- Thermodynamic analysis of F1-ATPase rotary catalysis using high-speed imaging
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F1-ATPase (F1) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F1 rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 μs. At a high concentration of ATP, F1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (Pi). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, Pi release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG? = 53-56 kJ mol-1, the contributions of the enthalpy (ΔH?), and entropy (ΔS?) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH? and ΔS?. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.
- Watanabe, Rikiya,Minagawa, Yoshihiro,Noji, Hiroyuki
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- Molecular identification of N-acetylaspartylglutamate synthase and β-citrylglutamate synthase
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The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.
- Collard, Francois,Stroobant, Vincent,Lamosa, Pedro,Kapanda, Coco N.,Lambert, Didier M.,Muccioli, Giulio G.,Poupaert, Jacques H.,Opperdoes, Fred,Van Schaftingen, Emile
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- Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1
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Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH2, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values kcat (0.052 ± 0.001 s-1), KMgATP (1.2 ± 0.1 μM), KiMgATP (1.3 ± 0.2 μM), KFAK-tide (5.6 ± 0.4 μM), and K iFAK-tide (6.1 ± 1.1 μM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the βγ-bridge:β-nonbridge positional oxygen exchange of [γ-18O4]ATP in the presence of 1 mM [γ- 18O4]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of kx/kcat = 0.14 ± 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on kcat/KFAK-tide, while kcat and kcat/KMgATP were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on kcat/Km for MgATP but not on kcat/K m for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (kcat) is rate-limited by both reversible phosphoryl group transfer (kforward ≈ 0.2 s-1 and kreverse ≈ 0.04 s-1) and a slow step (kconf ≈ 0.1 s-1) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.
- Schneck, Jessica L.,Briand, Jacques,Chen, Stephanie,Lehr, Ruth,McDevitt, Patrick,Zhao, Baoguang,Smallwood, Angela,Concha, Nestor,Oza, Khyati,Kirkpatrick, Robert,Yan, Kang,Villa, James P.,Meek, Thomas D.,Thrall, Sara H.
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- Coupling of proteolysis to ATP hydrolysis upon Escherichia coli Lon protease functioning: I. Kinetic aspects of ATP hydrolysis
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Some aspects of the Escherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by an elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.
- Melnikov,Tsirulnikov,Rotanova
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- The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids
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Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K m for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of SUR2B was less active than that of SUR2A. We further found that the NBDs of SUR2B interact, and that the activity of full-length SUR cannot be predicted from that of either the isolated NBDs or NBD mixtures. Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted in ATPase activity resembling that of NBD2 of SUR2A rather than that of NBD2 of SUR2B: this might indicate that these amino acids are responsible for the lower ATPase activity of SUR2B and the isolated NBD2 of SUR2B. We suggest that the lower ATPase activity of SUR2B may result in enhanced duration of the MgADP-bound state, leading to channel activation.
- De Wet, Heidi,Fotinou, Constantina,Amad, Nawaz,Dreger, Matthias,Ashcroft, Frances M.
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- Zn(II) enhances nucleotide binding and dephosphorylation in the presence of a poly(ethylene imine) dendrimer
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The interaction of a second-generation poly(ethylene imine) dendrimer (L) with the nucleotides AMP, ADP and ATP was analysed by means of potentiometric titrations (0.1 M Me4NCl, 298.1 K), in the absence and in the presence of Zn2+, to perform speciation of the systems and determination of complex stability constants. Protonated forms of L interact with anionic forms of the nucleotides giving rise to stable anion complexes. In the presence of Zn2+, ternary complexes (ion-pair complexes) become the main species in solution, the presence of each partner enhancing the binding of the other one (positive cooperativity effect). The stability constants determined by the same method and under the same experimental conditions for the formation of PO43-, P2O7 4- and P3O105- complexes with L showed a strict similarity between the binding of AMP, ADP and ATP and their inorganic counterparts, indicating that the main interaction of nucleotides with protonated ligand forms takes places through their inorganic tails. Dephosphorylation of ATP to form ADP and phosphate was monitored by means of 31P NMR spectra at pH 3 and 9 in the presence of L and of its Zn 2+ complexes. The rate of ATP dephosphorylation is enhanced by about 2 times at pH 3 and 5 times at pH 9 in the presence of L and by about 4 times at pH 3 and 6 times at pH 9 in the presence of its metal complex, relative to the uncatalysed process, the dephosphorylation reactions being much faster in the acidic media where successive cleavage of ADP to give AMP and phosphate was also observed. Depending on the pH, the observed dephosphorylation enhancements have been interpreted in terms of formation of phosphoramidate intermediates, formation of ATP-Zn2+ coordinative bonds and positive allosteric effects due to metal ion coordination.
- Bazzicalupi, Carla,Bianchi, Antonio,Giorgi, Claudia,Valtancoli, Barbara
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- A zinc(II)-based receptor for ATP binding and hydrolysis
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A protonated Zn(II) complex with a terpyridine-containing pentaamine macrocycle catalyses ATP hydrolysis in the presence of a second metal ion, which acts as cofactor assisting the phosphoryl transfer from ATP to an amine group of the receptor. The Royal Society of Chemistry 2005.
- Bazzicalupi, Carla,Bencini, Andrea,Bianchi, Antonio,Danesi, Andrea,Giorgi, Claudia,Lodeiro, Carlos,Pina, Fernando,Santarelli, Samuele,Valtancoli, Barbara
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- A general framework for inhibitor resistance in protein kinases
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Protein kinases control virtually every aspect of normal and pathological cell physiology and are considered ideal targets for drug discovery. Most kinase inhibitors target the ATP binding site and interact with residue of a hinge loop connecting the small and large lobes of the kinase scaffold. Resistance to kinase inhibitors emerges during clinical treatment or as a result of in vitro selection approaches. Mutations conferring resistance to ATP site inhibitors often affect residues that line the ATP binding site and therefore contribute to selective inhibitor binding. Here, we show that mutations at two specific positions in the hinge loop, distinct from the previously characterized "gatekeeper," have general adverse effects on inhibitor sensitivity in six distantly related kinases, usually without consequences on kinase activity. Our results uncover a unifying mechanism of inhibitor resistance of protein kinases that might have widespread significance for drug target validation and clinical practice.
- Balzano, Deborah,Santaguida, Stefano,Musacchio, Andrea,Villa, Fabrizio
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- Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase: Product inhibition, cooperativity, and magnesium activation
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In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min-1, the Km for ATP by 0.104 ± 0.024 mM, and the Ki for ADP by 0.037 ± 0.007 mM. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light.
- Hazra, Suratna,Henderson, J. Nathan,Liles, Kevin,Hilton, Matthew T.,Wachter, Rebekka M.
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- Magnesium coordination controls the molecular switch function of DNA mismatch repair protein MutS
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The DNA mismatch repair protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-protein switches and the regulation of their "on" and "off" states by guanine exchange factors. Although GDP release in monomeric GTPases is accelerated by guanine exchange factorinduced removal of magnesium from the catalytic site, we found that release of ADP from MutS is not influenced by the metal ion in this manner. Rather, ADP release is induced by the binding of mismatched DNA at the opposite end of the protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium influences switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-proteins and MutS conceptually employ the same efficient use of the high energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.
- Lebbink, Joyce H. G.,Fish, Alexander,Reumer, Annet,Natrajan, Ganesh,Winterwerp, Herrie H. K.,Sixma, Titia K.
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- Lipopolysaccharide biosynthesis without the lipids: Recognition promiscuity of Escherichia coli heptosyltransferase i
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Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k cat/Km of 2.7 × 106 M-1 s -1, compared to a value of 2.2 × 105 M-1 s-1 for the Kdo2-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.
- Czyzyk, Daniel J.,Liu, Cassie,Taylor, Erika A.
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- Purification and characterization of mouse mevalonate pyrophosphate decarboxylase
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Mevalonate pyrophosphate decarboxylase (MPD) in mouse liver was purified by affinity chromatography. The purified enzyme was a homodimer of 46-kDa subunits and had an isoelectric point of 5.0. Kinetic analysis revealed an apparent Km value of 10 μM for mevalonate pyrophosphate. The enzyme required ATP as a phosphate acceptor and Mg as a divalent cation, which could be substituted with Mn or Co. Its optimum pH was 4.0-7.0. A comparison with MPD from various other sources revealed the mouse MPD to have essentially the same properties as rat MPD, expect for the optimum pH range. An excess of rabbit anti-rat MPD antibody deleted approximately 80% of the MPD activity in the crude extract of mouse liver. These results suggested that the homodimer of 46-kDa subunits represents the major active form of MPD in mice.
- Michihara, Akihiro,Akasaki, Kenji,Yamori, Yukio,Tsuji, Hiroshi
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- Supramolecular catalysis in the hydrolysis of ATP facilitated by macrocyclic polyamines: mechanistic studies
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Protonated polyaza macrocycles that bind anionic substrates can perform reactions on the bound species and provide information on the various factors contributing to catalysis.They form stable complexes wtih adenosine triphosphate, adenosine diphosphate, and pyrophosphate, and these supramolecular assemblies catalyze the solvolysis of the bound substrates. 31P NMR was found to be a useful technique both for the kinetic analysis and for the detection of unstable intermediates formed in the reaction.The 24-membered macrocyclic polyamine, 1,4,7,13,16,19-hexaaza-10,22-dioxocyclotetradecane (N6O2,1), catalyzed the hydrolysis of ATP at both pH 3 and 7 with calculated entropies of activation of -11 and -8.7 eu.The reaction is intensive to ionic strength; however, both sodium and chloride ions depress the kobsd at pH 7.The reaction at neutral pH is characterized by nucleophilic catalysis with the formation of the symmetrical monophosphorylated derivative 2 of macrocycle 1; a Δs of -26 eu was found for the solvolysis of 2 at pH 7.A series of related 22- to 32-membered rings containing up to 10 groups demonstrated specific structural requiments for effective cataysis of the hydrolytic reactions.Analogues of 1 wherein the oxygen atoms are either replaced by amino groups (N8,4) or removed (N2C4, 6) enhanced the rate of ATP hydrolysis at pH 3 by a factor of 300.Clear evidence for electrostatic and nucleophilic catalysis coupled with potential sites for acid and base catalysis make such macrocycles fruitful materials for the study of the mechanisms of molecular catalysis in polyphosphate hydrolysis, as well as models for the processes that may occur in enzymes utilizing ATP and related species as substrates or phosphoryl donors.
- Hosseini, Mir Wais,Lehn, Jean-Marie,Maggiora, Linda,Mertes, Kristin Bowman,Mertes, Mathias P.
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- Structural and enzymatic characterization of the choline kinase LicA from Streptococcus pneumoniae
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LicA plays a key role in the cell-wall phosphorylcholine biosynthesis of Streptococcus pneumonia. Here we determined the crystal structures of apo-form LicA at 1.94 ? and two complex forms LicA-choline and LicA-AMP-MES, at 2.01 and 1.45 ? resolution, respectively. The overall structure adopts a canonical protein kinase-like fold, with the active site located in the crevice of the N- and C- terminal domains. The three structures present distinct poses of the active site, which undergoes an open-closed-open conformational change upon substrate binding and product release. The structure analyses combined with mutageneses and enzymatic assays enabled us to figure out the key residues for the choline kinase activity of LicA. In addition, structural comparison revealed the loop between helices α7 and α8 might modulate the substrate specificity and catalytic activity. These findings shed light on the structure and mechanism of the prokaryotic choline kinase LicA, and might direct the rational design of novel anti-pneumococcal drugs.
- Wang, Lei,Jiang, Yong-Liang,Zhang, Jing-Ren,Zhou, Cong-Zhao,Chen, Yuxing
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- Purification of breast cancer resistance protein ABCG2 and role of arginine-482
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Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wildtype and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding. Birkhaeuser Verlag, 2006.
- Pozza,Perez-Victoria,Sardo,Ahmed-Belkacem,Di Pietro
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- Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis.
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The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.
- Tokunaga,Shiraishi,Odachi,Mizukami,Tokunaga,Philo,Arakawa,Ishibashi,Tanaka,Takagi
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- Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity
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Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AKeco. Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of kcat. Addition of the osmolyte TMAO to AKeco results in population shift toward the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent, and in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response in KM. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AKeco, there exists ample flexibility to obtain a specificity constant (kcat/KM) that commensurate with the exerted cellular selective pressure.
- Aden, Joergen,Wolf-Watz, Magnus,Verma, Abhinav,Schug, Alexander
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p. 16562 - 16570,9
(2012)
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- YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity
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The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (Km) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.
- Rao, Feng,See, Rui Yin,Zhang, Dongwei,Toh, Delon Chengxu,Ji, Qiang,Liang, Zhao-Xun
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- ATP cleavage by cone tetraguanidinocalix[4]arene
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The upper rim cone tetraguanidinocalix[4]arene 1 is a highly effective catalyst of ATP hydrolysis. The catalytically most active species is the triprotonated form of the catalyst. The three protonated guanidinium groups provide the electrostatic driving force for substrate binding and activation, while the neutral guanidine most likely acts as a nucleophilic catalyst.
- Salvio, Riccardo,Casnati, Alessandro,Mandolini, Luigi,Sansone, Francesco,Ungaro, Rocco
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- Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells
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The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1–4]+ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH. Complexes [Eu.1]+ and [Eu.3]+ bearing hydrogen bond donor groups in the pendant arms showed excellent discrimination between ATP, ADP and monophosphate species. Complex [Eu.3]+ showed relatively strong binding to ATP (logKa=5.8), providing a rapid, long-lived luminescent signal that enabled its detection in a highly competitive aqueous medium containing biologically relevant concentrations of Mg2+, ADP, GTP, UTP and human serum albumin. This EuIII complex responds linearly to ATP within the physiological concentration range (1–5 mm), and was used to continuously monitor the apyrase-catalyzed hydrolysis of ATP to ADP in vitro. We demonstrate that [Eu.3]+ can permeate mammalian (NIH-3T3) cells efficiently and localize to the mitochondria selectively, permitting real-time visualization of elevated mitochondrial ATP levels following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as depleted ATP levels upon treatment with potassium cyanide under glucose starvation conditions.
- Mailhot, Romain,Traviss-Pollard, Thomas,Pal, Robert,Butler, Stephen J.
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- Bacillus subtilis DEAD protein YdbR possesses ATPase, RNA binding, and RNA unwinding activities
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The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His6 purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a ΔydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37°C, but more severely at 22°C.
- Ando, Yoshinari,Nakamura, Kouji
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- Compound and preparation method thereof, and preparation method of nucleoside oligophosphate
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The invention relates to the technical field of nucleotide synthesis, in particular to a compound and a preparation method thereof, and a preparation method of nucleoside oligophosphate. The preparation method of the compound comprises the following steps: putting a nucleoside phosphate raw material, imidazole and an activating agent into a first solvent for a first reaction. According to the invention, the water-soluble raw material and the activating agent are subjected to a reaction to prepare the compound, and the compound can be used as a raw material for synthesizing nucleoside oligophosphate, so that rapid and stable synthesis of nucleoside oligophosphate is facilitated. The phosphate raw material and the compound prepared in the invention are subjected to a second reaction in a second solvent, and then desalination is performed to prepare nucleoside oligophosphate; compared with the prior art, triethylamine conversion does not need to be carried out on the nucleoside phosphate raw material, the utilization rate of the nucleoside phosphate raw material is increased, and the reaction period is shortened.
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Paragraph 0188-0196
(2021/11/10)
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- ATP regeneration by a single polyphosphate kinase powers multigram-scale aldehyde synthesisin vitro
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ATP recycling systems are required to avoid the addition of stoichiometric quantities of cofactor and facilitate industrial implementation of ATP-dependent enzymes. One factor that limits the biocatalytic application of these enzymes is the lack of a scalable AMP to ATP regeneration system. Whole-cells or a combination of purified enzymes are often exploited for ATP regeneration from AMP, whereas cell free systems comprising a single crude enzyme preparation would be preferred. To establish such a system, we focussed on polyphosphate kinases (PPKs) to find a single enzyme that could be used to power ATP-consuming reactions. Screening of some previously reported PPKs revealed limitations of these biocatalysts for scale-up purposes. As such, a panel of novel putative PPK2-III enzymes was constructed and compared to characterised enzymes belonging to the same class. Multidimensional small-scale screening revealed that PPK12 (from an unclassifiedErysipelotrichaceaebacterium) displays enhanced expression levels, ATP formation rates, polyphosphate tolerance and stability under a variety of harsh conditions. The carboxylic acid reductase (CAR) catalysed reduction of carboxylates to aldehydes was chosen as a model reaction to test the applicability of PPK12 as a bifunctional biocatalyst for ATP regeneration from AMP. The implementation of the identified ATP-recycling enzyme provided the first example of cell free multigram-scale aldehyde synthesis employing enzymes and a single PPK2-III, paving the way for affordable scalable ATP regeneration technologies.
- Tavanti, Michele,Hosford, Joseph,Lloyd, Richard C.,Brown, Murray J. B.
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supporting information
p. 828 - 837
(2021/02/09)
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- Intrinsic Apyrase-Like Activity of Cerium-Based Metal–Organic Frameworks (MOFs): Dephosphorylation of Adenosine Tri- and Diphosphate
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Apyrase is an important family of extracellular enzymes that catalyse the hydrolysis of high-energy phosphate bonds (HEPBs) in ATP and ADP, thereby modulating many physiological processes and driving life activities. Herein, we report an unexpected discovery that cerium-based metal–organic frameworks (Ce-MOFs) of UiO-66(Ce) have intrinsic apyrase-like activity for ATP/ADP-related physiological processes. The abundant CeIII/CeIV couple sites of Ce-MOFs endow them with the ability to selectively catalyse the hydrolysis of HEPBs of ATP and ADP under physiological conditions. Compared to natural enzymes, they could resist extreme pH and temperature, and present a broad range of working conditions. Based on this finding, a significant inhibitory effect on ADP-induced platelet aggregation was observed upon exposing the platelet-rich plasma (PRP) to the biomimetic UiO-66(Ce) films, prefiguring their wide application potentials in medicine and biotechnology.
- Gu, Jinlou,Li, Chunzhong,Li, Ke,Yang, Jian
-
supporting information
p. 22952 - 22956
(2020/10/23)
-
- RECOMBINANT SMNPP5 AND METHODS OF USE
-
Described herein are methods and compositions for reducing coagulation, e.g., in a subject having a coagulation disease or disorder. Aspects of the invention relate to administering to a subject a recombinant SmNPP-5 protein or pharmaceutical composition described herein. Other aspects of the invention relate to methods for producing a recombinant SmNPP-5 protein.
- -
-
Paragraph 00196
(2019/10/04)
-
- Class III Polyphosphate Kinase 2 Enzymes Catalyze the Pyrophosphorylation of Adenosine-5′-Monophosphate
-
Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.
- Ogawa, Marin,Uyeda, Atsuko,Harada, Kazuo,Sato, Yu,Kato, Yasuhiko,Watanabe, Hajime,Honda, Kohsuke,Matsuura, Tomoaki
-
p. 2961 - 2967
(2019/08/26)
-
- DUAL-ACTIVITY NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE INHIBITORS
-
The present disclosure describes NAMPT modulatory compounds, and methods of identifying NAMPT modulatory compounds. The present disclosure also describes methods of testing NAMPT modulatory compounds for NTPase activity, cell mobility modulatory activity, and cell metastasis modulatory activity.
- -
-
Paragraph 00147-00150
(2018/05/24)
-
- SbnI is a free serine kinase that generates O-phospho-L-serine for staphyloferrin B biosynthesis in Staphylococcus aureus
-
Staphyloferrin B (SB) is an iron-chelating siderophore produced by Staphylococcus aureus in invasive infections. Proteins for SB biosynthesis and export are encoded by the sbnABCDEFGHI gene cluster, in which SbnI, a member of the ParB/Srx superfamily, acts as a heme-dependent transcriptional regulator of the sbn locus. However, no structural or functional information about SbnI is available. Here, a crystal structure of SbnI revealed striking structural similarity to an ADP-dependent free serine kinase, SerK, from the archaea Thermococcus kodakarensis. We found that features of the active sites are conserved, and biochemical assays and31P NMR and HPLC analyses indicated that SbnI is also a free serine kinase but uses ATP rather than ADP as phosphate donor to generate the SB precursor O-phospho- L-serine (OPS). SbnI consists of two domains, and elevated B-factors in domain II were consistent with the open-close reaction mechanism previously reported for SerK. Mutagenesis of Glu20 and Asp58 in SbnI disclosed that they are required for kinase activity. The only known OPS source in bacteria is through the phosphoserine aminotransferase activity of SerC within the serine biosynthesis pathway, and we demonstrate that an S. aureus serC mutant is a serine auxotroph, consistent with a function in L-serine biosynthesis. However, the serC mutant strain could produce SB when provided L-serine, suggesting that SbnI produces OPS for SB biosynthesis in vivo. These findings indicate that besides transcriptionally regulating the sbn locus, SbnI also has an enzymatic role in the SB biosynthetic pathway.
- Verstraete, Meghan M.,Perez-Borrajero, Cecilia,Brown, Kirstin L.,Heinrichs, David E.,Murphy, Michael E. P.
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p. 6147 - 6160
(2018/04/27)
-
- PROCESS FOR PREPARATION OF NICOTINAMIDE RIBOSIDE (NR) AND COSMETIC COMPOSITION COMPRISING (NR AND A PHOSPHATE-BINDING AGENT
-
Disclosed herein is a process for preparing nicotinamide riboside (NR) from an NR precursor and a phosphate-binding agent in a solvent. The reaction-derived mixture comprising NR may be further used without further processing in a variety of products, particularly in a cosmetic product.
- -
-
Page/Page column 28
(2017/09/15)
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- Detailed Mechanism of Phosphoanhydride Bond Hydrolysis Promoted by a Binuclear ZrIV-Substituted Keggin Polyoxometalate Elucidated by a Combination of 31P, 31P DOSY, and 31P EXSY NMR Spectroscopy
-
A detailed reaction mechanism is proposed for the hydrolysis of the phosphoanhydride bonds in adenosine triphosphate (ATP) in the presence of the binuclear ZrIV-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (ZrK 2:2). The full reaction mechanism of ATP hydrolysis in the presence of ZrK 2:2 at pD 6.4 was elucidated by a combination of 31P, 31P DOSY, and 31P EXSY NMR spectroscopy, demonstrating the potential of these techniques for the analysis of complex reaction mixtures involving polyoxometalates (POMs). Two possible parallel reaction pathways were proposed on the basis of the observed reaction intermediates and final products. The 1D 31P and 31P DOSY spectra of a mixture of 20.0 mM ATP and 3.0 mM ZrK 2:2 at pD 6.4, measured immediately after sample preparation, evidenced the formation of two types of complexes, I1A and I1B, representing different binding modes between ATP and the ZrIV-substituted Keggin type polyoxometalate (ZrK). Analysis of the NMR data shows that at pD 6.4 and 50 °C ATP hydrolysis in the presence of ZrK proceeds in a stepwise fashion. During the course of the hydrolytic reaction various products, including adenosine diphosphate (ADP), adenosine monophosphate (AMP), pyrophosphate (PP), and phosphate (P), were detected. In addition, intermediate species representing the complexes ADP/ZrK (I2) and PP/ZrK (I5) were identified and the potential formation of two other intermediates, AMP/ZrK (I3) and P/ZrK (I4), was demonstrated. 31P EXSY NMR spectra evidenced slow exchange between ATP and I1A, ADP and I2, and PP and I5, thus confirming the proposed reaction pathways.
- Luong, Thi Kim Nga,Shestakova, Pavletta,Absillis, Gregory,Parac-Vogt, Tatjana N.
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p. 4864 - 4873
(2016/06/01)
-
- Gallium(III)-containing, sandwich-type heteropolytungstates: Synthesis, solution characterization, and hydrolytic studies toward phosphoester and phosphoanhydride bond cleavage
-
The gallium(III)-containing heteropolytungstates [Ga4(H2O)10-- (β-XW9O33)2]6- (X = AsIII, 1; SbIII, 2) were synthesized in aqueous acidic medium by reaction of Ga3+ ions with the trilacunary, lone-pair-containing [XW9O33]9-. Polyanions 1 and 2 are isostructural and crystallized as the hydrated sodium salts Na6[Ga4(H2O)10(β-AsW9O33)2]·28H2O (Na-1) and Na6[Ga4(H2O)10(β-SbW9O33)2]·30H2O (Na-2) in the monoclinic space group P21/c, with unit cell parameters a = 16.0218(12) ?, b = 15.2044(10) ?, c = 20.0821(12) ?, and β = 95.82(0)°, as well as a = 16.0912(5) ?, b = 15.2178(5) ?, c = 20.1047(5) ?, and β = 96.2(0)°, respectively. The corresponding tellurium(IV) derivative [Ga4(H2O)10(β-TeW9O33)2]4- (3) was also prepared, by direct reaction of sodium tungstate, tellurium(IV) oxide, and gallium nitrate. Polyanion 3 crystallized as the mixed rubidium/sodium salt Rb2Na2[Ga4(H2O)10(β- TeW9O33)2]·28H2O (RbNa-3) in the triclinic space group P with unit cell parameters a = 12.5629(15) ?, b = 13.2208(18) ?, c = 15.474(2) ?, α = 80.52(1)°, β = 84.37(1)°, and γ = 65.83(1)°. All polyanions 1-3 were characterized in the solid state by single-crystal XRD, FT-IR, TGA, and elemental analysis, and polyanion 2 was also characterized in solution by 183W NMR and UV-vis spectroscopy. Polyanion 2 was used as a homogeneous catalyst toward adenosine triphosphate (ATP) and the DNA model substrate 4-nitrophenylphosphate, monitored by 1H and 31P NMR spectroscopy. The encapsulated gallium(III) centers in 2 promote the Lewis acidic synergistic activation of the hydrolysis of ATP and DNA model substrates at a higher rate in near-physiological conditions. A strong interaction of 2 with the P-O bond of ATP was evidenced by changes in chemical shift values and line broadening of the 31P nucleus in ATP upon addition of the polyanion.
- Kandasamy, Balamurugan,Vanhaecht, Stef,Nkala, Fiona Marylyn,Beelen, Tessa,Bassil, Bassem S.,Parac-Vogt, Tatjana N.,Kortz, Ulrich
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p. 9204 - 9211
(2016/09/28)
-
- Photo-electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode
-
A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD+. This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.
- Sabir, Nadeem,Khan, Nazimuddin,V?lkner, Johannes,Widdascheck, Felix,Del Pino, Pablo,Witte, Gregor,Riedel, Marc,Lisdat, Fred,Konrad, Manfred,Parak, Wolfgang J.
-
p. 5844 - 5850
(2016/01/25)
-
- KINASE INHIBITORS AND METHODS OF USE THEREOF
-
The present invention provides compounds of formula I, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting kinase (e.g., GSK3 (e.g., GSK3α or GSK3β) or CK1) activity. The present invention further provides methods of using the compounds described herein for treating kinase-mediated disorders, such as neurological diseases, psychriatic disorders, metabolic disorders, and cancer.
- -
-
Page/Page column
(2014/05/07)
-
- Monitoring ATP hydrolysis and ATPase inhibitor screening using 1H NMR
-
We present a versatile method to characterize ATPase and kinase activities and discover new inhibitors of these proteins. The proton NMR-based assay directly monitors ATP turnover and is easy to implement, requires no additional reagents and can potentially be applied to GTP. We validated the method's accuracy, applied it to the monitoring of ATP turnover by actin and to the screening of ATPase inhibitors, and showed that it is also applicable for the monitoring of GTP hydrolysis.
- Guo, Bingqian,Gurel, Pinar S.,Shu, Rui,Higgs, Henry N.,Pellegrini, Maria,Mierke, Dale F.
-
supporting information
p. 12037 - 12039
(2015/02/19)
-
- Spatiotemporal effects of a bioautocatalytic chemical wave revealed by time-resolved mass spectrometry
-
Mass spectrometry has been implemented as an on-line detection tool to monitor transmission of chemical signals due to natural processes such as diffusion and convection as well as a bienzymatic autocatalytic process. It was found that the enzyme-accelerated chemical wave propagates faster than the chemical wave propelled by other processes. The two enzymes (pyruvate kinase and adenylate kinase), involved in the process, work co-operatively catalysing production of adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and induce formation of a front propagating along a high-aspect-ratio drift cell towards the ion source of an ion trap mass spectrometer. Isotopically labelled 13C-ATP was used as the trigger of the accelerated chemical wave. Using this substrate, one could easily distinguish between the two chemical waves (passive and accelerated) in a single experiment, reducing the bias due to the inherent experimental instabilities. We think that-following further improvements-the process described in this report may find applications in bioengineered systems, in which chemical signals need to be transmitted over macroscopic distances.
- Ting, Hsu,Urban, Pawel L.
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p. 2103 - 2108
(2014/01/06)
-
- Molecular characterization and mutational analysis of recombinant diadenosine 5′,5″-P1,P4-tetraphosphate hydrolase from Plasmodium falciparum
-
Asymmetrical diadenosine 5′,5″-P1,P 4-tetraphosphate hydrolase (EC 3.6.1.17) from human malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity, and characterized for the first time as a biological target for chemotherapeutic agents against malaria. Plasmodium falciparum Ap4A (PfAp4A) hydrolase not only catalyzes diadenosine 5′,5″-P1,P4-tetraphosphate (Ap4A) to ATP and AMP, but also diadenosine 5′,5″-P1,P 5-pentaphosphate (Ap5A) to ATP and ADP. Marked enzyme heat stability corresponding to the highest level of activity was observed at 60°C. The recombinant enzyme showed maximal activity in the presence of 5 mM Mg2+ ions. Kinetic analysis revealed the values of Km and Kcat as 0.6 μM and 2.5 min-1, respectively. Comparative protein modeling indicated an additional space in the substrate binding site of the parasitic enzyme compared with that of humans. Mutagenic analysis of the amino acid residue (Pro133) forming the additional space revealed a 5-fold increase in the wild-type Km value when replaced by a smaller (Ala) residue. Furthermore, catalytic activity was markedly affected by introducing a larger residue (Phe), thus creating the potential to develop a specific inhibitor of PfAp4A hydrolase.
- Osman, Waleed,Endo, Satoshi,Oh-hashi, Kentaro,Kitamura, Yoshiaki,Kitade, Yukio
-
experimental part
p. 1191 - 1196
(2012/08/07)
-
- Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4
-
Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45 °C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.
- Chen, Min,Chen, Lei-Lei,Zou, Yang,Xue, Mengyang,Liang, Min,Jin, Lan,Guan, Wan-Yi,Shen, Jie,Wang, Wenjun,Wang, Lei,Liu, Jun,Wang, Peng George
-
experimental part
p. 2421 - 2425
(2011/12/15)
-
- Isolation and identification of diadenosine 5′,5?-P 1,P4-tetraphosphate binding proteins using magnetic bio-panning
-
We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap4A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap4A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap4A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.
- Guo, Wei,Azhar, M. Ameruddin,Xu, Yuhong,Wright, Michael,Kamal, Ahmed,Miller, Andrew D.
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p. 7175 - 7179
(2012/02/02)
-
- Recognition and catalytic hydrolysis of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid
-
Interactions among Cd2+, glutamic acid (Glu), and adenosine 5′-triphosphate (ATP) have been studied by potentiometric pH titration, IR, Raman, fluorescence, and NMR methods. In the Cd2+-ATP binary system, the main interaction sites are the α-, β-, and γ-phosphate groups, N-1, and/or N-7. Cd2+ binds to the N-1 site at relatively low pH and binds to the N-7 site of adenosine ring of ATP with increasing pH. In the Cd2+-Glu-ATP ternary system, ATP mainly binds to Cd2+ by the triphosphate chain. Oxygens of Glu coordinate with Cd2+ to form a complex to catalyze ATP hydrolysis. Hydrolysis of ATP catalyzed by the CdGlu complex was studied at pH 7.0 and 80°C by 31P-NMR spectrometry. Kinetics studies showed that the rate constant of ATP hydrolysis was 0.0199 min-1 in the ternary system, which is 9.9-fold faster than that in the ATP solution (2.01 10-3 min-1). Hydrolysis occurs through an addition-elimination reaction mechanism with Cd2+ regulating the recognition and catalytic hydrolysis of ATP; water participates in the hydrolysis reaction of ATP at different steps with different functions in the ternary system.
- Zhou, Jin-Ying,Lu, Gong-Xuan
-
experimental part
p. 3441 - 3453
(2012/07/14)
-
- Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3′-phosphoadenosine-5′-phosphosulfate
-
Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3′-phosphoadenosine- 5′-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.
- Zhou, Xianxuan,Chandarajoti, Kasemsiri,Pham, Truong Quang,Liu, Renpeng,Liu, Jian
-
experimental part
p. 771 - 780
(2012/02/14)
-
- Catalytic reversibility of Pyrococcus horikoshii trehalose synthase: Efficient synthesis of several nucleoside diphosphate glucoses with enzyme recycling
-
The trehalose synthase (TreT) from Pyrococcus horikoshii represented reversible catalysis in alternative synthesis of trehalose and nucleoside 5′-diphosphate-glucose (NDP-Glc), depending on the substrates involved. TreT from P. horikoshii had differential preferences on NDP-Glc as a donor for trehalose synthesis, in which guanosine 5′-diphosphate (GDP)-Glc was the most favored in terms of reaction specificity, kcat/Km. Uridine 5′-diphosphate (UDP)- and adenosine 5′-diphosphate (ADP)-Glcs were employed with less preferences. This enzyme reversely cleaved trehalose to transfer the glucosyl moiety to various NDPs, efficiently producing NDP-Glcs. Although ADP-Glc was the least favorable donor, the counterpart, ADP, was the most favorable acceptor for the reverse synthesis of NDP-Glc in k cat/Km. GDP and UDP were less preferred, compared to ADP. In a batch reaction of 12 h, the molar yield of NDP-Glc per NDP used was decreased approximately in the order of ADP-Glc > GDP-Glc > cytidine 5′-diphosphate (CDP)-Glc or UDP-Glc. The overall productivity of the enzyme was largely improved in a gram scale for NDP-Glcs using repetitive batch reactions with enzyme recycling. Thus, it is suggested that TreT from P. horikoshii may be useful for the regeneration of NDP-Glc from NDP, especially for ADP-Glc from ADP, with trehalose as a glucose resource.
- Ryu, Soo-In,Kim, Jeong-Eun,Kim, Eun-Joo,Chung, Seung-Kyung,Lee, Soo-Bok
-
experimental part
p. 128 - 134
(2011/09/20)
-
- Cloning of glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars
-
Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for D-glucuronic acid with a Km of 0.7 mM. It requires ATP as phosphate donor (K m 0.56mM). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.
- Pieslinger, Anja Maria,Hoepflinger, Marion Christine,Tenhaken, Raimund
-
experimental part
p. 2902 - 2910
(2011/02/22)
-
- Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
-
The continuous enzymatic gas/solid bio-reactor serves both for the production of volatile fine chemicals and flavors on an industrial scale and for thermodynamically controlled investigation of substrate and water effects on enzyme preparations for research purposes. Here, we comparatively investigated the molecular effects on the operational stability of NADPH-dependent Lactobacillus brevis alcohol dehydrogenase and an NADH-dependent variant thereof, LbADH G37D, in the gas/solid bioreactor. The reference reaction is the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the purpose of regeneration of the redox cofactor. It could be clearly shown that not the thermostability of the cofactor, but the thermostability of the proteins in the solid dry state govern the order of magnitude of the operational stability of both purified enzymes in the gas/solid reactor at low thermodynamic activity of water and substrate. However, at higher thermodynamic activity the operational stability in the gas/solid reactor is overlaid by stabilizing and destabilizing effects of the substrates that require further investigation. We demonstrated first evidence that the substrate affinity of the two variants in the gas/solid reactor is similar to the affinity in aqueous medium. We could also show that partial unfolding of the proteins with subsequent aggregation are the factors governing protein thermo-in-stability both in the dissolved and in the dry state. Thus, stability investigations of enzymes in the dry state are suggested to predict their basal level of operational stability in gas/solid reactions.
- Kulishova, Liliya,Dimoula, Kerasina,Jordan, Max,Wirtz, Astrid,Hofmann, Diana,Santiago-Schuebel, Beatrix,Fitter, Joerg,Pohl, Martina,Spiess, Antje C.
-
experimental part
p. 271 - 283
(2011/02/22)
-
- Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5′-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP)
-
Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5′-phosphosulfate (APS or SO4-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.
- Guranowski, Andrzej,Wojdyla, Anna Maria,Zimny, Jaroslaw,Wypijewska, Anna,Kowalska, Joanna,Jemielity, Jacek,Davis, Richard E.,Bieganowski, Pawel
-
experimental part
p. 93 - 98
(2010/12/18)
-
- Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins
-
Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily. They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their hydrolysis and/or phosphorolysis liberating either the corresponding 5'-NMP or 5'-NDP, respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine 5'-phosphosulfate (1), adenosine 5'-phosphoramidate (2), adenosine 5'-phosphorothioate (3), adenosine 5'-phosphorofluoride (4), diadenosine 5',5'-P1,P3-triphosphate (5), di(7-methylguanosine) 5',5'-P1,P3-triphosphate (6) and adenosine 5'-hypophosphate (7). Preferences for the recognition of these compounds as substrates by individual proteins differed. All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and Trypanosoma brucei HIT-45, cleaved (1), (2), (4), (5) and (6). Caenorhabditis elegans decapping protein DcpS degraded (1), (5), (6) and poorly (4). A. thaliana aprataxin-like protein and Hint4 hydrolyzed only (1), (2) and (4), in that order of efficiency. Velocities of those reactions and some K m values were determined. Applicability of this study to the metabolism of certain nucleotidyl-derivatives is discussed.
- Guranowski, Andrzej,Wojdyla, Anna Maria,Zimny, Jaroslaw,Wypijewska, Anna,Kowalska, Joanna,Lukaszewicz, MacIej,Jemielity, Jacek,Darzynkiewicz, Edward,Jagiello, Agata,Bieganowski, Pawel
-
experimental part
p. 888 - 893
(2010/08/04)
-
- Dual Src and Abl inhibitors target wild type Abl and the AblT315I Imatinib-resistant mutant with different mechanisms
-
The tyrosine kinase Src and its close homolog Abl, both play important roles in chronic myelogenous leukemia (CML) progression and Imatinib resistance. No clinically approved inhibitors of the drug-resistant AblT315I exist to date. Here, we present a thorough kinetic analysis of two potent dual Src-Abl inhibitors towards wild type Src and Abl, and the AblT315I mutant. Our results show that the most potent compound BO1 shows only a modest loss of potency (fourfold) towards the AblT315I mutant in vitro and was an ATP-competitive inhibitor of wild type Abl but it acted as a non-competitive inhibitor in the case of AblT315I.
- Crespan, Emmanuele,Radi, Marco,Zanoli, Samantha,Schenone, Silvia,Botta, Maurizio,Maga, Giovanni
-
experimental part
p. 3999 - 4008
(2010/08/06)
-
- Prodrug activation by Cryptosporidium thymidine kinase
-
Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of kcat= 0.28 s-1 and Km,ATP = 140 μM are similar to those of human TK1, the value of Km(thymidine) = 48 μM is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
- Sun, Xin E.,Sharling, Lisa,Muthalagi, Mani,Mudeppa, Devaraja G.,Pankiewicz, Krzysztof W.,Felczak, Krzysztof,Rathod, Pradipsinh K.,Mead, Jan,Striepen, Boris,Hedstrom, Lizbeth
-
body text
p. 15916 - 15922
(2011/04/15)
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- Mutant APH(2″)-IIa enzymes with increased activity against amikacin and isepamicin
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Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2″-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in Km values, while improved catalytic efficiency for amikacin is the result of both a decrease in Km values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides. Copyright
- Toth, Marta,Frase, Hilary,Chow, Joseph W.,Smith, Clyde,Vakulenko, Sergei B.
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experimental part
p. 1590 - 1595
(2011/11/12)
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- High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2′-5′-oligoadenylates
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Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2′-5′ oligoadenylate synthetases, which synthesize 2′-5′ adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2′-5′ adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2′-5′ adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail. Here, we report a method suitable to produce large amounts of 2-5As of defined lengths in vitro using porcine OAS1 (pOAS) and human OAS2 (hOAS). We have synthesized a broad spectrum of 2-5As at the milligram scale and report an HPLC-purification and characterisation protocol with quantified yield for 2-5A of various lengths.
- Morin,Rabah,Boretto-Soler,Tolou,Alvarez,Canard
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experimental part
p. 345 - 352
(2011/10/07)
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- Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1)
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Carnosine (β-alanyl-L-histidine) and homocarnosine (γ-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on β-alanine than on γ-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.
- Drozak, Jakub,Veiga-da-Cunha, Maria,Vertommen, Didier,Stroobant, Vincent,Van Schaftingen, Emile
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experimental part
p. 9346 - 9356
(2011/03/18)
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- Bimetallic Cu2+ complexes of bis-terpyridine ligands as catalysts of the cleavage of mRNA 5′-cap models. the effect of linker length and base moiety
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Ligands, where two terpyridine units are linked via an alkyl chain of three to five methylene units, have been synthesized. Their Cu2+ complexes have been studied as catalysts for the hydrolysis of the triphosphate bridge of three different dinucleoside triphosphates. The results show that the bimetallic complexes are up to 600 times more efficient catalysts than monomeric Cu2+-TerPy, and up to 5 × 105-fold rate enhancement in comparison to the uncatalysed reaction, is achieved. However, the catalytic activity strongly depends on the length of the linker and the base composition of the substrate. The differences can be attributed to interactions between the Cu2+-TerPy and nucleic acid base moieties as well as steric factors that may hinder the productive interaction between the substrate and the catalyst.
- Maanpaeae, Leena,Luzet, Vincent,Guillaume, Glemence,Taherpour, Sharmin,Maeki, Esa,Mikkola, Satu
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experimental part
p. 1853 - 1858
(2009/12/04)
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- Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase
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Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure-activity relationship of this series of inhibitors led to highly potent compounds.
- Desroy, Nicolas,Moreau, Francois,Briet, Sophia,Fralliec, Geraldine Le,Floquet, Stephanie,Durant, Lionel,Vongsouthi, Vanida,Gerusz, Vincent,Denis, Alexis,Escaich, Sonia
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body text
p. 1276 - 1289
(2009/07/11)
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- Acyclic phosphonate nucleotides and human adenylate kinases: Impact of a borano group on α-P position
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Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The α-borano or α-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the α-phosphate, resulting in substrate bad positioning. Copyright Taylor & Francis Group, LLC.
- Topalis,Alvarez,Barral,Munier-Lehmann,Schneider,Veron,Guerreiro,Mulard,El-Amri,Canard,Deville-Bonne
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p. 319 - 331
(2008/09/20)
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- Discovery and SAR of 1,3,4-thiadiazole derivatives as potent Abl tyrosine kinase inhibitors and cytodifferentiating agents
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A series of substituted benzoylamino-2-[(4-benzyl)thio]-1,3,4-thiadiazoles has been discovered as potent Abl tyrosine kinase inhibitors. Molecular docking simulations on the Abl tyrosine kinase were conducted in order to rationalize the SAR of the synthesized inhibitors. The most active compound identified from the enzymatic screening (6a) showed interesting inhibitory activity on Imatinib-sensitive murine myeloid 3B clone and Bcr-Abl-independent Imatinib-resistant leukemia cells. Surprisingly, 6a was also proved to act as differentiating inducers in human promyelocytic leukemia cells (HL-60).
- Radi, Marco,Crespan, Emmanuele,Botta, Giorgia,Falchi, Federico,Maga, Giovanni,Manetti, Fabrizio,Corradi, Valentina,Mancini, Manuela,Santucci, Maria Alessandra,Schenone, Silvia,Botta, Maurizio
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p. 1207 - 1211
(2008/09/20)
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- Cross-coupling reactions of unprotected halopurine bases, nucleosides, nucleotides and nucleoside triphosphates with 4-boronophenylalanine in water. Synthesis of (purin-8-yl)- and (purin-6-yl)phenylalanines
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An expeditious and highly efficient single-step methodology for the introduction of a phenylalanine moiety into position 8 and 6 of the purine scaffold was developed based on aqueous-phase Pd-catalysed Suzuki-Miyaura cross-coupling reactions of unprotected 4-boronophenylalanine with 8-bromo- or 6-chloropurines. The scope of the methodology was demonstrated by syntheses of unprotected (adenin-8-yl)phenylalanine base, nucleosides, nucleotides and nucleoside triphosphates as well as (purin-6-yl)phenylalanine base and nucleosides. All these products were obtained in high yields and in optically pure form. The Royal Society of Chemistry 2006.
- Capek, Petr,Pohl, Radek,Hocek, Michal
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p. 2278 - 2284
(2008/02/07)
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- Convenient synthesis of nucleoside-5′-diphosphates from the corresponding ribonucleoside-5′-phosphoroimidazole
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The reaction of ribonucleoside-5′-phosphoroimidazolide with a tributylammonium orthophosphate in anhydrous dimethylformamide at room temperature provides a general method for the synthesis of nucleoside-5′- diphosphate. The novelty of the approach is to use the triethylammonium salt of 5′-monophosphate nucleoside derivative prior to the imidazolate reaction with imidazole, triphenylphosphine, and 2,2′-dithiodipyridine. Deprotection, followed by displacement of the imidazole moiety using tributylammonium orthophosphate and a catalytic amount of zinc chloride in dimethylformamide gave the desired 5′-diphosphate products. The triethyl ammonium salt of 5′-diphosphate nucleosides was purified by flash chromatography using DEAE (diethylaminoethyl weak anion exchange resin) Sepharosa fast flow packed in an XK 50/60 column on an Akta FPLC (Fast Protein Liquid Chromatography). Synthesis procedures are reported for adenosine-5′-diphosphate, uridine-5′-diphosphate, cytidine-5′-diphosphate, and guanosine-5′-diphosphate. Yields for the displacement reactions ranged from 95 to 97%. Thus, this method offers the advantages of shorter reaction time, greater product yield, and a more cost-effective synthetic route. Copyright Taylor & Francis Group, LLC.
- Kore, Anilkumar R.,Parmar, Gaurang
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p. 3393 - 3399
(2007/10/03)
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