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Ternary Complex (ternary + complex)
Terms modified by Ternary Complex Selected AbstractsBiodegradable Hyaluronic Acid/N -Carboxyethyl Chitosan/Protein Ternary Complexes as Implantable Carriers for Controlled Protein ReleaseMACROMOLECULAR BIOSCIENCE, Issue 12 2005Hongliang Jiang Abstract Summary: An ampholytic N -carboxyethyl chitosan (CEC), with various isoelectric points (IPs), was synthesized by grafting acrylic acid on chitosan utilizing Michael's reaction. Compared to native chitosan, CEC has enhanced water solubility and dramatically accelerated enzymatic degradation; the rate of degradation is proportional to the degree of substitution (DS). The results from turbidimetric titration and fluorescence studies revealed that CEC formed complexes with either hyaluronic acid (HA) or bovine serum albumin (BSA) within a certain pH range. The HA/CEC/BSA ternary complexes could be prepared by colloid titration with quantitative yield and BSA entrapment. The rate of BSA release from the complexes was affected by pH, ionic strength, DS of CEC, and the molecular weight (MW) of HA. The endurance of BSA release from the complexes could be extended up to 20 d by formulating them with high-MW HA and CEC with low DS. BSA release profiles from HA/CEC-2/BSA complexes. [source] Behavior of PQQ Glucose Dehydrogenase on Prussian Blue-Modified Carbon ElectrodeELECTROANALYSIS, Issue 13 2008Valdas Laurinavicius Abstract Glucose sensitive biosensor containing pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase immobilized on Prussian blue (PB)-modified graphite electrode was designed. Properties of the biosensor were investigated in the cathodic and anodic response detection regions. It was shown, that anodic response of the biosensor is sum of two signals: direct electron transport from reduced PQQ to the electrode and by formation of the PQQ-oxygen-PB-carbon ternary complex. Cathodic response of the biosensor is based on the oxidation of the reduced PQQ by PB-oxygen-PB complex. Electrochemical regeneration of the enzyme does not produce free hydrogen peroxide. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Binding of ciprofloxacin by humic substances: A molecular dynamics studyENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2010Ludmilla Aristilde Abstract A comprehensive assessment of the potential impacts of antimicrobials released into the environment requires an understanding of their sequestration by natural particles. Of particular interest are the strong interactions of antimicrobials with natural organic matter (NOM), which are believed to reduce their bioavailability, retard their abiotic and biotic degradation, and facilitate their persistence in soils and aquatic sediments. Molecular dynamics (MD) relaxation studies of a widely used fluoroquinolone antibiotic, ciprofloxacin (Cipro), interacting with a model humic substance (HS) in a hydrated environment, were performed to elucidate the mechanisms of these interactions. Specifically, a zwitterionic Cipro molecule, the predominant species at circumneutral pH, was reacted either with protonated HS or deprotonated HS bearing Ca, Mg, or Fe(II) cations. The HS underwent conformational changes through rearrangements of its hydrophobic and hydrophilic regions and disruption of its intramolecular H-bonds to facilitate favorable intermolecular H-bonding interactions with Cipro. Complexation of the metal cations with HS carboxylates appeared to impede binding of the positively charged amino group of Cipro with these negatively charged HS complexation sites. On the other hand, an outer-sphere complex between Cipro and the HS-bound cation led to ternary Cipro,metal,HS complexes in the case of Mg,HS and Fe(II),HS, but no such bridging interaction occurred with Ca,HS. The results suggested that the ionic potential (valence/ionic radius) of the divalent cation may be a determining factor in the formation of the ternary complex, with high ionic potential favoring the bridging interaction. Environ. Toxicol. Chem. 2010;29:90,98. © 2009 SETAC [source] Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008Fumihiko Mouri Abstract High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1, protein. The ,131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S -transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes. [source] Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenaseFEBS JOURNAL, Issue 24 2007Katy Montin 6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho- d -erythronate (4PE) and 4-phospho- d -erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho- d -gluconate (6PG), the inhibitors 5-phospho- d -ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho- d -gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (Kd = 5 µm) and its analogues (Kd =1.3 µm and Kd = 2.8 µm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the Kd value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state. [source] Crystal structure of archaeal highly thermostable L -aspartate dehydrogenase/NAD/citrate ternary complexFEBS JOURNAL, Issue 16 2007Kazunari Yoneda The crystal structure of the highly thermostable l -aspartate dehydrogenase (l -aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidusl -aspDH was refined at a resolution of 1.9 Å with a crystallographic R -factor of 21.7% (Rfree = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidusl -aspDH/NAD/citrate ternary complex and the Thermotoga maritimal -aspDH/NAD binary complex showed that A. fulgidusl -aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritimal -aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidusl -aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two l -aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to l -aspDH and of the molecular basis of the high thermostability of a hyperthermophilic l -aspDH. [source] Functional analysis of disease-causing mutations in human galactokinaseFEBS JOURNAL, Issue 8 2003David J. Timson Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia. [source] Prostromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activatorFEBS JOURNAL, Issue 21 2000Begoña Arza Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 × 106 to 8.0 × 106 m,1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 × 106 m,1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis,Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 × 10,3 vs. 4.1 × 10,4 µm,1·s,1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 µm vs. 89 µm in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 × 10,2 vs. 3.6 × 10,2 s,1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 × 106 m,1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 × 106m,1) as compared to Glu-plasminogen (Ka of 1.2 × 106m,1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen. [source] Molecular Recognition of a Peptide by the Nickel(II) Complex of 1,4,7,10-Tetraazacyclododecane-2,9-dioneHELVETICA CHIMICA ACTA, Issue 3 2003Jian Gao The nickel(II) complex of the macrocycle 1,4,7,10-tetraazacyclododecane-2,9-dione (dota) was found to be efficient in the recognition of the dipeptide, glycyl-glycine (Gly-Gly) in aqueous solution. This (dota)NiII complex serves as a targeting molecule to form a stable ternary complex with the dipeptide at pH,8.3 in aqueous solution. The recognition constant (log K=19.20) and the recognition mechanism were investigated based on the potentiometric method. The single-crystal of a six-coordinated (dota)2NiII complex is also reported. [source] Catalytic hydrolysis of p -nitrophenyl picolinate by copper(II) and zinc(II) complexes of N-(2-deoxy-,-D-glucopyranosyl-2-salicylaldimino)INTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 6 2002Xiang Yan D-glucosamine Schiff base N-(2-deoxy-,-D-glucopyranosyl-2-salicylaldimino) and its Cu(II) and Zn(II) complexes were synthesized and characterized. The hydrolysis of p -nitrophenyl picolinate (PNPP) catalyzed by ligand and complexes was investigated kinetically by observing the rates of the release of p -nitrophenol in the aqueous buffers at 25°C and different pHs. The scheme for reaction acting mode involving a ternary complex composed of ligand, metal ion, and substrate was established and the reaction mechanisms were discussed by metal,hydroxyl and Lewis acid mechanisms. The experimental results indicated that the complexes, especially the Cu(II) complex, efficiently catalyzed the hydrolysis of PNPP. The catalytic reactivity of the Zn(II) complex was much smaller than the Cu(II) complex. The rate constant kN showing the catalytic reactivity of the Cu(II) complex was determined to be 0.299 s,1 (at pH 8.02) in the buffer. The pKa of hydroxyl group of the ternary complex was determined to be 7.86 for the Cu(II) complex. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 345,350, 2002 [source] Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopyJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2010Gloria Funari Abstract Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour-suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down-regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N-terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein,protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti-cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd. [source] The nuclear localization of SET mediated by imp,3/imp, attenuates its cytosolic toxicity in neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Dianbo Qu Abstract SET is a multi-functional protein in proliferating cells. Some of the proposed functions of SET suggest an important nuclear role. However, the nuclear import pathway of SET is also unknown and the function of SET in neurons is unclear. Presently, using cortical neurons, we report that the nuclear import of SET is mediated by an imp,/imp,-dependent pathway. Nuclear localization signal, 168KRSSQTQNKASRKR181, in SET interacts with imp,3, which recruits imp, to form a ternary complex, resulting in efficient transportation of SET into nucleus. By in vitro nuclear import assay based on digitonin-permeabilized neurons, we further demonstrated that the nuclear import of SET relies on Ran GTPase. We provide evidence that this nuclear localization of SET is important in neuronal survival. Under basal conditions, SET is predominately nuclear. However, upon death induced by genotoxic stress, endogenous SET decreases in the nucleus and increases in the cytoplasm. Consistent with a toxic role of SET in the cytoplasm, targeted expression of SET to the cytoplasm exacerbates death compared to wild type SET expression which is protective following DNA damage. Taken together, our results indicate that SET is imported into the nucleus through its association with imp,3/imp,, and that localization of SET is important in regulation of neuronal death. [source] Protein disulfide bond generation in Escherichia coli DsbB,DsbAJOURNAL OF SYNCHROTRON RADIATION, Issue 3 2008Kenji Inaba Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB,DsbA,ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA,DsbB oxidative and the DsbC,DsbD reductive pathways has also been proposed. [source] Tissue factor: a mini-reviewJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 3 2007Valéry Daubie Abstract Tissue factor (TF) is historically known as the trigger of the coagulation cascade. This integral membrane glycoprotein forms a ternary complex with factor VIIa (FVIIa) and zymogen factor (FX), which is then activated to factor Xa (FXa). The latter cleaves prothrombin into thrombin (FIIa), which in turn activates fibrinogen in fibrin monomers. What is less known is its additional non-haemostatic roles in inflammation, tumour growth and angiogenesis. This aspect will be developed here. TF, as a transmembrane protein, has a signalling effect requiring FVIIa. TF,FVIIa complex activates G protein-coupled receptor protease-activated receptor 2 (PAR-2) and therefore modulates various cellular processes, such as cell proliferation and survival, gene transcription and protein translation. In this review we will first highlight, using recent structural data, the ,potentially' active domain able to modulate the triggered intracellular response. We also will focus on the still emerging and promising results deciphering the diverse locations in which TF appears. We conclude with a description of an emerging and atypical use of tissue factor in platelet gel surgery for sinus augmentation. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of ibuprofen in pharmaceutical formulations using time-resolved terbium-sensitized luminescenceLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 4 2007Salma M. Z. Al-Kindy Abstract A sensitive and specific luminescence method for the determination of ibuprofen (IB) in pharmaceutical formulations in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb3+) by formation of ternary complex with IB in the presence of tri- n -octylphosphine oxide (TOPO) and Tween-20 as surfactant. The luminescence signal for Tb,IB,TOPO is monitored at ,ex = 229 nm and ,em = 545 nm. Optimum conditions for the formation of the complex in aqueous system, were 16 mmol/L TRIS buffer, pH 5.7, TOPO 200 µmol/L and 15 µmol/L of Tb3+, which allows for the determination of 9.7 × 10,7 , 9.7 × 10,6 mol/L IB with a detection limit of 1.2 × 10,7 mol/L. The relative standard deviations of the method were <1.4%, indicating excellent reproducibility. The proposed method was successfully applied for the assays of IB in pharmaceutical formulations with average recoveries of 100.3,102.5%. Copyright © 2007 John Wiley & Sons, Ltd. [source] Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexateMOLECULAR MICROBIOLOGY, Issue 6 2006Alice Dawson Summary The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the ,6-,6 loop and ,6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis. [source] Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seedsTHE PLANT JOURNAL, Issue 2 2006I. Rubio-Somoza Summary The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5,- GATAAGATA -3,) encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5,-TATCCAC-3,/5,-GTGGATA -3,) encoding a high-pI , -amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans -activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF. [source] Structures of alternatively spliced isoforms of human ketohexokinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Chi H. Trinh A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. [source] Structure of the G225P/G226P mutant of mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrateACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Urmi Dhagat 3(17),-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17,-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17,-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP+ and the inhibitor hexoestrol refined at 2.1,Å resolution is presented. Kinetic analysis and molecular-modelling studies of 17,- and 17,-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (Km) for both 3,- and 17,-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates. [source] Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex formsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Teruya Nakamura During replication, Escherichia coli MutT prevents the misincorporation of mutagenic 8-oxoguanine into nascent DNA strands opposite adenine by hydrolyzing 8-oxo-dGTP in nucleotide pools to 8-oxo-dGMP. E. coli MutT is the most widely investigated member of the Nudix hydrolase family, which is large and found in all organisms. By co-crystallization of MutT with 8-oxo-dGMP, a reaction product, crystals of the binary complex were obtained using ammonium sulfate as a precipitant. The crystals belong to space group P212121, with unit-cell parameters a = 37.9, b = 56.0, c = 59.4,Å. Assuming the presence of one protein,nucleotide complex in the asymmetric unit, the Matthews coefficient VM is 2.1,Å3,Da,1. Crystals of the ternary complex were prepared by soaking crystals of the binary complex in 1,mM MnCl2 solution. They diffracted to 1.96 and 2.56,Å resolution, respectively. [source] Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermediusACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Tatyana A. Muranova Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD+. Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an `open' and `closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD+ have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P212121, with approximate unit-cell parameters a = 106, b = 118, c = 320,Å and an octamer in the asymmetric unit, corresponding to a VM of 3.1,Å3,Da,1. The crystals of the non-productive ternary complex belong to space group P61 or P65, with approximate unit-cell parameters a = b = 117, c = 502,Å and an octamer in the asymmetric unit, corresponding to a VM of 3.0,Å3,Da,1. These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3,Å resolution, respectively, and are suitable for a full structure determination. [source] Crystallization and preliminary X-ray analysis of a d -alanyl- d -alanine ligase (EcDdlB) from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Sarah Batson A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and d -alanyl- d -alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and d -alanyl- d -alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5,Å and a = 51.2, b = 97.8, c = 110.1,Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4,Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation. [source] A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C -methyl- d -erythritol kinase and reassessment of the quaternary structureACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Justyna Kalinowska-T 4-Diphosphocytidyl-2C -methyl- d -erythritol kinase (IspE; EC 2.7.1.148) contributes to the 1-deoxy- d -xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C -methyl- d -erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C -methyl- d -erythritol. A triclinic crystal structure of the Escherichia coli IspE,ADP complex with two molecules in the asymmetric unit was determined at 2,Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the `triclinic dimer' is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization. [source] The Z isomer of 2,4-diaminofuro[2,3- d]pyrimidine antifolate promotes unusual crystal packing in a human dihydrofolate reductase ternary complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Vivian Cody The crystal structure of the ternary complex of human dihydrofolate reductase (hDHFR) with NADPH and the Z isomer of 2,4-diamino-5-[2-(2,-methoxyphenyl)propenyl]-furo[2,3- d]pyrimidine (Z1) shows that the Z isomer binds in the normal antifolate orientation in which the furo oxygen occupies the 8-amino position observed in the binding of 2,4-diaminopteridine antifolates such as methotrexate and with the methoxyphenyl moiety cis to and coplanar with the furo[2,3- d]pyrimidine ring. The hDHFR ternary complex crystallized in the orthorhombic space group P212121 and its structure was refined to 1.7,Å resolution. Although other hDHFR complexes crystallize in this space group, these data provide only the second example of an unusual packing arrangement in which the conserved active-site Arg70 forms a salt bridge to the side chain of Glu44 from a symmetry-related molecule. As a result, the conformations of Phe31 and Gln35 shift with respect to those observed in the structure of mouse DHFR bound to Z1, which crystallizes in the monoclinic space group P21 and shows that Gln35 interacts with Arg70. [source] Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Marc Bailly Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNAAsnvia a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNAAsn prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNAAsn formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNAAsn in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES,NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P21, with unit-cell parameters a = 115.9, b = 214.0, c = 127.8,Å, , = 93.3°. A complete data set was collected to 3,Å resolution. Here, the isolation and crystallization of the transamidosome from T. thermophilus and preliminary crystallographic data are reported. [source] Crystallization and preliminary X-ray analysis of d -2-hydroxyacid dehydrogenase from Haloferax mediterraneiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009J. Domenech d -2-Hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8,M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with ,-ketohexanoic acid and NAD+ grew under these conditions. Crystals of form I diffracted to beyond 3.0,Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 66.0, b = 119.6, c = 86.2,Å, , = 96.3°. Crystals of form II diffracted to beyond 2.0,Å resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6,Å, , = 109.1, , = 107.5, , = 95.9°. The calculated values for VM and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry. [source] E230Q mutation of the catalytic subunit of cAMP-dependent protein kinase affects local structure and the binding of peptide inhibitorBIOPOLYMERS, Issue 6 2006Man-Un Ung Abstract The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of nonconserved acidic residues,Glu127, Glu170, Glu203, Glu230, and Asp241,that are crucial for substrate recognition and binding. Studies have shown that the Glu230 to Gln mutant (E230Q) of the enzyme has physical properties similar to the wild-type enzyme and has decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method (Lin, J.-H., et al. J Am Chem Soc 2002, 24, 5632,5633) to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 428,439, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Trypanosoma brucei UDP-galactose-4,-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-,- d -galactoseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006Magnus S. Alphey The structure of the NAD-dependent oxidoreductase UDP-galactose-4,-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-,- d -galactose has been determined using diffraction data to 2.7,Å resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-,- d -galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180° with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site. [source] Self-Assembly of Two-Component Gels: Stoichiometric Control and Component SelectionCHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2009Andrew Abstract Two-component systems capable of self-assembling into soft gel-phase materials are of considerable interest due to their tunability and versatility. This paper investigates two-component gels based on a combination of a L -lysine-based dendron and a rigid diamine spacer (1,4-diaminobenzene or 1,4-diaminocyclohexane). The networked gelator was investigated using thermal measurements, circular dichroism, NMR spectroscopy and small angle neutron scattering (SANS) giving insight into the macroscopic properties, nanostructure and molecular-scale organisation. Surprisingly, all of these techniques confirmed that irrespective of the molar ratio of the components employed, the "solid-like" gel network always consisted of a 1:1 mixture of dendron/diamine. Additionally, the gel network was able to tolerate a significant excess of diamine in the "liquid-like" phase before being disrupted. In the light of this observation, we investigated the ability of the gel network structure to evolve from mixtures of different aromatic diamines present in excess. We found that these two-component gels assembled in a component-selective manner, with the dendron preferentially recognising 1,4 - diaminobenzene (>70,%), when similar competitor diamines (1,2- and 1,3-diaminobenzene) are present. Furthermore, NMR relaxation measurements demonstrated that the gel based on 1,4-diaminobenzene was better able to form a selective ternary complex with pyrene than the gel based on 1,4-diaminocyclohexane, indicative of controlled and selective ,,, interactions within a three-component assembly. As such, the results in this paper demonstrate how component selection processes in two-component gel systems can control hierarchical self-assembly. [source] | |||||||||||||||||||||||||||||||||||||||||||