cas 61-19-8, Adenosine 5'-monophosphate | Articles data

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Dynamic Exchange of Substituents in a Prebiotic Organocatalyst: Initial Steps towards an Evolutionary System
All evolutionary biological processes lead to a change in heritable traits over successive generations. The responsible genetic information encoded in DNA is altered, selected, and inherited by mutation of the base sequence. While this is well known at the biological level, an evolutionary change at the molecular level of small organic molecules is unknown but represents an important prerequisite for the emergence of life. Here, we present a class of prebiotic imidazolidine-4-thione organocatalysts able to dynamically change their constitution and potentially capable to form an evolutionary system. These catalysts functionalize their building blocks and dynamically adapt to their (self-modified) environment by mutation of their own structure. Depending on the surrounding conditions, they show pronounced and opposing selectivity in their formation. Remarkably, the preferentially formed species can be associated with different catalytic properties, which enable multiple pathways for the transition from abiotic matter to functional biomolecules.
Bechtel, Maximilian,Closs, Anna C.,Trapp, Oliver
supporting information

(2021/12/01)

Mouse long-chain acyl-CoA synthetase 1 is active as a monomer
Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.
Dykstra, Holly,Fisk, Chelsea,LaRose, Cassi,Waldhart, Althea,Meng, Xing,Zhao, Gongpu,Wu, Ning
(2021/02/01)

Reduced nicotinamide mononucleotide is a new and potent nad+ precursor in mammalian cells and mice
Nicotinamide adenine dinucleotide (NAD+) homeostasis is constantly compromised due to degradation by NAD+-dependent enzymes. NAD+ replenishment by sup-plementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.
Zapata-Pérez, Rubén,Tammaro, Alessandra,Schomakers, Bauke V.,Scantlebery, Angelique M. L.,Denis, Simone,Elfrink, Hyung L.,Giroud-Gerbetant, Judith,Cantó, Carles,López-Leonardo, Carmen,McIntyre, Rebecca L.,van Weeghel, Michel,Sánchez-Ferrer, álvaro,Houtkooper, Riekelt H.
p. 1 - 17
(2021/03/22)

Rhodamine-based fluorescent probe for sequential detection of Al3+ ions and adenosine monophosphate in water
Organic nanoparticles (N1) were prepared by dispersing thiophene-conjugated rhodamine derivative 1 in a buffer solution (10 mM TRIS, pH 7.4, containing 1% DMSO, v/v). N1 selectively recognized Al3+ ions through the “OFF-ON” switching mechanism of the spirolactam ring in rhodamine. The resulting N1·Al3+ complex recognized the biologically important molecule adenosine monophosphate (AMP) through a cation displacement process with a detection limit of 2 nM. N1 was capable of determining the concentration of Al3+ ions in environmental and biological samples. Portable test strips of N1 were prepared for the recognition of Al3+ ions and AMP for practical uses. Furthermore, it was demonstrated that the N1·Al3+ complex facilitated real-time monitoring of AMP concentration in the hydrolysis of ATP and ADP.
Kaur, Rajinder,Saini, Sanjeev,Kaur, Navneet,Singh, Narinder,Jang, Doo Ok
(2019/09/16)

Intrinsic Apyrase-Like Activity of Cerium-Based Metal–Organic Frameworks (MOFs): Dephosphorylation of Adenosine Tri- and Diphosphate
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.
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Paragraph 00196

(2019/10/04)

Characterization of acetyl-CoA synthetase kinetics and ATP-binding
Background: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.
Gallego-Jara, Julia,Terol, Gema Lozano,écija Conesa, Ana,Zambelli, Barbara,Cánovas Díaz, Manuel,de Diego Puente, Teresa
p. 1040 - 1049
(2019/04/04)

A Stark Contrast to Modern Earth: Phosphate Mineral Transformation and Nucleoside Phosphorylation in an Iron- and Cyanide-Rich Early Earth Scenario
Organophosphates were likely an important class of prebiotic molecules. However, their presence on the early Earth is strongly debated because the low availability of phosphate, which is generally assumed to have been sequestered in insoluble calcium and iron minerals, is widely viewed as a major barrier to organophosphate generation. Herein, we demonstrate that cyanide (an essential prebiotic precursor) and urea-based solvents could promote nucleoside phosphorylation by transforming insoluble phosphate minerals in a “warm little pond” scenario into more soluble and reactive species. Our results suggest that cyanide and its derivatives (metal cyanide complexes, urea, ammonium formate, and formamide) were key reagents for the participation of phosphorus in chemical evolution. These results allow us to propose a holistic scenario in which an evaporitic environment could concentrate abiotically formed organics and transform the underlying minerals, allowing significant organic phosphorylation under plausible prebiotic conditions.
Burcar, Bradley,Casta?eda, Alma,Lago, Jennifer,Daniel, Mischael,Pasek, Matthew A.,Hud, Nicholas V.,Orlando, Thomas M.,Menor-Salván, César
p. 16981 - 16987
(2019/11/11)

Synthesis of Terminal Ribose Analogues of Adenosine 5′-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2
TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5′-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.
Baszczyňski, Ond?ej,Watt, Joanna M.,Rozewitz, Monika D.,Guse, Andreas H.,Fliegert, Ralf,Potter, Barry V.L.
p. 6143 - 6157
(2019/05/24)

Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8
Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg2+. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90 °C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.
Del Arco, Jon,Martinez, María,Donday, Manuel,Clemente-Suarez, Vicente Javier,Fernández-Lucas, Jesús
p. 216 - 223
(2017/09/30)

Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-l-AMP) using TthAPRT and 1-(β-Dribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthPRT.
Fateev, Ilja V.,Sinitsina, Ekaterina V.,Bikanasova, Aiguzel U.,Kostromina, Maria A.,Tuzova, Elena S.,Esipova, Larisa V.,Muravyova, Tatiana I.,Kayushin, Alexei L.,Konstantinova, Irina D.,Esipov, Roman S.
p. 3098 - 3105
(2019/01/21)

Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase
Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-Pβ-Pγ) and phosphorylated (Enz-X-Pβ) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[18O4]-ATP in support of the formation of the Enz-X-Pβ-Pγintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [15N-amide]-l-glutamine in direct support of the Enz-X-Pβintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and β-l-aspartyl hydroxamate, in addition to l-glutamine.
Taylor, Zane W.,Chamberlain, Alexandra R.,Raushel, Frank M.
(2018/09/21)

cUMP hydrolysis by PDE3A
As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a KM value of ~143?μM and a Vmax value of ~42?μmol/min/mg. PDE3A hydrolyzed cAMP with a KM value of ~0.7?μM and a Vmax of ~1.2?μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (Ki?=?57?nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.
Berrisch, Stefan,Ostermeyer, Jessica,Kaever, Volkhard,K?lble, Solveig,Hilfiker-Kleiner, Denise,Seifert, Roland,Schneider, Erich H.
p. 269 - 280
(2017/02/18)

A unique choanoflagellate enzyme rhodopsin exhibits lightdependent cyclic nucleotide phosphodiesterase activity
Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising secondgeneration tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).
Yoshida, Kazuho,Tsunoda, Satoshi P.,Brown, Leonid S.,Kandori, Hideki
p. 7531 - 7541
(2017/05/12)

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.
p. 4864 - 4873
(2016/06/01)

Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila
Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires’ disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC50values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC50-value of 4.24?μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.
Fiene, Amelie,Baqi, Younis,Malik, Enas M.,Newton, Patrice,Li, Wenjin,Lee, Sang-Yong,Hartland, Elizabeth L.,Müller, Christa E.
p. 4363 - 4371
(2016/08/23)

SELECTIVE SOLVENT FREE PHOSPHORYLATION
A synthetic process is provided for the preparation of phosphorylated analogs of nicotinamide riboside ("NR") having the formula (I), or salts thereof, and reduced or modified derivatives thereof, having the formula (II), wherein X-, Y1, Y2, Z1, Z2, n, R1, R2, R3, R4, R5, R6, and R7 are as defined herein. The present disclosure also relates to the preparation of phosphorylated analogs of nicotinic acid riboside ("NAR") having the formula (I), or salts thereof, and reduced or modified derivatives thereof, having the formula (II). Generally solvent-free conditions are employed using appropriate mechano-chemical techniques as described. (I) (II)
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Paragraph 0251; 0252; 0253

(2016/12/22)

Fluorescent Sensing of Guanine and Guanosine Monophosphate with Conjugated Receptors Incorporating Aniline and Naphthyridine Moieties
Ethyne-linked naphthyridine-aniline conjugated molecules are selective sensors of decylguanine in dichloromethane and guanosine monophosphate in water (Kass = 16 000 M-1). The 2-acetamido-1,8-naphthyridine moiety binds with guanine in a DAA-ADD triply hydrogen-bonded motif. The aniline moiety enhances an electron-donating effect, and the substituent is tuned to attain extra hydrogen bonds, π-π stacking, and electrostatic interactions. The proposed binding modes are supported by a Job plot, ESI-MS, 1H NMR, UV-vis, and fluorescence spectral analyses.
Lu, Shao-Hung,Phang, Riping,Fang, Jim-Min
supporting information
p. 1724 - 1727
(2016/05/19)

Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence
Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.
Branchini, Bruce R.,Behney, Curran E.,Southworth, Tara L.,Fontaine, Danielle M.,Gulick, Andrew M.,Vinyard, David J.,Brudvig, Gary W.
supporting information
p. 7592 - 7595
(2015/07/02)

Synthesis of fluorophosphate nucleotide analogues and their characterization as tools for 19F NMR studies
To broaden the scope of existing methods based on 19F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for 19F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m7G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as 19F NMR hybridization probes. The compounds were characterized by 19F NMR and evaluated as 19F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).
Baranowski, Marek R.,Nowicka, Anna,Rydzik, Anna M.,Warminski, Marcin,Kasprzyk, Renata,Wojtczak, Blazej A.,Wojcik, Jacek,Claridge, Timothy D. W.,Kowalska, Joanna,Jemielity, Jacek
p. 3982 - 3997
(2015/07/02)

Structural and kinetic studies on adenylosuccinate lyase from Mycobacterium smegmatis and Mycobacterium tuberculosis provide new insights on the catalytic residues of the enzyme
Adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis, has been recognized as a drug target against microbial infections. In the present study, ASL from Mycobacterium smegmatis (MsASL) and Mycobacterium tuberculosis (MtbASL) were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at a resolution of 2.16 A. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. These structures suggest that His149 and either Lys285 or Ser279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. Most of the active site residues were found to be conserved, with the exception of Ser148 and Gly319 of MsASL. Ser148 is structurally equivalent to a threonine in most other ASLs. Gly319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria and their high GC containing genomes, as well as their dependence on other salvage pathways for the supply of purine nucleotides.
Banerjee, Sanchari,Agrawal, Monika J.,Mishra, Diptimayee,Sharan, Siddharth,Balaram, Hemalatha,Savithri, Handanhal S.,Murthy, Mathur R. N.
p. 1642 - 1658
(2014/05/06)

Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
p. 1575 - 1581
(2015/02/19)

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)

The reaction of activated RNA species with aqueous fluoride ion: A convenient synthesis of nucleotide 5′-phosphorofluoridates and a note on the mechanism
The chemistry of 5′-phosphorimidazolides of ribonucleosides is extended to include their reaction with alkali metal fluorides in aqueous solution. High yields of 5′-phosphorofluoridates are formed, especially with potassium fluoride, but no detectable oligomerization products were formed. A combination of HPLC, mass spectrometry, synthesis, kinetics, and NMR confirms the identities of the products. Judicious control of pH leads to higher yields in shorter reaction times. This new methodology contrasts favorably with other synthetic routes involving non-aqueous chemistry or aqueous chemistry with a nucleotide triphosphate.
Aldersley, Michael F.,Joshi, Prakash C.,Schwartz, Herbert M.,Kirby, Anthony J.
p. 1464 - 1466
(2014/03/21)

PDE7A1 hydrolyzes cCMP
The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC-MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135 ± 19 μM. The Vmax for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.
Monzel, Maike,Kuhn, Maike,B?hre, Heike,Seifert, Roland,Schneider, Erich H.
p. 3469 - 3474
(2015/03/31)

Kinetic mechanism of human histidine triad nucleotide binding protein 1
Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 106-108-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.
Zhou, Xin,Chou, Tsui-Fen,Aubol, Brandon E.,Park, Chin Ju,Wolfenden, Richard,Adams, Joseph,Wagner, Carston R.
p. 3588 - 3600
(2013/07/28)

A nucleotide dimer synthesis without protecting groups using montmorillonite as catalyst
A synthesis has been developed providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately 30% of the imidazolide is converted into each 2-5 dimer and 3-5 dimer with the rest hydrolyzed to the 5-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5-monophosphates.
Joshi, Prakash C.,Aldersley, Michael F.,Zagorevskii, Dmitri V.,Ferris, James P.
experimental part
p. 536 - 566
(2012/10/08)

Mechanism of N6-threonylcarbamoyladenonsine (t6A) biosynthesis: Isolation and characterization of the intermediate threonylcarbamoyl-AMP
Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t6A). In this work, t6A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of l-threonine, bicarbonate/CO2 and ATP to give the intermediate l-threonylcarbamoyl- AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and 1H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of l-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl2), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO2 and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t6A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t6A is somewhat lower than the initial rate from l-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate.
Lauhon, Charles T.
p. 8950 - 8963
(2013/01/15)

Synthesis and enhanced DNA cleavage activities of bis-tacnorthoamide derivatives
A new metal-free DNA cleaving reagent, bis-tacnorthoamide derivative 1 with two tacnorthoamide (tacnoa) units linked by a spacer containing anthraquinone, has been synthesized from triazatricyclo[5.2.1.04,10]decane and characterized by NMR and mass spectrometry. For comparison, the corresponding compounds mono-tacnorthoamide derivative 2 with one tacnorthoamide unit and 6 with two tacnorthoamide units linked by an alkyl (1,6-hexamethylene) spacer without anthraquinone have also been synthesized. The DNA-binding property investigated via fluorescence and CD spectroscopy suggests that compounds 1 and 2 have an intercalating DNA binding mode, and the apparent binding constants of 1, 2 and 6 are 1.3 × 107 M-1, 0.8 × 10 7 M-1 and 8 × 105 M-1, respectively. Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage activity promoted by 1, 2, 6 and parent tacnoa under physiological conditions, which gives rate constants kobs of 0.2126 ± 0.0055 h-1, 0.0620 ± 0.0024 h-1, 0.040 ± 0.0007 h-1 and 0.0043 ± 0.0002 h-1, respectively. The 50-fold and 15-fold rate acceleration over parent tacnoa is because of the anthraquinone moiety of compound 1 or 2 intercalating into DNA base pairs via a stacking interaction. Moreover, DNA cleavage reactions promoted by compound 1 give 5.3-fold rate acceleration over compound 6, which further demonstrates that the introduction of anthraquinone results in a large enhancement of DNA cleavage activity. In particular, DNA cleavage activity promoted by 1 bearing two tacnoa units is 3.3 times more effective than 2 bearing one tacnoa unit and the DNA cleavage by compound 1 was achieved effectively at a relatively low concentration (0.03 mM). This dramatic rate acceleration suggests the cooperative catalysis of the two positively charged tacnoa units in compound 1. The radical scavenger inhibition study and ESI-MS analysis of bis(2,4-dinitrophenyl) phosphate (BDNPP) and adenylyl(3′-5′) phosphoadenine (APA) cleavage in the presence of compound 1 suggest the cleavage mechanism would be via a hydrolysis pathway by cleaving the phosphodiester bond of DNA.
Wei, Li,Hu, Hong-Wen,Lu, Guo-Yuan,Shao, Ying,Zhou, Mi
supporting information
p. 8484 - 8492,9
(2012/12/13)

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)

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues
Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5′-O-sulfamoyl-N 6-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PPi exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.
Chen, Jesse J.,Tsu, Christopher A.,Gavin, James M.,Milhollen, Michael A.,Bruzzese, Frank J.,Mallender, William D.,Sintchak, Michael D.,Bump, Nancy J.,Yang, Xiaofeng,Ma, Jingya,Loke, Huay-Keng,Xu, Qing,Li, Ping,Bence, Neil F.,Brownell, James E.,Dick, Lawrence R.
experimental part
p. 40867 - 40877
(2012/06/30)

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)

Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis
Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4- en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola- 1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.
Capyk, Jenna K.,Casabon, Israel,Gruninger, Robert,Strynadka, Natalie C.,Eltis, Lindsay D.
experimental part
p. 40717 - 40724
(2012/06/29)

Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin
The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and l-luciferin (l-LH2) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and d-luciferin (d-LH2, from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (Ki = 0.88 ± 0.03 μM), L is a tight-binding uncompetitive inhibitor (Ki = 0.00490 ± 0.00009 μM) and l-LH2 acts as a mixed-type non-competitive-uncompetitive inhibitor (Ki = 0.68 ± 0.14 μM and αKi = 0.34 ± 0.16 μM). The Km values obtained for L-CoA, L and l-LH2 were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, respectively. L and l-LH2 are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA Ki supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.
Da Silva, Luis Pinto,Esteves Da Silva, Joaquim C. G.
experimental part
p. 1039 - 1045
(2012/03/08)

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)

Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill
Using a ball mill, rapid, atom-economic coupling between adenosine-5′-phosphoromorpholidate and phosphorylated ribose derivatives as their sodium or barium salts was achieved. Facile purification by reversed-phase HPLC enabled product isolation within hours.
Ravalico, Francesco,Messina, Ivano,Berberian, M. Victoria,James, Stuart L.,Migaud, Marie E.,Vyle, Joseph S.
supporting information; experimental part
p. 6496 - 6497
(2011/11/05)

Synthesis of oligoribonucleotides with phosphonate-modified linkages
Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2′-O-benzoyloxymethoxymethyl protected monomers is presented in both 3′→5′ and 5′→3′ directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3′- and 5′-phosphonate linkages are evaluated. The introduction of the 5′-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔTm -1.8 °C/mod.), whereas the introduction of the 3′-phosphonate units resulted in considerable destabilisation of the duplexes (ΔTm -5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
Pav, Ondej,Koiova, Ivana,Barvik, Ivan,Pohl, Radek,Budinsky, Milo,Rosenberg, Ivan
supporting information; experimental part
p. 6120 - 6126
(2011/10/10)

Protein thiocarboxylate-dependent methionine biosynthesis in Wolinella succinogenes
Thiocarboxylated proteins are important intermediates in a variety of biochemical sulfide transfer reactions. Here we identify a protein thiocarboxylate-dependent methionine biosynthetic pathway in Wolinella succinogenes. In this pathway, the carboxy terminal alanine of a novel sulfur transfer protein, HcyS-Ala, is removed in a reaction catalyzed by a metalloprotease, HcyD. HcyF, an ATP-utilizing enzyme, catalyzes the adenylation of HcyS. HcyS acyl-adenylate then undergoes nucleophilic substitution by bisulfide produced by Sir to give the HcyS thiocarboxylate. This adds to O-acetylhomoserine to give HcyS-homocysteine in a PLP-dependent reaction catalyzed by MetY. HcyD-mediated hydrolysis liberates homocysteine. A final methylation completes the biosynthesis. The biosynthetic gene cluster also encodes the enzymes involved in the conversion of sulfate to sulfide suggesting that sulfate is the sulfur source for protein thiocarboxylate formation in this system.
Krishnamoorthy, Kalyanaraman,Begley, Tadhg P.
experimental part
p. 379 - 386
(2011/03/16)

An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
Gillerman, Irina,Fischer, Bilha
p. 245 - 256
(2011/08/06)

Inhibition of dengue virus RNA synthesis by an adenosine nucleoside
We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin- 7-yl)-3-ethynyl-5-hydroxymethyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis. Copyright
Chen, Yen-Liang,Yin, Zheng,Duraiswamy, Jeyaraj,Schul, Wouter,Lim, Chin Chin,Liu, Boping,Xu, Hao Ying,Qing, Min,Yip, Andy,Wang, Gang,Chan, Wai Ling,Tan, Hui Pen,Lo, Melissa,Liung, Sarah,Kondreddi, Ravinder Reddy,Rao, Ranga,Gu, Helen,He, Handan,Keller, Thomas H.,Shi, Pei-Yong
experimental part
p. 2932 - 2939
(2011/10/09)

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors
A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-γ-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (μM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni2+, Mg2+, Mn2+, and Ca2+). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP). The Royal Society of Chemistry.
Florea, Mara,Nau, Werner M.
supporting information; experimental part
p. 1033 - 1039
(2010/06/20)

A reversed phase hplc method for the analysis of nucleotides to determine 5'-PDE enzyme activity
5'-Phosphodiesterase (5'-PDE) can be extracted from barley roots and used as a catalyst in the hydrolysis of RNA to produce 5'-nucleotides. The assay of enzyme activity is essential for the production of 5'PDE. To improve the conventional assays, we developed and validated a new method for the analysis of 5'-PDE enzyme activity using reversed phased high performance liquid chromatography (RP-HPLC). The method is based on the quantification of the four 5'-nucleotides namely cytidine 5'-monophosphate (5'-CMP), uridine 5'monophosphate (5'-UMP), guanosine 5'-mono-phosphate (5'-GMP) and adenosine 5'-mono-phosphate (5'AMP), produced in the enzymatic hydrolysis of yeast RNA. The optimal condition for the enzymatic hydrolysis of RNA to detect the enzyme activity was investigated. The results show that when the hydrolysis takes place at 70 °C for 30 min at pH 5.0, the hydrolysis reaction has highest yield for the four of the 5'-nucleotides. 5'-PDE demonstrated highest catalytic capability. These four 5'-nucleotides were utilized for the analysis of enzyme activity of 5'-PDE with our newly developed HPLC method. Excellent reproducibility, precision, and linearity were obtained for this HPLC method.
Hua, Jie,Huang, Ke-Long
experimental part
p. 167 - 174
(2011/11/29)

High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2′-5′-oligoadenylates
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
experimental part
p. 345 - 352
(2011/10/07)

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)

Synthesis and DNA cleavage activity of triazacrown-anthraquinone conjugates
1,4,7-Triazacyclononane (TACN), with DNA cleaving ability, was appended to anthraquinone via different spacers to construct the new compounds 1,8-[2,2′-(1,4,7-triazacyclonon)diethoxy] anthracene-9,10-dione hydrochloride (1) and 1,8-[2,2′-(1,4,7-triazacyclonon)dihexyloxy] anthracene-9,10-dione hydrochloride (2) as new agents for metal-free DNA cleavage. Fluorescence and CD spectroscopic studies suggest an intercalating DNA binding mode, and the apparent DNA binding constants of 1 and 2 are 3.93 × 107 and 6.07 × 107 M-1, respectively. Compound 2, bearing the longer spacer, exhibits the higher DNA binding ability. The apparent initial first-order rate constant (k obs) of DNA cleavage promoted by 1 and 2 (0.05 mM) in physiological media are 0.077 ± 0.0028 and 0.123 ± 0.0027 h-1, respectively. The 51-fold and 82-fold rate accelerations over parent TACN (the kobs is 0.0015 ± 0.00003 h-1 (0.05 mM) under the same conditions) are due to the anthraquinone moiety of compounds 1 and 2 intercalating into the DNA base pairs via stacking interactions. ESI-MS analysis of the dinucleotide cleavage promoted by 1 and 2, and radical scavenger inhibition studies suggest that the cleavage process is a hydrolytic mechanism.
Xu, Weihong,Yang, Xiaoli,Yang, Lian,Jia, Zhao-Li,Wei, Li,Liu, Fang,Lu, Guo-Yuan
experimental part
p. 2654 - 2661
(2011/01/12)

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)

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3
Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg2+, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.
Maruoka, Shintaro,Horita, Shoichiro,Lee, Woo Cheol,Nagata, Koji,Tanokura, Masaru
experimental part
p. 417 - 429
(2011/01/03)

What is the conformation of physiologically-active dinucleoside polyphosphates in solution? Conformational analysis of free dinucleoside polyphosphates by NMR and molecular dynamics simulations
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
experimental part
p. 4637 - 4652
(2010/12/19)

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)

Transition state of ADP-ribosylation of acetyllysine catalyzed by archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches
Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes bind NAD+, a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD+-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD+ with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1N-15N] = 1.024(2), [1′N-14C] = 1.014(4), [1′N- 3H] = 1.300(3), [2′N-3H] = 1.099(5), [4′N-3H] = 0.997(2), [5′N- 3H] = 1.020(5), [4′N-18O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.
Cen, Yana,Sauve, Anthony A.
supporting information; experimental part
p. 12286 - 12298
(2010/11/03)