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.

A two-dimensional molybdenum (V) phosphate with covalently bonded transition metal coordination complexes: hydrothermal synthesis and structure characterization of Na-2[{Mn(phen)(2)(H2O)}{Mn(phen)(2)}(3){Mn (Mo12O24)-O-V (HPO4)(6)(PO4)(2)(OH)(6)}] center dot 4H(2)O (phen=1,10-phenanthroline)

An organic-inorganic hybrid molybdenum phosphate, Na-2[{Mn(phen)(2)(H2O)} {Mn(phen)(2)}(3){(MnMo12O24)-O-v (HPO4)(6)(PO4)(2) (OH)(6)}] . 4H(2)O (phen=1,10-phenanthroline), involving molybdenum present in V oxidation state and covalently bonded transition metal coordination complexes, has been hydrothermally synthesized and structurally characterized by single-crystal X-ray diffraction. Deep brown-red crystals are formed in the triclinic system, space group P (1) over bar, a=16.581(l)Angstrom, b=18.354(1)Angstrom, c=24.485(2)Angstrom, alpha=80.589(l)degrees, beta=71.279(1)degrees, gamma=67.084(1)degrees, V=6493.8(8)Angstrom(3), Z=2, lambda(MoKalpha)=0.71073Angstrom (R(F)=0.0686 for 29,053 reflections). Data were collected on a Bruker Smart Apex CCD diffractometer at 293 K in the range of 1.76 < theta < 28.06degrees using omega-2theta scans technique. The structure of the title compound may be considered to be based on {Mo6O12(HPO4)(3)(PO4)(OH)(3)} units bonded together with {Mn(phen)(2)} subunits into a two-dimensional network. Two types of tunnels are observed in the solid of the title compound.

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.

The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.

Molecular Structure of the mineral woodhouseite CaAl3(PO4,SO4)2(OH)6

The mineral woodhouseite CaAl3(PO4,SO4)2(OH)6 is a hydroxy phosphate-sulphate mineral belonging to the beudantite subgroup of alunites, and has been characterised by Raman spectroscopy, complimented with infrared spectroscopy. Bands at various wavenumbers were assigned to the different vibrational modes of woodhouseite, which were then associated to the molecular structure of the mineral. Bands were primarily assigned to phosphate and sulphate stretching and bending modes. Two symmetric stretching modes for both phosphate and sulphate supported the concept of non-equivalent phosphate and sulphate units in the mineral structure. Bands in the OH stretching region enabled hydrogen bond distances to be calculated.

Molecular structure of the mineral svanbergite SrAl3(PO 4,SO4)2(OH)6 - A vibrational spectroscopic study

The mineral svanbergite SrAl 3(PO 4,SO 4) 2(OH) 6 is a hydroxy phosphate-sulphate mineral belonging to the beudantite subgroup of alunites and has been characterised by vibrational spectroscopy. Bands at various wavenumbers were assigned to the different vibrational modes of svanbergite, which were then associated with the structure of the mineral. Bands were primarily assigned to phosphate and sulphate stretching and bending modes. Two symmetric stretching modes for both phosphate and sulphate supported the concept of non-equivalent phosphate and sulphate units in the mineral structure. Bands in the OH stretching region enabled hydrogen bond distances to be calculated. Comparison of the hydrogen bond distances and the calculated hydrogen bond distances from the structure models indicates that hydrogen bonding in svanbergite occurs between the two OH units rather than OH to SO42- units.

Hydrothermal synthesis and structural characterization of a novel P-V-O layered compound [H(2)en](2)[H3O](6)[Co(H2O)(2)(VO)(8)(OH)(4)(PO4)8]

A new compound [H(2)en](2)[H3O](6)[Co(H2O)(2)(VO)(8)(OH)(4)(PO4)(8)] has been hydrothermally synthesized. Single crystal X-ray analysis indicates that this compound crystallizes in a monoclinic system, space group P2(1)/n with a=1.438 5(3) nm, b=1.012 2(2) nm, c=1.832 5(4) nm, beta=90.21degrees, V=2.668 2 (9) nm(3), Z = 2, D-c = 2.112 g/cm(3), R = 0.055, wR = 0.149 7, S = 1.037. The structure of [H(2)en](2)[H3O](6)[Co(H2O)(2)(VO)(8)(OH)(4)(PO4)(8)] is characterized by P-V-O layers constructed by [(VO)4 (OH)(2)(PO4)(4)](6-) non-symmetric units. The P-V-O layers are pillared by [Co(H2O)(2)](2+) group, resulting in the channels within which the protonated diaminoethane and H3O+ are located.

Synthesis and enzymatic evaluation of the guanosine analogue 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG): insights into the phosphorolysis reaction mechanism based on the blueprint transition state: SN1 or SN2?

A modified experimental procedure for the synthesis of MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside) 1 has been successfully performed and its full characterization is presented. High resolution ESI(+)-MSMS indicates both the nucleoside bond cleavage as the main fragmentation in the gas phase and a possible SN1 mechanism. Ab initio transition state calculations based on the blue print transition state support this mechanistic rationale and discard an alternative SN2 mechanism. Assays using purine nucleoside phosphorylase (PNP) enzyme (human and M. tuberculosis sources) indicate its efficiency in the phosphorolysis of MESG and allow the quantitative determination of inorganic phosphate in real time assay.

Anhydrous 1:1 proton-transfer compounds of isonipecotamide with picric acid and 3,5-dinitrosalicylic acid: 4-carbamoylpiperidinium 2,4,6-trinitrophenolate and two polymorphs of 4-carbamoylpiperidinium 2-carboxy-4,6-dinitrophenolate

The structures of the anhydrous 1:1 proton-transfer compounds of isonipecotamide (4-carbamoylpiperidine) with picric acid and 3,5-dinitrosalicylic acid, namely 4-carbamoylpiperidinium 2,4,6-trinitrophenolate, C6H13N2O8+ C6H2N3O7- (I) and 4-carbamoylpiperidinium 2-carboxy-4,6-dinitrophenolate, C6H13N2O8+ C7H3N2O7-: two forms, the monoclinic alpha-polymorph (II) and the triclinic beta-polymorph (III) have been determined at 200 K. All compounds form hydrogen-bonded structures, one-dimensional in (II), two-dimensional in (I) and three-dimensional in (III). In (I), the cations form centrosymmetric cyclic head-to-tail hydrogen-bonded homodimers [graph set R2/2(14)] through lateral duplex piperidinium N---H...O(amide) interactions. These dimers are extended into a two-dimensional network structure through further interactions with anion phenolate-O and nitro-O acceptors, including a direct symmetric piperidinium N-H...O(phenol),O(nitro) cation--anion association [graph set R2/1(6)]. The monoclinic polymorph (II) has a similar R2/1(6) cation-anion hydrogen-bonding interaction to (I) but with an additional conjoint symmetrical R1/2(4) interaction as well as head-to-tail piperidinium N-H...O(amide) O hydrogen bonds and amide N-H...O(carboxyl) hydrogen bonds, give a network structure which include large R3/4(20) rings. The hydrogen bonding in the triclinic polymorph (III) is markedly different from that of monoclinic (II). The asymmetric unit contains two independent cation-anion pairs which associate through cyclic piperidinium N-H...O,O'(carboxyl) interactions [graph set R2/1(4)]. The cations also show the zig-zag head-to-tail piperidinium N-H...O(amide) hydrogen-bonded chain substructures found in (II) but in addition feature amide N-H...O(nitro) and O(phenolate) and amide N-H...O(nitro) associations. As well there is a centrosymmetric double-amide N-H...O(carboxyl) bridged bis(cation-anion) ring system [graph set R2/4(8)] in the three-dimensional framework. The structures reported here demonstrate the utility of the isonipecotamide cation as a synthon with previously unrecognized potential for structure assembly applications. Furthermore, the structures of the two polymorphic 3,5-dinitrosalicylic acid salts show an unusual dissimilarity in hydrogen-bonding characteristics, considering that both were obtained from identical solvent systems.

Years 2 to 6 students' ability to generalise : models, representations and theory for teaching and learning

Over the last three years, in our Early Algebra Thinking Project, we have been studying Years 3 to 5 students’ ability to generalise in a variety of situations, namely, compensation principles in computation, the balance principle in equivalence and equations, change and inverse change rules with function machines, and pattern rules with growing patterns. In these studies, we have attempted to involve a variety of models and representations and to build students’ abilities to switch between them (in line with the theories of Dreyfus, 1991, and Duval, 1999). The results have shown the negative effect of closure on generalisation in symbolic representations, the predominance of single variance generalisation over covariant generalisation in tabular representations, and the reduced ability to readily identify commonalities and relationships in enactive and iconic representations. This chapter uses the results to explore the interrelation between generalisation and verbal and visual comprehension of context. The studies evidence the importance of understanding and communicating aspects of representational forms which allowed commonalities to be seen across or between representations. Finally the chapter explores the implications of the studies for a theory that describes a growth in integration of models and representations that leads to generalisation.

2-(4-Aminophenyl)-1-phenyldiazenium 2,4,6-trinitrophenolate

In the structure of the title salt, C12H12N3+ C6H2N3O7-, the diazenyl group of the 4-(phenyldiazenyl)aniline molecule is protonated and forms a hydrogen bond with the phenolate O acceptor of the picrate anion. Structure extension occurs through two symmetrical inter-ion three-centre amine N---H...O,O'(nitro) hydrogen-bonding associations [graph set R2/1(4)] giving a convoluted two-dimensional network structure.

2,4,6-Trinitro-N-[4-(phenyldiazenyl)phenyl)phenyl]aniline

The title compound, C18H12N6O6 was prepared from the reaction of 4-(phenyldiazenyl)aniline (aniline yellow) with picrylsulfonic acid. The dihedral angle formed by the two benzene rings of the diphenyldiazenyl ring system 6.55(13)deg. and that formed by the rings of the picrate-aniline ring system is 48.76(12)deg. The molecule contains an intramolecular aniline-nitro N-H...O hydrogen bond.

2,3-Dimethoxy-10-oxostrychnidinium 2-(2,4,6-trinitroanilino)benzoate monohydrate : a 1:1 proton-transfer salt of brucine with o-picraminobenzoic acid

In the structure of the 1:1 proton-transfer compound of brucine with 2-(2,4,6-trinitroanilino)benzoic acid C23H27N2O4+ . C13H7N4O8- . H~2~O, the brucinium cations form the classic undulating ribbon substructures through overlapping head-to-tail interactions while the anions and the three related partial water molecules of solvation (having occupancies of 0.73, 0.17 and 0.10) occupy the interstitial regions of the structure. The cations are linked to the anions directly through N-H...O(carboxyl) hydrogen bonds and indirectly by the three water molecules which form similar conjoint cyclic bridging units [graph set R2/4(8)] through O-H...O(carbonyl) and O(carboxyl) hydrogen bonds, giving a two-dimensional layered structure. Within the anion, intramolecular N-H...O(carboxyl) and N H...O(nitro) hydrogen bonds result in the benzoate and picrate rings being rotated slightly out of coplanarity inter-ring dihedral angle 32.50(14)\%]. This work provides another example of the molecular selectivity of brucine in forming stable crystal structures and also represents the first reported structure of any form of the guest compound 2-(2,4,6-trinitroanilino)benzoic acid.