- Analysis of the Mildiomycin Biosynthesis Gene Cluster in Streptoverticillum remofaciens ZJU5119 and Characterization of MilC, a Hydroxymethyl cytosyl-glucuronic Acid Synthase
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Mildiomycin (MIL) is a peptidyl-nucleoside antibiotic produced by Streptoverticillum remofaciens ZJU5119 that exhibits strong inhibitory activity against powdery mildew. The entire MIL biosynthesis gene cluster was cloned and expressed in Streptomyces lividans 1326. Systematic gene disruptions narrowed down the cluster to 16 functional ORFs and identified the boundaries of the gene cluster. A putative cytosylglucuronic acid (CGA) synthase gene, milC, was disrupted in Sv. remofaciens and heterologously expressed in E. coli. An in vitro assay revealed that purified MilC could utilize either cytosine or hydroxymethylcytosine as substrate to yield CGA or hydroxymethyl-CGA (HM-CGA), respectively. MilG is believed to be a key enzyme in the MIL biosynthesis pathway and contains the CXXXCXXC motif characteristic of members of the radical S-adenosyl methionine (SAM) superfamily. Disruption of milG leads to accumulation of HM-CGA. Labeling experiments with 13C6-L-arginine indicated that decarboxylation at C5 of the pyranoside ring was coupled with the attachment of 5-guanidino-2,4-dihydroxyvalerate side chain through C-C bond formation. In contrast, exogenous 13C6-labeled 4-hydroxy-L-arginine was not incorporated into the MIL structure. Comparative analysis of the 16 MIL ORFs with counterparts involved in the biosynthesis of the structurally similar compound blasticidin S, along with the results above, provide insight into the complete MIL biosynthetic pathway.
- Wu, Jun,Li, Li,Deng, Zixin,Zabriskie, T. Mark,He, Xinyi
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Read Online
- Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics
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Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6′-N-alkyl-5′-C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5′-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2′-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5′S,6′S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.
- Cui, Zheng,Liu, Xiaodong,Overbay, Jonathan,Cai, Wenlong,Wang, Xiachang,Lemke, Anke,Wiegmann, Daniel,Niro, Giuliana,Thorson, Jon S.,Ducho, Christian,Van Lanen, Steven G.
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p. 7239 - 7249
(2018/05/29)
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- Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs
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Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.
- Nakajima, Kazuki,Kizuka, Yasuhiko,Yamaguchi, Yoshiki,Hirabayashi, Yoshio,Takahashi, Kazuo,Yuzawa, Yukio,Taniguchi, Naoyuki
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p. 401 - 407
(2017/11/17)
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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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- METHOD FOR PRODUCING P1,P4-DI(URIDINE 5'-)TETRAPHOSPHATE
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A method for producing P1,P4-di(uridine 5'-)tetraphosphate (UP4U) that can avoid reduction of the synthetic efficiency without using UTP free is developed. A method for producing UP4U comprising reacting a phosphoric acid-activating compound represented by formula [II] or [III] with a phosphoric acid compound selected from the group consisting of UMP, UDP, UTP and a pyrophosphoric acid or a salt thereof (excluding UTP free) in water or a hydrophilic organic solvent, in the presence of a metal ion selected from the group consisting of an iron (II) ion, an iron (III) ion, a trivalent aluminum ion, a trivalent lanthanum ion, and a trivalent cerium ion. where, in the formula [II], R1 represents a uridyl group binding to the 5'-position, X represents a heterocyclic group, and n represents an integer of 1 or 2, where, in the formula [III], X represents a heterocyclic group selected from the group consisting of an imidazolyl group, a benzimidazolyl group, and a 1,2,4-triazolyl group.
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Page/Page column 0056
(2015/11/24)
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- Biosynthesis of CMP-legionaminic acid from fructose-6-P
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The sialic acid-like sugar legionaminic acid is found as a virulence-associated surface glyco-conjugate in Legionella pneumophila and Campylobacter coli. In this work, we have purified and biochemically characterized eleven candidate biosynthetic enzymes from C. jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates and provides a facile means for the efficient large-scale synthesis of an important sialic acid mimic and novel precursors.
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Paragraph 0082
(2015/09/22)
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- Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions
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Regioselective O-β-D-glucosylation of flavonoid core structures is used in plants to create diverse natural products. Their prospective application as functional food and pharmaceutical ingredients makes flavonoid glucosides interesting targets for chemical synthesis, but selective instalment of a glucosyl group requires elaborate synthetic procedures. We report glycosyltransferase-catalysed cascade reactions for single-step highly efficient O-β-D-glucosylation of two major dihydrochalcones (phloretin, davidigenin) and demonstrate their use for the preparation of phlorizin (phloretin 2′-O-β-d-glucoside) and two first-time synthesised natural products, davidioside and confusoside, obtained through selective 2′- and 4′-O-β-d-glucosylation of the dihydroxyphenyl moiety in davidigenin, respectively. Parallel biocatalytic cascades were established by coupling uridine 5′-diphosphate (UDP)-glucose dependent synthetic glucosylations catalysed by herein identified dedicated O-glycosyltransferases (OGTs) to UDP dependent conversion of sucrose by sucrose synthase (SuSy; from soybean). The SuSy reaction served not only to regenerate the UDP-glucose donor substrate for OGT (up to 9 times), but also to overcome thermodynamic restrictions on dihydrochalcone β-d-glucoside formation (up to 20% conversion and yield enhancement). Using conditions optimised for overall coupled enzyme activity, target 2′-O- or 4′-O-β-d-glucoside was obtained in ≥88% yield from reactions consisting of 5 mM dihydrochalcone acceptor, 100 mM sucrose, and 0.5 mM UDP. Davidioside and confusoside were isolated and their proposed chemical structures confirmed by NMR. OGT-SuSy cascade transformations present a green chemistry approach for efficient glucosylation in natural products synthesis. the Partner Organisations 2014.
- Gutmann, Alexander,Bungaruang, Linda,Weber, Hansjoerg,Leypold, Mario,Breinbauer, Rolf,Nidetzky, Bernd
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supporting information
p. 4417 - 4425
(2014/11/08)
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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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supporting information
p. 84 - 87
(2014/01/23)
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- 2,3,6-Trideoxy sugar nucleotides: Synthesis and stability
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The synthesis and characterization of highly challenging 2,3,6-trideoxy sugar nucleotides were described for the first time. The study of their hydrolysis kinetics in aqueous buffers provided insight into their application as glycosyl donors.
- Wu, Mingxuan,Meng, Qingqing,Ge, Min,Bai, Linquan,Zhou, Huchen
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p. 5799 - 5801
(2011/12/14)
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- Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
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Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an l-methionine:uridine-5′-aldehyde aminotransferase; (ii) LipP, a 5′-amino-5′-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-d-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.
- Chi, Xiuling,Pahari, Pallab,Nonaka, Koichi,Van Lanen, Steven G.
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supporting information; experimental part
p. 14452 - 14459
(2011/11/04)
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- A novel fluorescent assay for T-synthase activity
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Loss of T-synthase (uridine diphosphate galactose:Nacetylgalactosaminyl- α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
- Ju, Tongzhong,Xia, Baoyun,PAryal, Rajindra,Wang, Wenyi,Wang, Yingchun,Ding, Xiaokun,Mi, Rongjuan,He, Miao,Richard D. Cummings
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experimental part
p. 352 - 362
(2011/10/09)
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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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- Bimetallic Cu2+ complexes of bis-terpyridine ligands as catalysts of the cleavage of mRNA 5′-cap models. the effect of linker length and base moiety
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Ligands, where two terpyridine units are linked via an alkyl chain of three to five methylene units, have been synthesized. Their Cu2+ complexes have been studied as catalysts for the hydrolysis of the triphosphate bridge of three different dinucleoside triphosphates. The results show that the bimetallic complexes are up to 600 times more efficient catalysts than monomeric Cu2+-TerPy, and up to 5 × 105-fold rate enhancement in comparison to the uncatalysed reaction, is achieved. However, the catalytic activity strongly depends on the length of the linker and the base composition of the substrate. The differences can be attributed to interactions between the Cu2+-TerPy and nucleic acid base moieties as well as steric factors that may hinder the productive interaction between the substrate and the catalyst.
- Maanpaeae, Leena,Luzet, Vincent,Guillaume, Glemence,Taherpour, Sharmin,Maeki, Esa,Mikkola, Satu
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experimental part
p. 1853 - 1858
(2009/12/04)
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- Optimization of UDP-N-acetylmuramic acid synthesis
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UDP-N-acetylmuramic acid (UDP-MurNAc) is a substrate of MurC, an important enzyme in the intracellular pathway of bacterial peptidoglycan biosynthesis. Various approaches towards preparation of UDP-Mur/VAc have been published but these synthetic preparations were shown to include many problematic steps. An optimization study with the focus on muramyl phosphate and UMP-morpholidate coupling was performed, resulting in a synthetic procedure enabling robust and easily reproducible production on a multi-gram scale.
- Humljan, Jan,Starcevic,Car,Stefanic Anderluh,Kocjan,Jenko,Urleb
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p. 102 - 106
(2008/09/21)
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- An enzyme module system for in situ regeneration of deoxythymidine 5′-diphosphate (dTDP)-activated deoxy sugars
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A highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5′-diphosphate (dTDP)-activated deoxy sugars and furthermore enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-β-L-rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So cel2. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP-α-D-glucose, dTDP-β-L-rhamnose, uridine diphosphate (UDP)-α-D-glucose (Glc), and dTDP-6-deoxy-4-keto-α-D-glucose. The synthesis of glucosyl-sorangioside with in situ regeneration of dTDP-Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one-pot for the in situ regeneration of dTDP-activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl-sorangioside and glucosyl-sorangioside, demonstrating the in situ regeneration of dTDP-β-L-rhamnose and dTDP-a-D-glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4-ketoreductase RmlD could be integrated in the one-pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP-activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one-pot.
- Rupprath, Carsten,Kopp, Maren,Hirtz, Dennis,Mueller, Rolf,Elling, Lothar
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p. 1489 - 1496
(2008/09/18)
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- P2Y6 RECEPTOR AGONISTS FOR TREATING LUNG DISEASES
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This invention is directed to a method of enhancing or facilitating the clearance of the lung mucus secretions in a subject. This invention is also directed to a method of facilitating the hydration of the lung mucus secretions in a subject. This invention is further directed to a method of preventing or treating diseases or conditions associated with impaired lung or airway function in a human or other mammal. The method comprises administering to a subject a pharmaceutical composition comprising a therapeutic effective amount of P2Y6 receptor agonist compound, wherein said amount is effective to activate the P2Y6 receptors on the luminal surface of lung epithelia. The P2Y6 receptor agonist compounds useful for this invention include mononucleoside 5'-diphosphates, dinucleoside monophosphate, dinucleoside diphosphates, or dinucleoside triphosphates of general Formula (I), or salts, solvates, hydrates thereof.
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Page/Page column 19-20
(2008/06/13)
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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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- Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3
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We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca 2+>Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis-Menten kinetics with apparent Kms of 11, 10, 19 and 27 μM, respectively. In agreement with the Km values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.
- Lavoie, Elise G.,Kukulski, Filip,Levesque, Sebastien A.,Lecka, Joanna,Sevigny, Jean
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p. 1917 - 1926
(2007/10/03)
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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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p. 743 - 751
(2007/10/03)
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- Structure-activity studies of glucose transfer: Determination of the spontaneous rates of hydrolysis of uridine 5′-diphospho-α-D-glucose (UDPG) and uridine 5′-diphospho-α-D-glucuronic acid (UDPGA)
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The pH-rate profiles for the hydrolysis of uridine 5′-diphospho-α-D-glucose (UDPG) and uridine 5′-diphospho-α-D-glucuronic acid (UDPGA) in aqueous solution have been measured. The results obtained and a comparison with other data suggests that the mechanism of hydrolysis of each activated glycosyl-donor at pH 1-4 probably involves the slow ionisation, via an SN1 process, of the neutral molecule to a glycosyl ion and UDP. From these data, the catalytic power (kcat/kuncat) of the glycosyltransferases has been estimated for the first time to be in the order of 1011-13.
- Bedford, Colin T.,Hickman, Alan D.,Logan, Christopher J.
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p. 2339 - 2345
(2007/10/03)
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- Sialic acid biosynthesis: Stereochemistry and mechanism of the reaction catalyzed by the mammalian UDP-N-acetylglucosamine 2-epimerase
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The bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase, catalyzes the first two steps in the biosynthesis of the sialic acids in mammals. The epimerase domain converts UDP-GlcNAc into ManNAc and UDP. This paper demonstrates that α-ManNAc is the first formed anomer and therefore the reaction proceeds with a net retention of configuration at C-1. Studies in deuterated buffer show that solvent-derived deuterium is quantitatively incorporated into the C-2 position of the product during catalysis, but it is not incorporated into the remaining pool of substrate. This indicates that the inversion of stereochemistry is ultimately brought about by the removal and replacement of a proton at C-2 and is consistent with a two-base mechanism. Studies with 18O-labeled UDP-GlcNAc show that the anomeric oxygen of the glycosyl phosphate bond departs with the UDP product and therefore the net hydrolysis reaction involves C-O bond cleavage. Incubation of the putative intermediate, 2-acetamidoglucal, with the enzyme resulted in a slow hydration reaction to give the product, ManNAc. Additional kinetic isotope effect and positional isotope exchange (PIX) experiments address the nature of the rate-determining step of the reaction and show that C-H bond cleavage is not rate limiting. Overall, these results support a reaction mechanism involving an anti-elimination of UDP to give 2-acetamidoglucal, followed by a synaddition of water.
- Chou, Wayne K.,Hinderlich, Stephan,Reutter, Werner,Tanner, Martin E.
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p. 2455 - 2461
(2007/10/03)
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- Synthesis of 5-fluoro N-acetylglucosamine glycosides and pyrophosphates via epoxide fluoridolysis: Versatile reagents for the study of glycoconjugate biochemistry
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Numerous carbohydrate-processing enzymes facilitate catalysis via stabilization of positive charges on or near the C-1, C-4, C-5, or C-6 positions. Substrate analogues differing only in the substitution of a fluorine for the axial C-5 hydrogen would possess reduced electron density at these positions and could be useful mechanistic probes of these enzymes. Introduction of this 5-fluoro substituent after radical halogenation was problematic because of the incompatibility of many protecting groups to the radical halogenation and the instability of the subsequent 5-fluoro hexosamines. Thus, to allow easy access to a wide variety of 5-fluoro glycosides and glycosyl phosphates, a versatile method for the introduction of the 5-fluoro group has been developed, the key step being the fluoridolysis of C-5, 6 epoxides. By use of this method, two fluorinated carbohydrates, uridine 5′-diphospho-5-fluoro-N-acetylglucosamine and octyl 5-fluoro-N-acetylglucosamine, have been synthesized. Initial biochemical investigations of these compounds show that 5-fluoro analogues are useful probes of transition-state charge development in several enzyme-catalyzed reactions.
- Hartman, Matthew C. T.,Coward, James K.
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p. 10036 - 10053
(2007/10/03)
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- Characterization of Recombinant Sucrose Synthase 1 from Potato for the Synthesis of Sucrose Analogues
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The characteristics and the application of recombinant sucrose synthase 1 (SuSy1) from potato for the synthesis of sucrose analogues are described. With UDP-Glc as donor substrate SuSy1 accepts a variety of ketoses, e.g., 1-deoxy-1-fluoro-D-fructose (6; 100%), L-sorbose (7, 55%), and D-xylulose (8; 42%), as well as aldoses, e.g., D-talose (15; 36%), D-idose (16; 24%), D-lyxose (12; 48%), L-arabinose (13; 36%), and D-ribose (14; 7%). Kinetic analyses revealed that the non-natural acceptors 6 (kcat/ Km = 3.5 s-1 mM-1), 7 (kcat/Km = 1.10-2 s-1 mM-1), 8 (kcat/Km = 2.10-2 s-1 mM-1), and 12 (kcat/Km = 2.10-2 s-1 mM-1) were relatively poor substrates when compared to D-fructose (5; kcat/Km = 34.1 s-1 mM-1). It is concluded that the configuration and/or presence of the hydroxymethyl group at C5 determine the affinity of the ketoses for SuSy1. The acceptance of aldoses can be explained by their flexible chair conformations, which lead to isosteric hydroxy groups recognized by SuSy1. The preparative synthesis of sucrose analogues yielded 1′-deoxy-1′-fluoro-β-D-fructofuranosyl-α-D- glucopyranoside (1), [13C1]-β-D-fructofuranosyl-α-D-glucopyranoside (2), α-D-glucopyranosyl-α-L-sorbofuranoside (3), and α-D-glucopyranosyl-α-D-lyxopyranoside (4), in a 0.1-1.0 g scale. The sucrose analogues 1, 3, and 4 were not hydrolyzed by invertase, which makes them valuable tools for studies on signal transduction pathways and sugar transport in plants.
- Roemer, Ulrike,Nettelstroth, Nadja,Koeckenberger, Walter,Elling, Lothar
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p. 655 - 661
(2007/10/03)
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- Rate enhancements brought about by uridine nucleotides in the reduction of NAD+ at the active site of UDP-galactose 4-epimerase
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UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glucose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible oxidation to the 4-keto- glucopyranosyl ring by reaction with the enzyme-bound NAD+. The UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the enzyme that enhances the chemical reactivity of NAD+ toward reducing agents. This is modeled by UMP-dependent reductive inactivation of the epimerase-NAD+ complex by various sugars as well as by borohydrides. The present work shows that UDP also activates the reduction of epimerase-bound NAD+. Furthermore, the reduction of epimerase-NAD+ by glucose at a very slow rate can be observed under anaerobic conditions in the absence of a uridine nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD+ by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of the rate enhancements brought about by UMP and UDP to be estimated. The rate enhancements by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol-1, respectively, in the activation energy. A decrease of 4.0 kcal mol-1 in the activation energy for reduction by NaBH3CN was brought about by UMP-binding. The maximum increases in the reduction potential of epimerase-NAD+ induced by UMP- and UDP-binding are estimated to be 120 and 90 mV, respectively. The results are well correlated with the perturbations of the nicotinamide-13C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220-12230). (C) 2000 Academic Press.
- Liu, Yijeng,Arabshahi, Abolfazl,Frey, Perry A.
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- Enzymatic production of pyrimidine nucleotides using corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: Enzymatic production of CDP-choline from orotic acid and choline chloride (Part I)
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Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CCTase; encoded by the CKIgene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.
- Fujio,Akihiko
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p. 956 - 959
(2007/10/03)
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- Identification of enzymes catalyzing two-step phosphorylation of cidofovir and the effect of cytomegalovirus infection on their activities in host cells
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Cidofovir [CDV; (S)-1-(3-hydroxy-2-phosphonomethoxyethyl)-cytosine] is an acyclic nucleotide analog with potent and selective in vitro and in vivo activities against a broad spectrum of herpesviruses and other DNA viruses. We studied the mechanism of enzymatic synthesis of CDV diphosphate, the putative antiviral metabolite of CDV. The phosphorylation is two-step process catalyzed by several enzymes. An enzymatic activity phosphorylating CDV to its monophosphate derivative was purified from human liver and identified as pyrimidine nucleoside monophosphate kinase (EC 2.7.4.14.). CDV (K(m) = 2.10 ± 0.18 mM and V(max) = 1.10 ± 0.05 μmol/min/mg) was found to be a substantially weaker substrate for purified enzyme than CMP, UMP, or dCMP. Pyrimidine nucleoside monophosphate kinase was used for preparative enzymatic synthesis of CDV monophosphate. Pyruvate kinase (EC 2.7.1.40), creatine kinase (EC 2.7.3.2), and nucleoside diphosphate kinase (EC 2.7.4.6) were found to catalyze CDV diphosphate synthesis from CDV monophosphate, whereas phosphoglycerate kinase (EC 2.7.2.3) and succinyl-CoA synthetase (EC 6.2.1.4) did not. Based on V(max)/K(m) (phosphorylation efficiency) values determined with enzymes purified from human sources, the most efficient phosphorylation of CDV monophosphate is catalyzed by pyruvate kinase. After infection of human lung fibroblasts with cytomegalovirus, the intracellular activities of pyrimidine nucleoside monophosphate kinase, pyruvate kinase, creatine kinase, and nucleoside diphosphate kinase increased 2-, 1.3-, 3-, and 5-fold, respectively. The metabolism of [3H]CDV in mock- and cytomegalovirus-infected cells was examined. The intracellular levels of CDV monophosphate and CDV diphosphate increased ~20- and 8-fold, respectively, in cytomegalovirus-infected cells, presumably due to the stimulation of CDV uptake and higher activities of phosphorylating enzymes.
- Cihlar, Tomas,Chen, Ming S.
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p. 1502 - 1510
(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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