Potassium nickel(II) gallium phosphate hydrate, K[NiGa2(PO4)(3)(H2O)(2)]

The title compound, potassium nickel(II) digallium tris-( phosphate) dihydrate, K[NiGa2(PO4)(3)(H2O)(2)], was synthesized hydrothermally. The structure is constructed from distorted trans-NiO4(H2O)2 octahedra linked through vertices and edges to GaO5 trigonal bipyramids and PO4 tetrahedra, forming a three-dimensional framework of formula [NiGa2(PO4)(3)(H2O)(2)](-). The K, Ni and one P atom lie on special positions (Wyckoff position 4e, site symmetry 2). There are two sets of channels within the framework, one running parallel to the [10 (1) over bar] direction and the other parallel to [001]. These intersect, forming a three-dimensional pore network in which the water molecules coordinated to the Ni atoms and the K+ ions required to charge balance the framework reside. The K+ ions lie in a highly distorted environment surrounded by ten O atoms, six of which are closer than 3.1 angstrom. The coordinated water molecules are within hydrogen-bonding distance to O atoms of bridging Ga-O-P groups.

Synthesis and crystal structure of a 3-D zinc phosphate, [C5N2H14][Zn-2(PO3(OH))(3)], containing (4.8) net sheets

A new 3-D zinc phosphate, [C5N2H14][Zn-2(PO3(OH))(3)], has been synthesised under solvothermal conditions in the presence of 1-methylpiperazine. The structure, determined by single-crystal X-ray diffraction at 293 K (RMM = 520.9, orthorhombic, space group P2(1)2(1)2(1); a = 10.0517(2) &ANGS;, b = 10.4293(2) &ANGS; and c = 14.9050(5) &ANGS;; V = 1562.52 &ANGS;(3); Z = 4; R(F) = 2.60%, wR(F) = 2.93%), consists of vertex linked ZnO4 and PO3(OH) tetrahedra assembled into (4.8) net sheets which in turn are linked through further PO3(OH) units to generate a 3-D framework. 1-Methylpiperazinium cations reside within the 3-D channel system, held in place by a strong network of hydrogen bonds. The (4.8) net sheets occur in a number of zeolite structures e.g. ABW and GIS and related zinc phosphate phases. © 2004 Academie des sciences. Published by Elsevier SAS. All rights reserved.

Catena-Poly[bis(ethane-1,2-diammonium) [manganese(II)-di-mu-phosphato-kappa O-4 : O ']]: a one-dimensional manganese phosphate

The title compound,{(C2H10N2)(2)[Mn(PO4)(2)]}(n), contains anionic square-twisted chains of formula [Mn(PO4)(2)](4-) constructed from corner-sharing four-membered rings of alternating MnO4 and PO4 units. The Mn and P atoms have distorted tetrahedral coordination and the Mn atom lies on a twofold axis. The linear manganese-phosphate chains are held together by hydrogen-bonding interactions involving the framework O atoms and the H atoms of the ethane-1,2-diammonium cations, which lie in the interchain spaces.

Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after Delta(9)-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716

Rationale: Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability. Objectives: The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution. Methods: Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats. Results: Delta(9)-tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a nonsignificant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent. Conclusions: These data are consistent with endocannabinoid involvement in the mediation of food palatability.

Reaction of (±)-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate {(±)-phosH} with copper(II), cobalt(II) and iron(II) compounds; Identification by x-ray diffraction of [Co(CH3OH)4(H2O)2][(+)-phos][(−)-phos]. 2CH3OH·H2O

[Cu2(μO2CCH3)4(H2O)2], [CuCO3·Cu(OH)2], [CoSO4·7H2O], [Co((+)-tartrate)], and [FeSO4·7H2O] react with excess racemic (±)- 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate {(±)-PhosH} to give mononuclear CuII, CoII and FeII products. The cobalt product, [Co(CH3OH)4(H2O)2]((+)-Phos)((−)-Phos) ·2CH3OH·H2O (7), has been identified by X-ray diffraction. The high-spin, octahedral CoII atom is ligated by four equatorial methanol molecules and two axial water molecules. A (+)- and a (−)-Phos− ion are associated with each molecule of the complex but are not coordinated to the metal centre. For the other CoII, CuII and FeII samples of similar formulation to (7) it is also thought that the Phos− ions are not bonded directly to the metal. When some of the CuII and CoII samples are heated under high vacuum there is evidence that the Phos− ions are coordinated directly to the metals in the products.

The Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate KINETIC AND STRUCTURAL ANALYSIS OF THE ALLOSTERIC ATP INHIBITION

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 angstrom. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.

Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

The effect of BMP-2 on the osteoconductive properties of beta-tricalcium phosphate in rat calvaria defects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol.

Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.

Suicide nucleophilic attack: Reactions of benzohydroxamate anion with bis(2,4-dinitrophenyl) phosphate

(Chemical Equation Presented) The reaction between the benzohydroxamate anion (BHO-) and bis(2,4-dinitrophenyl)phosphate (BDNPP) has been examined kinetically, and the products were characterized by mass and NMR spectroscopy. The nucleophilic attack of BHO- follows two reaction paths: (i) at phosphorus, giving an unstable intermediate that undergoes a Lossen rearrangement to phenyl isocyanate, aniline, diphenylurea, and O-phenylcarbamyl benzohydroxamate; and (ii) on the aromatic carbon, giving an intermediate that was detected but slowly decomposes to aniline and 2,4-dinitrophenol. Thus, the benzohydroxamate anion can be considered a self-destructive molecular scissor since it reacts and loses its nucleophilic ability. © 2009 American Chemical Society.