Mn²⁺ ions at 37°C. Mn²⁺ ions could be substituted by other metal ions. In all cases, Mn²⁺ ions proofed to be the most effective (Mn²⁺ > Co²⁺ > Ca²⁺ > Mg²⁺ > Cu²⁺ > Ni²⁺ > EDTA). The enzyme shows substrate specificity for Type I disaccharide (1) with a K _M of 114 μM. In addition, the H. pylori α-1,4 fucosyltransferase efficiently transfers GDP-activated l-fucose derivatives to Galβ1-3GlcNAc-OR (1). Interestingly, the presence of organic solvents such as DMSO and methanol up to 20% in the reaction medium does not affect significantly the enzyme activity. However, at the same concentration of dioxane, activity is totally abolished.     

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k _cat of the Fe(III)Ni(II) derivative (approximately 60 s⁻¹) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Release of Metal Ions from Orthodontic Appliances: An In Vitro Study

In this paper, we report the results of an in vitro experiment on the release of metal ions from orthodontic appliances composed of alloys containing iron, chromium, nickel, silicon, and molybdenum into artificial saliva. The concentrations of magnesium, aluminum, silicon, phosphorus, sulfur, potassium, calcium, titanium, vanadium, manganese, iron, cobalt, copper, zinc, nickel, and chromium were significantly higher in artificial saliva in which metal brackets, bands, and wires used in orthodontics were incubated. In relation to the maximum acceptable concentrations of metal ions in drinking water and to recommended daily doses, two elements of concern were nickel (573 vs. 15 μg/l in the controls) and chromium (101 vs. 8 μg/l in the controls). Three ion release coefficients were defined: α, a dimensionless multiplication factor; β, the difference in concentrations (in micrograms per liter); and γ, the ion release coefficient (in percent). The elevated levels of metals in saliva are thought to occur by corrosion of the chemical elements in the alloys or welding materials. The concentrations of some groups of dissolved elements appear to be interrelated.     

Identification of a Novel Vanadium-binding Protein by EST Analysis on the Most Vanadium-rich Ascidian, Ascidia gemmata

Ascidians are known to accumulate extremely high levels of vanadium in their blood cells (up to 350 mM). The branchial sac and the intestine are thought to be the first tissues to contact the outer environment and absorb vanadium ions. The concentration of vanadium in the branchial sac and the intestine of the most vanadium-rich ascidian Ascidia gemmata were determined to be 32.4 and 11.9 mM, respectively. Using an expressed sequence tag (EST) analysis of a cDNA library from the intestine of A. gemmata, we determined 960 ESTs and found 55 clones of metal-related gene orthologs, 6 redox-related orthologs, and 18 membrane transporter orthologs. Among them, two genes, which exhibited significant similarity to the vanadium-binding proteins of other vanadium-rich ascidian species, were designated AgVanabin1 and AgVanabin2. Immobilized metal ion affinity chromatography revealed that recombinant AgVanabin1 bound to metal ions with an increasing affinity for Cu(II) > Zn(II) > Co(II) and AgVanabin2 bound to metal ions with an increasing affinity for Cu(II) > Fe(III) > V(IV). To examine the use of AgVanabins for a metal absorption system, we constructed Escherichia coli strains that expressed AgVanabin1 or AgVanabin2 fused to maltose-binding protein and secreted into the periplasmic space. We found that the strain expressing AgVanabin2 accumulated about 13.5 times more Cu(II) ions than the control TB1 strain. Significant accumulation of vanadium was also observed in the AgVanabin2-expressing strain as seen by a 1.5-fold increase.