Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

NMR study of the complexation of D-gulonic acid with tungsten(VI) and molybdenum(VI)

By using multinuclear (1H, 13C, 17O, 95Mo, 183W) magnetic resonance spectroscopy (1D and 2D), D-gulonic acid is found to form ten and seven complexes, respectively, with tungsten(VI) and molybdenum(VI), in aqueous solution, depending on pH and metal-ligand molar ratios. Two isomeric 1:2 (metal-ligand) complexes involving the carboxylate and the adjacent OH group are present in the pH range 2-9. At intermediate and high pH, molybdate forms a 2:1 tetradentate complex involving the four secondary hydroxyl groups, whereas tungstate forms one 2:1 terdentate species. At low and intermediate pH values, three 2:1 complexes are found for both metals, involving the carboxylate group and three secondary hydroxyl groups, as well as a 5:2 species involving the carboxylate group and all the secondary hydroxyl groups; the concentration of this species increases in time mainly at the expense of 2:1 and 1:2 complexes. Tungstate can also form two additional species, probably a 5:2 species involving the carboxylate group and all the hydroxyl groups, and a 2:1 pentadentate species involving the carboxylate group and all the secondary hydroxyl groups. In alkaline solutions, tungstate is able to form an additional 2:1 pentadentate complex involving all the hydroxyl groups.     

Substrate specificity of ribose-5-phosphate isomerases from <Emphasis Type="Italic">Clostridium difficile</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for d-ribose isomerization was optimal at pH 7.5 and 40°C, while that from Thermotoga maritima for l-talose isomerization was optimal at pH 8.0 and 70°C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, l-lyxose, d-gulose, and l-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.     

Screening of biologically active monosaccharides: growth inhibitory effects of d-allose,d-talose,and l-idose against the nematode Caenorhabditis elegans

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.     

Crystal structures, antioxidation and DNA binding properties of Eu(III) complexes with Schiff-base ligands derived from 8-hydroxyquinoline-2-carboxyaldehyde and three aroylhydrazines

Eu(III) and every newly synthesized ligand can form a binuclear Eu(III) complex with a 1:1 metal to ligand stoichiometry and nine-coordinate at Eu(III) center. Every ligand acts as a dibasic tetradentate ligand, binding to Eu(III) through the phenolate oxygen atom, nitrogen atom of quinolinato unit, the CN group (methylene) and ⁻O-CN- group (enolized and deprotonated from OC-NH- group) of the aroylhydrazine side chain. One DMF (N,N-dimethylformamide) molecule is binding orthogonally to the ligand-plane from one side to the metal ion, while another DMF and a nitrate anion (bidentate) are binding from the other. Dimerization of the monomeric unit occurs through the phenolate oxygen atoms leading to a central planar four-membered (EuO)₂ ring. On the other hand, all the ligands and Eu(III) complexes may be used as potential anticancer drugs, binding to Calf thymus DNA through intercalations at the order of magnitude 10⁵-10⁷ M⁻¹. All the ligands and Eu(III) complexes are strong scavengers of hydroxyl radicals and superoxide radicals, but Eu(III) complex containing active phenolic hydroxyl group shows stronger scavenging effects for hydroxyl radicals than others, and Eu(III) complex containing N-heteroaromatic substituent shows stronger scavenging effects for superoxide radicals than others.     

Crystal structures, antioxidation and DNA binding properties of Yb(III) complexes with Schiff-base ligands derived from 8-hydroxyquinoline-2-carbaldehyde and four aroylhydrazines

X-ray crystal and other structural analyses indicate that Yb(III) and all four newly synthesized ligands can form a binuclear Yb(III) complex with a 1:1 metal to ligand stoichiometry by octacoordination at the Yb(III) center. Investigations of DNA binding properties show that all the ligands and Yb(III) complexes can bind to Calf thymus DNA through intercalations with the binding constants at the order of magnitude 10⁵–10⁷ M⁻¹, but Yb(III) complexes present stronger affinities to DNA than ligands. All the ligands and Yb(III) complexes may be used as potential anticancer drugs. Investigations of antioxidation properties show that all the ligands and Yb(III) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals but Yb(III) complexes show stronger scavenging effects for hydroxyl radicals than ligands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Selective benzoylation of some N-acetyl-N-aryl-β-D-xylopyranosylamines

Selectivity in the benzoylation of N-acetyl-N-p-methoxyphenyl-, -p-bromophenyl-, and -p-chlorophenyl-β-D-xylopyranosylamines has been demonstrated. The structures of the products was shown by using periodate oxidation and n.m.r. spectroscopy. The relative reactivity of the hydroxyl groups was HO-3 ≈ HO-4 > HO-2. The β-D-xylopyranosylamine derivatives were shown to possess the ⁴C¹ conformation.     

Effects of Temperature,Nucleotides and Sodium Molybdate on Activation and DNA Binding of Estrogen,Glucocorticoid, Progesterone and Androgen Receptors In MCF-7 Human Cancer Cells

Abstract

We studied the effects of temperature, ribonucleotides and sodium molybdate on the activation and DNA cellulose binding of estrogen, glucocorticoid, progesterone and androgen receptor complexes in MCF-7 cells. Using DNA cellulose binding as a measure of receptor activation, we found that ribonucleotides activated all four of these receptor complexes. Temperature also activated glucocorticoid receptor complexes efficiently but activated progesterone and androgen receptor complexes less well. Temperature did not activate estrogen receptor complexes. Sodium molybdate blocked either ATP or temperature induced activation of glucocorticoid, progesterone and androgen receptor complexes but only partially blocked estrogen activation. Sodium molybdate also prevented the formation of multiple forms of estrogen and glucocorticoid receptor complexes seen on DEAE cellulose and hydroxylapatite chromatography of crude cytosol. The mechanism by which ribonucleotide enhances and molybdate inhibits activation are discussed.     

Synthesis, crystal structure, cytotoxic activities and DNA-binding properties of new binuclear copper(II) complexes bridged by N,N′-bis(N-hydroxyethylaminoethyl)oxamide

Two new μ-oxamido-bridged binuclear copper(II) complexes with formulae of [Cu₂(heae)(pic)₂] (1) and [Cu₂(heae)(Me₂phen)₂](ClO₄)₂ · H₂O (2), where heae and pic stand for the anion of N,N′-bis(N-hydroxyethylaminoethyl)oxamide and 2,4,6-trinitrophenol, respectively, and Me₂phen represents 2,9-dimethyl-1,10-phenanthroline; have been synthesized and characterized by elemental analyses, molar conductivity measurements, IR and electronic spectra studies. The crystal structures of the two binuclear copper(II) complexes have been determined by X-ray single-crystal diffraction. In both the two binuclear complexes the central two copper(II) atoms are bridged by trans-heae. In complex 1 the coordination environment around each copper(II) atom can be described as a distorted octahedral geometry, while in complex 2 each copper(II) atom displays a square-pyramid stereochemistry. Hydrogen bonding and π-π stacking interactions link the binuclear copper(II) complex 1 or 2 into a 3D infinite network. The cytotoxicities of the two binuclear copper(II) complexes were tested by Sulforhodamine B (SRB) assays against human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549. Both of the two binuclear copper(II) complexes exhibit potent cytotoxic effects against SMMC-7721 and A549 cell lines. The interactions of the two binuclear complexes with herring sperm DNA (HS-DNA) are investigated by using absorption and emission spectra and electrochemical techniques and viscometry. The results suggest that both the two binuclear copper(II) complexes interact with HS-DNA in the mode of intercalation with the intrinsic binding constants of 1.73 × 10⁵ M⁻¹ (1) and 1.92 × 10⁶ M⁻¹ (2). The influence of structural variation of the terminal ligands in the binuclear complexes on DNA-binding properties is preliminarily discussed.     

Structural, magnetic, electrochemical, catalytic, DNA binding and cleavage studies of new macrocyclic binuclear copper(II) complexes

The macrocyclic symmetrical and a series of unsymmetrical binuclear copper(II) complexes have been synthesized by using mononuclear complex [CuL] [3,3′-((1E,7E)-3,6-dioxa-2,7-diazaocta-1,7-diene-1,8-diyl)bis(3-formyl-5-methyl-2-diolato)copper(II)]. Another compartment of the [CuL] have been condensed with various diamines like 1,2-bis(aminooxy)ethane (L¹), 1,2-diamino ethane(L^2a), 1,3-diamino propane(L^2b), 1,4-diamino butane(L^2c), 1,2-diamino benzene(L^2d), 1,8-diamino naphthalene(L^2e) and characterized by elemental, spectroscopic, and X-ray crystallographic methods. The influence of the coordination geometry and the ring size of the binucleating ligands on the electronic, redox, magnetic, catecholase activity, DNA binding and cleavage properties have been studied. The molecular structures of the symmetrical binuclear complex [Cu₂L¹(H₂O)₂](ClO₄)₂ (1) and unsymmetrical binuclear complex [Cu₂L^2b(ClO₄)(H₂O)]ClO₄ (2b) were determined by X-ray crystallography. Both of them were discrete binuclear species in which each Cu(II) ions are in distorted square pyramid. The Cu?Cu distances vary from 3.0308 (2b) to 3.0361 Å (1). Electrochemical studies evidenced that two quasi-reversible one electron-transfer reduction waves −0.91 to −1.01 V, −1.26 to −1.55 V) for binuclear complexes are obtained in the cathodic region. Cryomagnetic investigation of the binuclear complexes reveals a weak antiferromagnetic spin exchange interaction between the Cu(II) ions within the complexes (−2J = 104.4-127.5 cm⁻¹). The initial rate (V_in) for the oxidation of 3,5-di-tert-butylcatechol to o-quinone by the binuclear Cu(II)complexes are in the range 3.6 × 10⁻⁵ to 7.3 × 10⁻⁵ Ms⁻¹. The binuclear Cu(II) complexes are avid binders to calf thymus DNA. The complexes display significant oxidative cleavage of circular plasmid pBR322 DNA in the presence of mercaptoethanol using the singlet oxygen as a reactive species. The aromatic diamine condensed macrocyclic ligands of copper(II) complexes display better DNA interaction and significant chemical nuclease activity than the aliphatic diamine condensed macrocyclic Cu(II) complexes.     

Influence of the orientation of hydroxyl groups on the puckering modes of furanoid rings

The influence of the orientations of HO-2 and HO-3 on the puckering of the ring in (2S,3R)-tetrahydrofuran-2,3-diol (1) and (3R)-tetrahydrofuran-3-ol (2) has been investigated by consistent force-field methods. In the E or W regions of the pseudorotation cycle, there are no local energy minima. The local minima in the N and S regions are affected by different orientations of the hydroxyl groups, with up to 12.2 kJ.mol^?1 for 1 and 6.9 kJ.mol^?1 for 2. If statistical weights of different rotamers are used to calculate N/S equilibrium constants, both conformational states are equally populated. However, if the orientations of the hydroxyl groups are restricted to simulate behaviour of these molecular fragments in DNA double-helices, the equilibrium shifts towards N. The results indicate that, in the vapor state, a unique one-dimensional N-S energy-barrier does not exist and that there is a spectrum of barriers depending on the orientation of the hydroxyl groups.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1^+/+, HO-1^+/−, and HO-1^−/− mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1^−/− and HO-1^+/− than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1^−/− lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.     

Influence of borate complexation on the electrophoretic behavior of 2-AA derivatized saccharides in capillary electrophoresis

A complete separation with baseline resolution of the 2-AA derivatized saccharides, including mono-, di-, and oligosaccharides, was achieved using 50 mM sodium phosphate-150 mM borate solution, pH 7.0 as running buffer by capillary electrophoresis. It was thought to be a result of the inclusion of 150 mM borate in the running electrolyte solution. The formation of borate complexes was observed by means of ¹¹B and ¹³C NMR spectroscopy and the electrophoretic mobilities of the various derivatives were calculated. It was found that steric factors play an important role in the stability of the formed borate complexes, which depends strongly on the configuration of the three vicinal hydroxyl groups at C-2, C-3, and C-4. 2-AA-Glc mainly forms stable 1,2-diester complexes with borate and 2-AA-Mal can form stable 1,2-monoesters. In turn, for 2-AA-Rib the formation of complexes is difficult to take place. The results implied that the configurational difference between the hydroxyl groups could cause the difference in formation of borate complexes leading to significant difference among saccharide molecules in their migration time on CE analysis.     

Spectroscopic characterization of mononuclear, binuclear, and insoluble polynuclear oxovanadium(IV)-Schiff base complexes and their oxidation catalysis

The objective was to prepare mononuclear, binuclear, and insoluble polynuclear oxovanadium(IV)-Schiff base complexes and to use them for sulfoxidation and epoxidation of organic substrates. [VO(salen)] (complex 1) with tetradentate salen(salicylideneethylenediamine) being coordinated in the equatorial plane of oxovanadium(IV), [VO(salap)] (complex 2), and [(VO)₂(sal₂-dhdabp)] (complex 3) with tridentate salap(salicylideneorthoaminophenol) and sal₂-dhdabp(salicylidene-3,3^′-dihydroxy-4,4^′-diaminobiphenyl) being bound, respectively, in the equatorial plane, of which polynuclear complexes were constituted as monomer units, were prepared and spectroscopically characterized. A sulfide and olefins were oxidized by use of complexes 1 and 2 (mononuclear), complex 3 (binuclear), and the polynuclear complexes (poly-1 and poly-3) synthesized with 1 and 3, respectively. The reaction rates for poly-1 and -3 were a little lower than those of the corresponding 1 and 3. On oxidation of sulfides, poly-3 exhibited lowering of activity by about 15% in three cycles, while poly-1 showed significant lose of activity with each use. Poly-3 was efficient for the oxidation of the olefins only in the first cycle. It was suggested that the loss of activity depends not only on the coordination geometry of the oxovanadium complex, but also on the kind of the substrate.     

Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by Azotobacter vinelandii

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe³⁺. Thus, competition between Fe³⁺ and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn²⁺, and Mn²⁺. We found that all five of these metal ions partially inhibited uptake of ⁵⁵Fe-protochelin and ⁵⁵Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.     

Dinitrosyl Iron Complexes with Thiol-Containing Ligands Exist in Living Organisms Mainly in the Binuclear Form

The levels of the mononitrosyl iron complex with diethyldithiocarbamate that form in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite, or the vasodilating drug isosorbide dinitrate (Isoket®) have been assessed by electron paramagnetic resonance (EPR). The levels of the complex in mice that received binuclear dinitrosyl iron complexes with thiol-containing ligands or S-nitrosoglutathione do not change after the treatment of liver preparations with the strong reducing agent dithionite, in contrast to those formed after nitrite or isosorbide dinitrate administration, whose levels sharply increase after the same treatment. It is inferred that in the latter case an EPR-active mononitrosyl iron complex with diethyldithiocarbamate is produced with the absence or presence of dithionite in the reaction of NO formed from nitrite with Fe²⁺-diethyldithiocarbamate and Fe³⁺-diethyldithiocarbamate complexes, respectively. In the former case, the mononitrosyl iron complex with diethyldithiocarbamate is produced by transition of iron-mononitrosyl fragments from already present iron-dinitrosyl groups of binuclear dinitrosyl complexes, whose content is three to four times higher than the content of the mononuclear form of these complexes in the tissue. The results we obtained indicate that when dinitrosyl iron complexes with thiol-containing ligands, either introduced into the body or produced with the participation of endogenous NO, appear in animal tissues in vivo, these complexes are presented in these tissues mainly in their diamagnetic, EPR-silent binuclear form.

     

Square-planar copper(II) complexes with tetradentate amido-carboxylate ligands. Crystal structure of Na2[Cu(obap)]2 · 2H2O. Strain analysis and spectral assignments of complexes

Novel N-N-N-O-type of tetradentate ligands H₃obap (H₃obap = oxamido-N-aminopropyl-N′-benzoic acid) and H₃maeb (H₃maeb = malamido-N-aminoethyl-N′-benzoic acid) and the corresponding square-planar copper(II) complexes have been prepared and characterized. The obap³⁻ and maeb³⁻ ligands coordinate to the copper(II) ion via four ligating atoms (three deprotonated atoms: one carboxylate oxygen and two deprotonated amide nitrogens; one amine nitrogen) with in-plane square chelation. A four coordinate, square-planar geometry has been established crystallographically for the binuclear Na₂[Cu(obap)]₂ · 2H₂O complex. Structural data correlating the square-planar geometry of the [Cu(obap)]⁻ unit and an extensive strain analysis are discussed in relation to the information obtained for similar complexes. The infrared and electronic absorption spectra of the complexes are discussed in comparison to the related complexes of known geometries. Antibacterial activity of ligands and copper(II) complexes towards common Gram-negative and Gram-positive bacteria are reported as well.     

Metal ions in biological processes. Volume 15. Zinc and its role in biology and nutrition. : Edited by Helmut Sigel. (Institute of Inorganic Chemistry,University of Basel.) Marcel Dekker,Inc., New York,N.Y. and Basle,Switzerland, 1983. 520 pp., bound,illustrated. SFr. 198.

Seven new complexes where the chelate ring was nucleophilically substituted were synthesized by the reactions of asymmetrical tetradentate Schiff base complexes of nickel(II) and copper(II) with benzenethiols and thiolycollic ehtyl ester. They were characterized by means of elemental analyses, electronic and ¹H-NMR spectra. Kinetics of the nucleophilic ring-substitution reactions were spectrophotometrically studied in a benzene solution. A four-centered intermediate is proposed for the reactions.