- Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones
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Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering ZnII to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for ZnII metallochaperone activity. ZnII chelator competition experiments reveal one high-affinity (KZn1 ≈ 1010-1011 M-1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with ZnII more effective than CoII. Both GTPases bind the product, GDP, more tightly in the apoprotein than the ZnII-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.
- Jordan, Matthew R.,Wang, Jiefei,Weiss, Andy,Skaar, Eric P.,Capdevila, Daiana A.,Giedroc, David P.
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- What is the conformation of physiologically-active dinucleoside polyphosphates in solution? Conformational analysis of free dinucleoside polyphosphates by NMR and molecular dynamics simulations
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Dinucleoside polyphosphates, or dinucleotides (NpnN′; N, N′ = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of NpnN′ Na+ salts (N = A, G, U, C; N′ = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up4/5U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in NpnN′s was found to be anti/high anti and the preferred conformation around the C4′-C5′, C5′-O5′ bonds was found to be gauche-gauche (gg). The ribose moiety in NpnN′s showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap2A and Ap4A Na+ salts supported the experimental results. In addition, three modes of base-stacking were found for Ap2/4A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free NpnN′s (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of NpnN′s does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.
- Stern, Noa,Major, Dan Thomas,Gottlieb, Hugo Emilio,Weizman, Daniel,Fischer, Bilha
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- One-Pot Synthesis of α,γ-Dinucleoside 5'-Triphosphates, G5'pppG and A5'pppA, Using S,S'-Bis(4-chlorophenyl)phosphorodithioate
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S,S'-Bis(4-chlorophenyl) phosphorodithioate was useful for the synthesis of α,γ-dinucleoside 5'-triphosphates, G5'pppG and A5'pppA starting from the corresponding unprotected nucleoside 5'-phosphates under neutral conditions. G5'pppG was used for the synthesis of m7G5'pppG by means of the N7-methylation of one of two guanine moieties of G5'pppG.
- Fukuoka, Koichiro,Suda, Fuminori,Suzuki, Ryo,Ishikawa, Masahide,Hata, Tsujiaki
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- Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain
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Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the β-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. Database: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.
- Ince, Semra,Zhang, Ping,Kutsch, Miriam,Krenczyk, Oktavian,Shydlovskyi, Sergii,Herrmann, Christian
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- Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8)
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Background: Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell-cell recognition and host-pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme. Methods: We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein-O-fucosyltransferase (cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations. Results: Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the β-phosphate and the fucose moiety at the same time. Conclusions: Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques. General significance: The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors.
- K?tzler, Miriam P.,Blank, Simon,Bantleon, Frank I.,Spillner, Edzard,Meyer, Bernd
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- The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O- and S-glycosylation
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The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the thuS gene cluster was reconstituted in vitro, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward Bacillus thuringiensis.
- Wang, Huan,Oman, Trent J.,Zhang, Ran,Garcia De Gonzalo, Chantal V.,Zhang, Qi,Van Der Donk, Wilfred A.
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- THE PREPARATION OF ADENOSINE 5'-PYROPHOSPHATE BY A NON-ENZYMIC METHOD.
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1. A non-enzymic method for the preparation of adenosine 5'-diphosphate
- DAWSON,FORD,EICHBERG
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- Phosphorylation of ganciclovir phosphonate by cellular GMP kinase determines the stereoselectivity of anti-human cytomegalovirus activity
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A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S- enantiomer was less active. We show that enantiomeric selectivity of antiviral activity for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoslective inhibition of DNA polymerase from human cytomegalovirus.
- Miller, Wayne H.,Beauchamp, Lilia M.,Meade, Eric,Reardon, John E.,Biron, Karen K.,Smith, Albert A.,Goss, Charles A.,Miller, Richard L.
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- Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with G(i)-proteins in HL-60 leukemia cell membranes
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G proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP) bound form, G-protein α-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their α-subunits. Like GTP, inosine 5' -triphosphate (ITP) and xanthosine 5' triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L- phenylaline (fMLP) and leukotriene B4 (LTB4,) effectively activated GTP and ITP hydrolysis by G(i) proteins. fMLP and LTB4, were at least l0-fold more potent activators of ITPase than of GTPasc. Complement C5a effectively activated the GTPase of G(i)-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP stimulated GTPase had a lower K(m) value than the fMLP-stimulated ITPase, whereas the opposite was true for the V(max) values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by G(i)-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.
- Klinker, Jan F.,Seifert, Roland
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- Biochemical properties of the human guanylate binding protein 5 and a tumor-specific truncated splice variant
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The human guanylate binding protein 5 (hGBP5) belongs to the family of interferon-γ-inducible large GTPases, which are well known for their high induction by pro-inflammatory cytokines. The cellular role of this protein family is unclear at this point, but there are indications for antiviral and antibacterial activity of hGBP1. hGBP5 exists in three splice variants, forming two different proteins, of which the tumor-specific one is C-terminally truncated by 97 amino acids, and therefore lacks the CaaX motif for geranylgeranylation. Here we present biochemical data on the splice variants of hGBP5. We show that, unlike hGBP1, hGBP5a/b and hGBP5ta do not bind GMP or produce any GMP during hydrolysis despite the fact the residues involved in GMP production from hGBP1 are conserved in hGBP5. Hydrolysis of GTP is concentration-dependent and shows weak self-activation. Thermodynamic studies showed strongly negative entropic changes during nucleotide binding, which reflect structural ordering in the protein during nucleotide binding. These structural changes were also observed during changes in the oligomerization state. We observed only a minor influence of the C-terminal truncation on hydrolysis, nucleotide binding and oligomerization of hGBP5. Based on these similarities we speculate that the missing C-terminal part, which also carries the geranylgeranylation motif, is the reason for the dysregulation of hGBP5′s function in lymphoma cells.
- Wehner, Mark,Herrmann, Christian
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- Enzymatic synthesis of UTPγS, a potent hydrolysis resistant agonist of P2U-purinoceptors
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1 The defective Cl- secretion characteristic of cystic fibrosis airway epithelial cells can be bypassed by an alternative Ca2+ dependent Cl- secretory pathway that is activated by extracellular nucleotides, e.g. uridine-5′triphosphate (UTP), acting on P2U purinoceptors. Since UTP is susceptible to hydrolysis by nucleotidases and phosphatases present in the airways, the identification of stable P2U-purinoceptor agonists would be of therapeutic relevance. 2 Uridine-5′-O-(3-thiotriphosphate) (UTPγS) was synthesized by nucleoside diphosphate kinase-catalyzed transfer of the γ-phosphorothioate from guanosine-5′-O-(3-thiotriphosphate) (GTPγS) or adenosine-5′-O-(3-thiotriphosphate) (ATPγS) to UDP. Formation of UTPγS was illustrated by observation of transfer of 35S from [35S]-GTPγS and transfer of 3H from [3H]-UDP. The chemical identity of high performance liquid chromatography (h.p.l.c.)-purified UTPγS was confirmed by nuclear magnetic resonance analysis. 3 Human 1321N1 astrocytoma cells stably expressing the phospholipase C-coupled human P2U-purinoceptor were utilized to lest the activity of UTPγS. UTPγS (EC50 = 240 nM) was essentially equipotent to UTP and ATP for stimulation of inositol phosphate formation. 4 Unlike [3H]-UTP, [3H]-UTPγS was not hydrolyzed by alkaline phosphatase, acid phosphatase, or apyrase. Moreover, no hydrolysis was detected during a 1 h incubation with human nasal epithelial cells. 5 UTPγS was equally potent and efficacious with UTP for stimulation of Cl- secretion by human nasal epithelium from both normal donors and cystic fibrosis patients. Based on its high potency and resistance to hydrolysis, UTPγS represents a promising compound for treatment of cystic fibrosis.
- Lazarowski, Eduardo R.,Watt, William C.,Stutts, M. Jackson,Brown, H. Alex,Boucher, Richard C.,Harden, T. Kendall
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- Catalysis of Hydrolysis and Nucleophilic Substitution at the P-N Bond of Phosphoimidazolide-Activated Nucleotides in Phosphate Buffers
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Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 (*) pH (*) 8.6.This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phophate.The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate.Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities.The results show that both H2PO4(-) and HPO4(2-) participate in both hydrolysis and nucleophilic substitution.Statistically corrected bimolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile.The low Bronsted value β = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before.The βnuc = 0.35 calculated for water, H2PO4(-), HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR(-) with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (βnuc = 0.25).With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phophate, assuming the concentrations of phosphate, Pj, on prebiotic Earth were similar to those in the present oceans (j> ca. 2.25 μM).
- Kanavarioti, Anastassia,Rosenbach, Morgan T.
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- Molecular Cloning of cDNA for BRab from the Brain of Bombyx mori and Biochemical Properties of BRab Expressed in Escherichia coli
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From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rabl family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTPγS and [3H]-GDP with association constants of 1.5 × 106M-1 and 0.58 × 106 M-1, respectively. The binding of [35S]-GTPγS was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgC12, bound [35S]-GTPγS and [3H]-GDP were exchanged with GTPyS most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
- Uno, Tomohide,Ueno, Mayumi,Nakajima, Ayumi,Shirai, Yasuhito,Aizono, Yasuo
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- Helicobacter hepaticus Hh0072 gene encodes a novel α1-3- fucosyltransferase belonging to CAZy GT11 family
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Lewis x (Lex) and sialyl Lewis x (SLex)-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is α1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus α1-3- fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Lex, sialyl Lex and their derivatives. Unlike all other α1-3/4- fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with α1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first α1-3-fucosyltransferase identified in the GT11 family.
- Zhang, Lei,Lau, Kam,Cheng, Jiansong,Yu, Hai,Li, Yanhong,Sugiarto, Go,Huang, Shengshu,Ding, Li,Thon, Vireak,Wang, Peng G,Chen, Xi
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- Photo-electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode
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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.
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- Borate-nucleotide complex formation depends on charge and phosphorylation state
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Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 μM nucleotide and 500 μM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3
- Kim, Danny H.,Faull, Kym F.,Norris, Andrew J.,Eckhert, Curtis D.
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- Structural analysis of the activation of ribavirin analogs by NDP kinase: Comparison with other ribavirin targets
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Ribavirin used in therapies against hepatitis C virus (HCV) is potentially efficient against other viruses but presents a high cytotoxicity. Several ribavirin triphosphate analogs modified on the ribose moiety were synthesized and tested in vitro on the RNA polymerases of HCV, phage T7, and HIV-1 reverse transcriptase. Modified nucleotides with 2′-deoxy, 3′-deoxy, 2′,3′-dideoxy, 2′,3′-dideoxy-2′,3′-dehydro, and 2′,3′-epoxy-ribose inhibited the HCV enzyme but not the other two polymerases. They were also analyzed as substrates for nucleoside diphosphate (NDP) kinase, the enzyme responsible for the last step of the cellular activation of antiviral nucleoside analogs. An X-ray structure of NDP kinase complexed with ribavirin triphosphate was determined. It demonstrates that the analog binds as a normal substrate despite the modified base and confirms the crucial role of the 3′-hydroxyl group in the phosphorylation reaction. The 3′-hydroxyl is required for inhibition of the initiation step of RNA synthesis by HCV polymerase, and both sugar hydroxyls must be present to inhibit elongation. The 2′deoxyribavirin is the only derivative efficient in vitro against HCV polymerase and properly activated by NDP kinase.
- Gallois-Montbrun, Sarah,Chen, Yuxing,Dutartre, Helene,Sophys, Magali,Morera, Solange,Guerreiro, Catherine,Schneider, Benoit,Mulard, Laurence,Janin, Joel,Veron, Michel,Deville-Bonne, Dominique,Canard, Bruno
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- DUAL-ACTIVITY NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE INHIBITORS
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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.
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Paragraph 00128; 00133; 00134; 00136; 00138; 00140-00142
(2018/05/24)
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- Efficient enzymatic synthesis of guanosine 5′-diphosphate-sugars and derivatives
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An N-acetylhexosamine 1-kinase from Bifidobacterium infantis (NahK-15697), a guanosine 5′-diphosphate (GDP)-mannose pyrophosphorylase from Pyrococcus furiosus (PFManC), and an Escherichia coli inorganic pyrophosphatase (EcPpA) were used efficiently for a one-pot three-enzyme synthesis of GDP-mannose, GDP-glucose, their derivatives, and GDP-talose. This study represents the first facile and efficient enzymatic synthesis of GDP-sugars and derivatives starting from monosaccharides and derivatives.
- Li, Lei,Liu, Yonghui,Wan, Yue,Li, Yanhong,Chen, Xi,Zhao, Wei,Wang, Peng George
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supporting information
p. 5528 - 5530
(2013/11/19)
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- Enzymatic and molecular characterization of arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26
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Not only in bacteria but also in plant cells, guanosine- 3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrola
- Ito, Daisuke,Kato, Takahiro,Maruta, Takanori,Tamoi, Masahiro,Yoshimura, Kazuya,Shigeoka, Shigeru
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p. 2236 - 2241
(2013/02/25)
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- Catalytic reversibility of Pyrococcus horikoshii trehalose synthase: Efficient synthesis of several nucleoside diphosphate glucoses with enzyme recycling
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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
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experimental part
p. 128 - 134
(2011/09/20)
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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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- 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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- Macrocyclic amines as catalysts of the hydrolysis of the triphosphate bridge of the mRNA 5′-cap structure
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The reactions of a 5′-cap model compound P 1-(7-methylguanosine) P3-guanosine 5′,5′-triphosphate, m7GpppG, were studied in the presence of three different macrocyclic amines (2-4) under neutral conditions. The only products observed in the absence of the macrocycles resulted from the base-catalysed imidazole ring-opening and the acid-catalysed cleavage of the N7-methylguanosine base, whereas in the presence of these catalysts hydrolysis of the triphosphate bridge predominated. The latter reaction yielded guanosine 5′-monophosphate, guanosine 5′-diphosphate, 7-methylguanosine 5′-monophosphate and 7-methylguanosine 5′-diphosphate as the initial products, indicating that both of the phosphoric anhydride bonds were cleaved. The overall catalytic activity of all three macrocycles was comparable. The hydrolysis to guanosine 5′-diphosphate and 7-methylguanosine 5′-monophosphate was slightly more favoured than the cleavage to yield guanosine 5′-monophosphate and 7-methylguanosine diphosphate. All the macrocycles also enhanced the subsequent hydrolysis of the nucleoside diphosphates, 2 being more efficient than 3 and 4.
- Zhang, Zhibo,Loennberg, Harri,Mikkola, Satu
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p. 3404 - 3409
(2007/10/03)
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- A synthetic pentasaccharide with GTPase activity.
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The design, synthesis, and preliminary evaluation of the first example of synthetic pentasaccharide (1) that shows marked rate enhancement and specificity for the hydrolysis of GTP to GDP and orthophosphate (OP) are reported. At the concentration ratios of GTP/1 = 3.6 and GTP/Mg(2+) = 1 (pH 7.1, 50 degrees C), a rate enhancement of about 500-fold was obtained.[reaction: see text]
- Solomon,Fridman,Zhang,Baasov
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p. 4311 - 4314
(2007/10/03)
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- Hydrolytic Susceptibilities of Modified 5'-mRNA Cap Analogues to the Yellow Lupin Ap4A Hydrolases
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Several new dinucleoside tri- and tetraphosphates, 5'-mRNA cap analogues, have been tested as potential substrates for two enzymes: highly specific homogeneous Ap3A and (asymmetrical) Ap4A hydrolase from yellow lupin seeds.The Ap3A hydrolase cleaves all examined dinucleotide triphosphates yielding in most cases GMP or m7GMP and modified diphosphates as the main hydrolysis products (75-100percent).Only m7Gp3G has been hydrolyzed randomly.Compounds with unmodified guanosine have been degraded at comparable rate as natural substrate Ap3A.For the analogues modified in guanosine or sugar moiety significant differences have been observed in the rate of hydrolysis.Ap4A hydrolase seems to be more specific enzyme than Ap3A hydrolase.Dinucleoside tetraphosphates containing guanosine or 7-methylguanosine have been degraded more slowly than Ap4A.In contrast, di- and trimethylated compounds have been hydrolyzed with the rate 2-fold higher in comparison with Ap4A.For all methylated dinucleoside tetraphosphates, GTP and methylated nucleoside monophosphates have been found as the main hydrolysis products.
- Bojarska, Elzbieta,Stepinski, Janusz,Guranowski, Andrzej,Starzynska, Elzbieta,Chlebicka, Lidia,et al.
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p. S192 - S196
(2007/10/03)
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- Nucleoside-Triphosphatase Activity of an ATP-Dependent Enzyme, N-Methylhydantoin Amidohydrolase
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N-Methylhydantoin amidohydrolase, which catalyzes ATP-dependent hydrolysis of N-methylhydantoin to N-carbamoylsarcosine, was found to hydrolyze several nucleoside triphosphates to nucleoside diphosphates not only in the presence but also in the absence of amide substrates.Amide substrates, such as N-methylhydantoin and dihydrouracil, seem to be absolutely necessary for hydrolysis of ATP and dATP.However, N-methylhydantoin inhibited the hydrolysis of nucleoside triphosphates other than ATP and dATP.The kinetic data suggest that the presence of an amide substrate changes the affinity of the enzyme toward nucleoside triphosphates.
- Ogawa, Jun,Nirdnoy, Warawadee,Yamada, Hideaki,Shimizu, Sakayu
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p. 1737 - 1739
(2007/10/02)
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- Mg(II) ION-MEDIATED CONVERSION OF MONO- AND OLIGONUCLEOTIDES TO 5'-POLYPHOSPHATES IN AQUEOUS SOLUTION
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5'-Polyphosphates of mono- and oligonucleotides were prepared from the corresponding 5'-monophosphates with phosphorotriimidazolide or phosphorotribenzimidazolide mediated by Mg(II) or Mn(II) ion in aqueous solution.
- Sawai, Hiroaki,Inaba, Yoshiko,Hirano, Atsushi,Wakai, Hiromichi,Shimazu, Masamitsu
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p. 4801 - 4804
(2007/10/02)
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- Synthesis of Nucleotide 5'-Diphosphates from 5'-O-Tosyl Nucleosides
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Procedures are described for the synthesis of nucleoside 5'-diphosphates, methanediphosphonates, and difluoromethanediphosphonates.The general strategy involves protection of the nucleosides as amidine, 2',3'-methoxymethylidene, and 3'-(tert-butyldimethylsilyl) derivatives prior to tosylation with tosyl chloride and (N,N-dimethylamino)pyridine.Deprotection, followed by displacement of the tosyl moiety with the tris(tetra-n-butylammonium) pyrophosphate, methanediphosphonate, or difluoromethanediphosphonate salts gave the desired products.The ammonium salts of the nucleotides were purified by flash chromatography on cellulose or medium pressure ion-exchange chromatography on DEAE Fractogel.Syntheses are reported for UDP (18), CDP (19), TDP (20), GDP (21), ADP (23), 2',3'-isopropylidene-ADP (22), adenosine 5'-methanediphosphonate (24), adenosine 5'-difluoromethanediphosphonate (25), and deoxyadenosine 5'-methanediphosphonate (27).In addition ATP (26) was prepared by treatment of 5'-O-tosyladenosine with tetrakis(tetra-n-butylammonium) thiophosphate.Yields for the displacement reactions ranged from 43percent to 93percent.
- Davisson, V. Jo,Davis, Darrell R.,Dixit, Vyas M.,Poulter, C. Dale
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p. 1794 - 1801
(2007/10/02)
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