Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain. Zn(II) release activates DDAH-1

L-N(omega),N(omega)-dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.     

Zn(II) metabolism in prokaryotes

It is difficult to over-state the importance of Zn(II) in biology. It is a ubiquitous essential metal ion and plays a role in catalysis, protein structure and perhaps as a signal molecule, in organisms from all three kingdoms. Of necessity, organisms have evolved to optimise the intracellular availability of Zn(II) despite the extracellular milieu. To this end, prokaryotes contain a range of Zn(II) import, Zn(II) export and/or binding proteins, some of which utilise either ATP or the chemiosmotic potential to drive the movement of Zn(II) across the cytosolic membrane, together with proteins that facilitate the diffusion of this ion across either the outer or inner membranes of prokaryotes. This review seeks to give an overview of the systems currently classified as altering Zn(II) availability in prokaryotes.     

The crystal structure of Zn(II)-free Treponema pallidum TroA, a periplasmic metal-binding protein, reveals a closed conformation

We previously demonstrated that Treponema pallidum TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone. To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 A and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa). apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4 degrees tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix. This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 A, resulting in an unfavorable interaction of I251 with D66. To avoid this contact, D66 shifts towards H68, one of the four Zn(II)-coordinating residues. The approach of this negative charge coincides with the flipping of the imidazole side chain of H68, resulting in the formation of a new hydrogen bond. The conformational change of H68, along with a slight rearrangement of D279, a C-terminal domain Zn(II)-coordinating residue, distorts the metal-binding site geometry, presumably causing the release of the bound metal ion. Ligand binding and release by TroA, and presumably by other members of the MBP cluster, differs from the "Venus flytrap" mechanism utilized by bacterial nonmetal solute-binding receptors.     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

真菌吸附铅锌的研究

正随着采矿业的迅速发展,越来越多的重金属通过多种途径进入土壤环境中,对生态环境造成了不可估量的破坏并严重威胁人类健康。铅锌在工业上具有非常重要的作用且其应用极为广泛,而他们具有的难去除、难迁移和生物累积等特性使得铅锌在环境中的污染尤为突出。通过微生物的生长代谢,有效降低土壤重金属毒性,是促进植物生长的重要步骤之一。同时也要求微生物自身具有抵抗重金属的功能,根际微生     

Study of the coordination behaviour of (3,5-diphenyl-1H-pyrazol-1-yl)ethanol against Pd(II), Zn(II) and Cu(II)

A new easily synthetic route with a 96% yield of ligand 2-(3,5-diphenyl-1H-pyrazol-1-yl)ethanol (L) is obtained. The reactivity of L against Pd(II), Zn(II) and Cu(II) leads to [PdCl₂(L)₂] (1), [ZnCl₂(L)] (2) and [CuCl(L′)]₂ (3) (L′ is the ligand L without alcoholic proton), respectively. According to the different geometries imposed by the metallic centre and the capability of L to present various coordination links, it has been obtained complexes with square planar (1 and 3) or tetrahedral (2) geometry and different nuclearity: monomeric (1 and 2) or dimeric (3). Complete characterisation by analytical and spectroscopic methods, resolution of L and 1-3 by single-crystal X-ray diffraction and magnetic studies for complex 3 are presented.     

c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines.

We have investigated the mechanism by which deregulated expression of c-Myc induces death by apoptosis in serum-deprived fibroblasts. We demonstrate that Myc-induced apoptosis in low serum is inhibited by a restricted group of cytokines, principally the insulin-like growth factors and PDGF. Cytokine-mediated protection from apoptosis is not linked to the cytokines' abilities to promote growth. Protection from apoptosis is evident in the post-commitment (mitogen-independent) S/G2/M phases of the cell cycle and also in cells that are profoundly blocked in cell cycle progression by drugs. Moreover, IGF-I inhibition of apoptosis occurs in the absence of protein synthesis, and so does not require immediate early gene expression. We conclude that c-Myc-induced apoptosis does not result from a conflict of growth signals but appears to be a normal physiological aspect of c-Myc function whose execution is regulated by the availability of survival factors. We discuss the possible implications of these findings for models of mammalian cell growth in vivo.     

LHRH interaction with Zn(II) ions

Luteinizing hormone-releasing hormone (LHRH), a hypothalamic neurohormone, forms a complex with Zn ions in solution. In order to explain the structure of this complex, the stability constants of Zn(II) complexes of LHRH and also pyroglutamyl-histidine-methylester, N-acetyl-histamine, and N-acetyl-histidine were established with the use of potentiometric technique. The nuclear magnetic resonance spectroscopy shows that the mode of coordination of Zn(II) to LHRH consists of binding to the imidazole nitrogen and the peptide oxygen of the His-Trp bond.     

Glucose metabolism is inhibited by caspases upon the induction of apoptosis

Rapidly proliferating cells, such as cancer cells, have adopted aerobic glycolysis rather than oxidative phosphorylation to supply their energy demand; this phenomenon is known as ‘the Warburg effect''. It is now widely accepted that during apoptosis the loss of energy production, orchestrated by caspases, contributes to the dismantling of the dying cell. However, how this loss of energy production occurs is still only partially known. In the present work, we established that during apoptosis the level of cellular ATP decreased in a caspase-dependent manner. We demonstrated that this decrease in ATP content was independent of any caspase modification of glucose uptake, ATP consumption or reactive oxygen species production but was dependent on a caspase-dependent inhibition of glycolysis. We found that the activity of the two glycolysis-limiting enzymes, phosphofructokinase and pyruvate kinase, were affected by caspases, whereas the activity of phosphoglycerate kinase was not, suggesting specificity of the effect. Finally, using a metabolomic analysis, we observed that caspases led to a decrease in several key metabolites, including phosphoserine, which is a major regulator of pyruvate kinase muscle isozyme activity. Thus, we have established that during apoptosis, caspases can shut down the main energy production pathway in cancer cells, leading to the impairment in the activity of the two enzymes controlling limiting steps of glycolysis.The activation of caspase proteases is fundamental to apoptotic cell death. Once activated, ‘executioner'' caspases, such as caspase-3, orchestrate the rapid dismantling of the cell. Apoptosis is a process that requires energy. ATP is required for caspase activation, enzymatic hydrolysis of molecules, bleb formation and chromatin condensation.¹ However, in contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed ‘the Warburg effect''.² This phenomenon induces an increase of glucose consumption and provides the basis for the most sensitive and specific imaging technique available for the diagnosis and staging of solid cancers: positron emission tomography scan of 2-[¹⁸F] fluoro-2-deoxy-glucose uptake.Glycolysis is a series of metabolic processes, catalyzed by one of ten specific enzymes, by which 1 mole of glucose is catabolized to 2 moles of pyruvate and 2 moles of NADH with a net gain of 2 moles of ATP. Glycolysis is tightly regulated by the three allosteric enzymes, hexokinase (HK), phosphofructokinase-1 (PFK) and pyruvate kinase (PK), which catalyze the irreversible steps. HK, the first enzyme of glycolysis, phosphorylates glucose into glucose-6-phosphate, preventing the molecule from leaking out of the cell.The most complex control over glycolytic flux is attributed to PFK, which catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate using MgATP as the phosphoryl donor.³ PFK1 is stimulated by fructose-2,6-bisphosphate (F-2,6-BP), ADP/AMP and ammonium ions, whereas citrate and ATP act as strong inhibitors.Another limiting step is controlled by the final enzyme of the glycolytic pathway, PK. Four PK isoforms exist in mammals; the L and R isoforms are expressed in liver and red blood cells, respectively, whereas the M1 (muscle) isoform is expressed in most adult tissues, and tumor cells have been shown to mainly express the embryonic M2 isoform.⁴In the presence of oxygen, mitochondria can oxidize pyruvate and NADH, resulting in the production of 36 moles of ATP (OXPHOS). However, even under normoxic conditions, most cancer cells will not perform OXPHOS but will instead reduce pyruvate to lactate. Although, aerobic glycolysis is an inefficient way to generate ATP, aerobic glycolysis seems to confer certain advantages to cancer cells, such as the ability to generate several intermediates that can be used by other metabolic pathways to produce nucleotides or lipids.⁵ However, the exact nature of the benefits conferred by glycolysis is still under debate.It is well established that caspase activation relies on ATP to proceed. However, it has been previously suggested that upon induction of apoptosis, ATP levels dramatically fall in a caspase-dependent manner.⁶ In the present report, we explored the role of caspases on glycolysis, the main energy-producing pathway used by cancer cells.     

Adsorption of Cu(II), Ni(II) and Zn(II) on modified jute fibres

The potential of a lignocellulosic fibre, jute, was assessed for adsorption of heavy metal ions like Cu(II), Ni(II) and Zn(II) from their aqueous solutions. The fibre was also used as adsorbent after chemically modifying it by two different techniques viz, loading of a dye with specific structure, C.I. Reactive Orange 13, and oxidising with hydrogen peroxide. Both the modified jute fibres gave higher metal ion adsorption. Thus, the dye loaded jute fibres showed metal ion uptake values of 8.4, 5.26 and 5.95 mg/g for Cu(II), Ni(II) and Zn(II), respectively, while the corresponding values for oxidised jute fibres were 7.73, 5.57 and 8.02 mg/g, as against 4.23, 3.37 and 3.55 mg/g for unmodified jute fibres. Adsorption isotherm models indicated best fit for Langmuir model for the modified jute fibres. The adsorption values decreased with lowering of pH. The desorption efficiency, regenerative and reuse capacity of these adsorbents were also assessed for three successive adsorption-desorption cycles. The adsorptive capacity was retained only when the caustic soda regeneration is carried out as an intermediate step after desorption. Possible mechanism has been given.     

Asexual growth of Babesia bovis is inhibited by specific sulfated glycoconjugates

In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.     

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1 . The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae . The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Protein damage by photo-activated Zn(II) N-alkylpyridylporphyrins

Destruction of unwanted cells and tissues in photodynamic therapy (PDT) is achieved by a combination of light, oxygen, and light-sensitive molecules. The advantages of PDT compared to other traditional treatment modalities, and the shortcomings of the currently used photosensitizers, have stimulated the search for new, more efficient photosensitizer candidates. Ability to inflict selective damage to particular proteins through photo-irradiation would significantly advance the design of highly specific photosensitizers. Achieving this objective requires comprehensive knowledge concerning the interactions of the particular photosensitizer with specific targets. Here, we summarize the effects of Zn(II) N-alkylpyridylporphyrin-based photosensitizers on intracellular (metabolic, antioxidant and mitochondrial enzymes) and membrane proteins. We emphasize how the structural modifications of the porphyrin side substituents affect their lipophilicity, which in turn influence their subcellular localization. Thus, Zn(II) N-alkylpyridylporphyrins target particular cellular sites and proteins of interest, and are more efficient than hematoporphyrin D, whose commercial preparation (Photofrin) has been clinically approved for PDT.     

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA,the soluble component of the ZnuABC transporter

BackgroundUnder conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake.MethodsAntibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15 Å resolution.ResultsTwo di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound.ConclusionsTwo molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella.General SignificanceThe data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.     

Cu(II) organizes beta-2-microglobulin oligomers but is released upon amyloid formation

beta-2-Microglobulin (beta2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause beta2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with beta2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature beta2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede beta2m amyloid formation.     

Effects of Zn(II) on galactosyltransferase activity

The enzyme -4-galactosyltransferase (GT) catalyzes the transfer of a galactosyl group from UDP-galactose to N-acetylglucosamine (GlcNAc) on glycoproteins. In the presence of -lactalbumin (-LA), galactosyltransferase catalyzes the transfer of galactose to glucose to yield lactose. It is known that, in the absence of -lactalbumin, Zn(II) competes with Mn(II) for the same binding site(s) in galactosyltransferase, resulting in an increase in the apparent Michaelis constant,K _m (app), for Mn(II)-activation of N-acetyllactosamine synthesis. In the presence of -lactalbumin (i.e., lactose synthase), the Mn(II)-activation is biphasic and the initial phase is inhibited by increasing concentrations of Zn(II). The Zn(II) inhibition of lactose synthase plateaus at [Zn(II)]:[-lactalbumin] 1:1, while for N-acetyllactosamine synthesis there is no plateau at all. The results suggest that Zn(II) binding to -lactalbumin effects lactose synthase. Kinetically, Zn(II) induces a decrease in both theK _m (app) andV _m for Mn(II), which results in an apparent increase, followed by a decrease, in lactose synthase activity at Mn(II) concentrations below saturation of the first [Mn(II)] binding site. Increasing Zn(II) also decreasesK _m (app) andV _m for both glucose and UDP-galactose in the lactose synthase reaction with either both Ca(II)- or apo--lactalbumin, further suggesting novel interactions between Zn(II)--lactalbumin and the lactose synthase complex, presumably mediated via a Zn(II)-induced conformational change upon binding to -lactalbumin. On the other hand, in N-acetyllactosamine synthesis, Zn(II) only slightly effectsK _m (app) for N-acetylglucosamine and has essentially no effect onK _m (app) orV _m for UDP-galactose.On leave from the Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia     

Biosorption of Zn(II) by Thiobacillus ferrooxidans

There have been a number of studies considering the possibility of removing and recovering heavy metals from diluted solutions. These are due, principally, because of the commercial value of some metals as well as in the environmental impact caused by them. The traditional methods for removing have several disadvantages when metals are present in concentrations lower than 100 mg/l. Biosorption, which uses biological materials as adsorbents, has been considered as an alternative method. In this work, variables like pH and biomass chemical pretreatment have been studied for its effect on the capacity for zinc biosorption by Thiobacillus ferrooxidans. Also, studies to determinate the time for zinc adsorption were carried out. Results indicate that a capacity as high as 82.61 mg of Zn(II)/g of dry biomass can be obtained at a temperature of 25v°C and that the biosorption process occurs in a time of 30 min.