Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms

The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o     

Synthesis,characterization, and electrocatalytic behaviour of hydrothermally grown nanostructured La2O3 and La2O3/K-complex

Rare earth metals play a conspicuous role in magnetic resonance imaging (MRI) for detecting cancerous cells. The alkali metal potassium is a neurotransmitter in the sodium–potassium pump in biomedical sciences. This unique property of rare earth metals and potassium drew our attention to carry forward this study. Therefore, in this work, previously synthesized potassium (K) complexes formed by the reflux of 4-N,N-dimethylaminobenzoic acid (DBA) and potassium hydroxide in methanol, and named [(μ2–4-N,N-dimethylaminobenzoate-κO)(μ2–4-N,N-dimethylaminobenzoic acid-κO)(4-N,N-dimethylaminobenzoic acid-κO) potassium(I) coordination polymer)] were treated hydrothermally with La₂O₃ nanomaterials to obtain a nanohybrid La₂O₃/K-complex. After that, the K-complex was analyzed using single-crystal X-ray diffraction and ¹H and ¹³C NMR spectroscopy. In addition, the structural and morphological properties of the as-prepared nanostructured La₂O₃/K-complex were also characterized, which involved an investigation using X-ray diffraction (XRD)spectroscopy, Fourier transform infrared (FTIR) spectroscopy, atomic force spectroscopy (AFM), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) analysis. After this, the electrochemical redox behaviour of the synthesized nanohybrid material was studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Therefore, the results from these studies revealed that the as-prepared material was a La₂O₃/K-complex that has a promising future role in sensing various analytes, as it showed effective electrocatalytic behaviour.     

Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-A resolution.

The crystal structure of a catalytically active fragment of glucoamylase-I from Aspergillus awamori var. X100 has been determined to a resolution of 2.2 A. Twelve of its 13 alpha-helices are arranged into an "alpha/alpha-barrel." An inner core of six mutually parallel alpha-helices are connected to each other through a peripheral set of six alpha-helices. The peripheral helices are parallel to each other, but approximately antiparallel to the inner core of alpha-helices. The putative active site lies in the packing void of the inner set of helices. The last 30 residues of the enzyme comprise a separate domain containing 10 sites of O-glycosylation. Each instance of O-glycosylation involves a serine or threonine side chain linked to the alpha-anomer of a single mannosyl residue. The O-glycosylated domain is in an extended conformation, wrapping around the "waist" of the alpha/alpha-barrel. Two additional sites of N-glycosylation contribute well ordered glycosyl chains that lie in proximity to the belt of O-glycosylation. The model developed for glucoamylase is a rare and valuable structural example of a glycoprotein and an exo-acting amylolytic enzyme.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Structure of inhibited fructose-1,6-bisphosphatase from Escherichia coli: distinct allosteric inhibition sites for AMP and glucose 6-phosphate and the characterization of a gluconeogenic switch

Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe(15), AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability.     

Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos

BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities.     

Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.     

Tryptophan fluorescence reveals the conformational state of a dynamic loop in recombinant porcine fructose-1,6-bisphosphatase

Wild-type porcine fructose-1,6-bisphosphatase (FBPase) has no tryptophan residues. Hence, the mutation of Try57 to tryptophan places a unique fluorescent probe in the structural element (loop 52-72) putatively responsible for allosteric regulation of catalysis. On the basis of steady-state kinetics, circular dichroism spectroscopy, and X-ray crystallography, the mutation has little effect on the functional and structural properties of the enzyme. Fluorescence intensity from the Trp57 mutant is maximal in the presence of divalent cations, fructose 6-phosphate and orthophosphate, which together stabilize an R-state conformation in which loop 52-72 is engaged with the active site. The level of fluorescence emission decreases monotonically with increasing levels of AMP, an allosteric inhibitor, which promotes the T-state, disengaged-loop conformation. The titration of various metal-product complexes of the Trp57 mutant with fructose 2,6-bisphosphate (F26P(2)) causes similar decreases in fluorescence, suggesting that F26P(2) and AMP individually induce similar conformational states in FBPase. Fluorescence spectra, however, are sensitive to the type of divalent cation (Zn(2+), Mn(2+), or Mg(2+)) and suggest conformations in addition to the R-state, loop-engaged and T-state, loop-disengaged forms of FBPase. The work presented here demonstrates the utility of fluorescence spectroscopy in probing the conformational dynamics of FBPase.