RESUMEN
UDP-Xyl, a nucleotide sugar involved in the biosynthesis of various glycoconjugates, is difficult to obtain and quite expensive. Biocatalysis using a one-pot multi-enzyme cascade is one of the most valuable biotransformation processes widely used in the industry. Herein, two enzymes, UDP-glucose (UDP-Glc) dehydrogenase (CGIUGD) and UDP-Xyl synthase (CGIUXS) from the Pacific oyster Crassostrea gigas, which are coupled together for the biotransformation of UDP-Xyl, were characterized. The optimum pH was determined to be pH 9.0 for CGIUGD and pH 7.5 for CGIUXS. Both enzymes showed the highest activity at 37 °C. Neither enzyme is metal ion-dependent. On this basis, a single factor and orthogonal test were applied to optimize the condition of biotransformation of UDP-Xyl from UDP-Glc. Orthogonal design L9 (33) was conducted to optimize processing variables of enzyme amount, pH, and temperature. The conversion of UDP-Xyl was selected as an analysis indicator. Optimum variables were the ratio of CGIUGD to CGIUXS of 2:5, enzymatic pH of 8.0, and temperature of 37 °C, which is confirmed by three repeated validation experiments. The UDP-Xyl conversion was 69.921% in a 1 mL reaction mixture by optimized condition for 1 h. This is the first report for the biosynthesis of UDP-Xyl from oyster enzymes.
Asunto(s)
Biocatálisis , Crassostrea/genética , Ligasas/química , Oxidorreductasas/química , Uridina Difosfato/síntesis química , Animales , Crassostrea/enzimología , Ligasas/genética , Oxidorreductasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Uridina Difosfato/químicaRESUMEN
Unnatural chemically modified nucleotide sugars UDP-4-N3-GlcNAc and UDP-4-N3-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N3-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N3-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.
Asunto(s)
Glicosaminoglicanos/análisis , Hexosaminas/síntesis química , Uridina Difosfato/síntesis química , Química Clic , Hexosaminas/química , Conformación Molecular , Uridina Difosfato/químicaRESUMEN
UDP-galactofuranose (UDP-Galf) is the donor substrate for both bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which are involved in the biosynthesis of mycobacterial galactan. In this paper, a group of UDP-Galf mimics were synthesized via reductive amination of a bicyclo[3.1.0]hexane-based amine by reacting with aromatic, linear, or uridine-containing aldehydes. These compounds were evaluated against GlfT2 using a coupled spectrophotometric assay, and were shown to be weak inhibitors of the enzyme.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Hexanos/química , Mycobacterium/enzimología , Uridina Difosfato/análogos & derivados , Diseño de Fármacos , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Mycobacterium/efectos de los fármacos , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacologíaRESUMEN
Two complementary methods for the synthesis of fluorinated exo-glycals have been developed, for which previously no general reaction had been available. First, a Selectfluor-mediated fluorination was optimized after detailed analysis of all the reaction parameters. A dramatic effect of molecular sieves on the course of the reaction was observed. The reaction was generalized with a set of biologically relevant furanosides and pyranosides. A second direct approach involving carbanionic chemistry and the use of N-fluorobenzenesulfonimide (NFSI) was performed and this method gave better diastereoselectivities. Assignment of the Z/E configuration of all the fluorinated exo-glycals was achieved based on the results of HOESY experiments. Furthermore, fluorinated exo-glycal analogues of UDP-galactofuranose were prepared and assayed against GlfT2, which is a key enzyme involved in the cell-wall biosynthesis of major pathogens. The fluorinated exo-glycals proved to be potent inhibitors as compared with a series of C-glycosidic analogues of UDP-Galf, thus demonstrating the double beneficial effect of the exocyclic enol ether functionality and the fluorine atom.
Asunto(s)
Compuestos de Diazonio/química , Inhibidores Enzimáticos/química , Galactosa/análogos & derivados , Galactosiltransferasas/antagonistas & inhibidores , Sulfonamidas/química , Uridina Difosfato/análogos & derivados , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Compuestos de Diazonio/síntesis química , Compuestos de Diazonio/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Galactosiltransferasas/metabolismo , Halogenación , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Tuberculosis/tratamiento farmacológico , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacologíaRESUMEN
Esters and anhydrides of phosphoric acid are essential in biology. It is very difficult to identify processes in life that do not involve these modifications and their transformation at some point. Consequently, phosphorylation chemistry is an essential methodology with significant impact on the biological sciences. This perspective gives an overview of some very recent achievements in synthetic phosphorylation chemistry and aims at identifying challenges that lie ahead.
Asunto(s)
Anhídridos/química , Ésteres/química , Fosfatos/síntesis química , Anhídridos/síntesis química , Nucleótidos de Desoxiguanina/síntesis química , Nucleótidos de Desoxiguanina/química , Fosfatos/química , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Monofosfato/químicaRESUMEN
We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.
Asunto(s)
N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Uridina Difosfato/síntesis química , Animales , Células CHO , Cricetinae , Difosfatos/química , Activación Enzimática , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosafosfatos/química , Espectroscopía de Resonancia Magnética , N-Acetilglucosaminiltransferasas/química , Proteínas Recombinantes/química , Solventes/química , Tetrazoles/química , Factores de Tiempo , Uridina Difosfato/análogos & derivados , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-D-galactopyranose into UDP-α-D-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-D-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.
Asunto(s)
Galactosa/análogos & derivados , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/química , Uridina Difosfato/análogos & derivados , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dominio Catalítico , Cristalografía por Rayos X , Deinococcus/enzimología , Deinococcus/genética , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Transferasas Intramoleculares/genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Modelos Moleculares , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Sondas Moleculares/farmacología , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacologíaRESUMEN
P2Y nucleotide receptors (P2Y-Rs) play important physiological roles. However, most of the P2Y-R subtypes are still lacking potent and selective agonists and antagonists. Based on data mining analysis of binding interactions in 44 protein-uridine nucleos(t)ides complexes, we designed uracil nucleotides, substituted at the C5/C6 position. All C6-substituted derivatives were inactive at the P2Y(2,4,6)-Rs, while out of the C5-substituted analogues, only 5-OMe-UD(T)P showed activity. To rationalize the data, the ionization and conformation of these analogues were evaluated. The pK(a) values of most analogues substituted at the C5/C6 positions were unaltered compared to UTP (pK(a) 9.42), except for 5-F-UTP nucleotide (pK(a) 7.85). C6-substituted analogues adopt the syn or high-syn conformations, which are disfavored by the receptors, while 5-OMe-UD(T)P adopt the favored anti conformation. Furthermore, 5-OMe-UDP adopts the S sugar puckering, which is the conformation preferred by the P2Y(6)-R, but not the P2Y(2)- or P2Y(4)-Rs. 5-OMe-UDP fulfills the conformational and H-bonding requirements of P2Y(6)-R, thus, making a potent P2Y(6)-R agonist (EC(50) 0.08 microM), more than UDP (EC(50) 0.14 microM).
Asunto(s)
Agonistas del Receptor Purinérgico P2 , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Receptores Purinérgicos P2/genética , Relación Estructura-Actividad , Transfección , Uridina Difosfato/farmacologíaRESUMEN
Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.
Asunto(s)
Nucleótidos/síntesis química , Agonistas del Receptor Purinérgico P2 , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/síntesis química , Adenosina Difosfato/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/química , Línea Celular Tumoral , Humanos , Nucleótidos/química , Receptores Purinérgicos P2/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntesis química , Uridina Trifosfato/químicaRESUMEN
UDP-galactopyranose mutase (UGM or Glf), which catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose, is implicated in the viability and virulence of multiple pathogenic microorganisms. Here we report the synthesis of high-affinity ligands for UGM homologues from Klebsiella pneumoniae and Mycobacterium tuberculosis. The potency of these compounds stems from their ability to access both the substrate binding pocket and an adjacent site.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Transferasas Intramoleculares/antagonistas & inhibidores , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato/análogos & derivados , Catálisis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Klebsiella pneumoniae/enzimología , Ligandos , Mycobacterium tuberculosis/enzimología , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacología , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/químicaRESUMEN
UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDP-Galp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a manner similar to that of UDP-Galp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the "open" mobile loop in UGM "closes" upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf and shed light on the mechanism of action of UGM.
Asunto(s)
Simulación por Computador , Galactosa/análogos & derivados , Transferasas Intramoleculares/química , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Modelos Químicos , Uridina Difosfato/análogos & derivados , Galactosa/síntesis química , Galactosa/química , Klebsiella pneumoniae/enzimología , Conformación Molecular , Unión Proteica , Estándares de Referencia , Uridina Difosfato/síntesis química , Uridina Difosfato/químicaRESUMEN
A series of UTP, UDP, and UMP derivatives and analogues were synthesized and evaluated at the human pyrimidinergic P2Y receptor subtypes P2Y2, P2Y4, and P2Y6 stably expressed in 1321N1 astrocytoma cells. Substituents at N3 of UTP were poorly tolerated by P2Y2 and P2Y4 receptors. In contrast, a large phenacyl substituent at N3 of UDP was well tolerated by the P2Y6 receptor, yielding a potent and selective P2Y6 receptor agonist (3-phenacyl-UDP, EC50=70 nM, >500-fold selective). The most potent and selective P2Y2 receptor agonist of the present series was 2-thio-UTP (EC50=50 nM, >or=30-fold selective vs P2Y4 and P2Y6). All modifications at the uracil base of UTP led to a decrease in potency at the P2Y4 receptor. A beta,gamma-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase- and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonists.
Asunto(s)
Agonistas del Receptor Purinérgico P2 , Nucleótidos de Uracilo/síntesis química , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular Tumoral , Humanos , Fosfatos de Inositol/biosíntesis , Purinas/síntesis química , Purinas/farmacología , Receptores Purinérgicos P2 , Receptores Purinérgicos P2Y2 , Relación Estructura-Actividad , Nucleótidos de Uracilo/farmacología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Uridina Difosfato/farmacología , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/síntesis química , Uridina Monofosfato/farmacología , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntesis química , Uridina Trifosfato/farmacologíaRESUMEN
The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor have been studied. Uridine 5'-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5'-diphosphate analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3',5'-diphosphate analogue, a 3'-diphosphate analogue, and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 = 0.15 microM) was equipotent to UDP, while substitutions of the 2'-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the beta-phosphate of 5'-UDP and analogues is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 microM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation (P = 126 degrees , 1'-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands.
Asunto(s)
Receptores Purinérgicos P2/efectos de los fármacos , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Línea Celular Tumoral , Humanos , Modelos Moleculares , Agonistas del Receptor Purinérgico P2 , Estereoisomerismo , Relación Estructura-Actividad , Uridina Difosfato/farmacologíaRESUMEN
Two non-natural fluorinated 2-N-acetamidosugar nucleotides, uridine 5'-diphosphate (UDP) 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-glucopyranose (UDP-4-FGlcNAc) 1 and its galacto isomer (UDP-4-FGalNAc) 2, were enzymatically constructed by treating chemically synthesized fluorinated 2-N-acetamidosugar 1-phosphates as the donor with UDP 2-acetamido-2-deoxy-alpha-D-glucopyranose pyrophosphorylase in the presence of uridine 5'-triphosphate (UTP).
Asunto(s)
Acetilgalactosamina/síntesis química , Acetilglucosamina/síntesis química , Nucleotidiltransferasas/metabolismo , Uridina Difosfato/síntesis química , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Conformación de Carbohidratos , Escherichia coli/enzimología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , Uridina Trifosfato/químicaRESUMEN
The "epimerisation" of UDP-GlcNAc to ManNAc, the first step in the biosynthesis of sialic acids, is catalyzed by UDP-GlcNAc 2-epimerase. In this paper we report the synthesis of transition state based inhibitors of this enzyme. To mimic the assumed first transition state of this reaction (TS 1), we designed and synthesized the novel UDP-exo-glycal derivatives 1-4. We also report herein the synthesis of 5 and 6, the first C-glycosidic derivatives of 2-acetamidoglucal, and the synthesis of the ketosides 7 and 8, which were designed as bis-substrate analogue and bis- product analogue, respectively, to mimic the second step of the reaction via the assumed second transition state TS 2.
Asunto(s)
Carbohidrato Epimerasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hexosaminas/química , Imitación Molecular , Ácidos Siálicos/química , Estereoisomerismo , Uridina Difosfato/químicaRESUMEN
UDP-galactose mutase is a flavoenzyme that catalyzes the isomerization of UDP-galactopyranose into UDP-galactofuranose, a key step in the biosynthesis of important bacterial oligosaccharides. Several mechanisms for this unique ring-contraction have been proposed, one of them involving a putative 1,4-anhydrogalactopyranose as an intermediate in the reaction. The purpose of this study was to probe the mutase binding site with conformationally restricted analogues of its substrate. Thus, we describe the straightforward synthesis of two C-glycosidic UDP-galactose derivatives: analogue 1, presenting a galactose moiety locked in a bicyclic (1,4)B boat conformation, and UDP-C-Galf 2, where the galactose residue is locked in the conformation of the mutase substrate. The two molecules were found to be inhibitors of UDP-galactose mutase at levels depending on the redox state of the enzyme. Strong inhibition of the native enzyme, but a low one of the reduced mutase, were observed with UDP-C-Galf 2, whereas 1 displayed intermediate inhibition levels under both native and reducing conditions. These data provide evidence of a significant conformational difference of the mutase binding pocket in the reduced enzyme and in the native one, the enzyme switching from a low Galf-affinity state (reduced enzyme) to a very strong one (native enzyme). It is remarkable that the mutase binds the boat-locked analogue 1 with similar affinities in both its conformational states. These results support a mechanism involving the formation of 1,4-anhydrogalactopyranose as a low-energy intermediate. An alternative explanation would be that the distortion of the galactose moiety during the cycle contraction transiently brings the carbohydrate into a conformation close to a (1,4)B boat.
Asunto(s)
Galactosa/análogos & derivados , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Uridina Difosfato/análogos & derivados , Bacterias/enzimología , Secuencia de Bases , Sitios de Unión , Catálisis , Galactosa/síntesis química , Galactosa/química , Galactosa/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Estereoisomerismo , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/metabolismoRESUMEN
[reaction: see text] A reliable and efficient synthetic route to UDP-alpha-D-galactofuranose (UDP-Galf) has been developed. Reaction of UMP-N-methylimidazolide with Galf 1-phosphate proceeds rapidly to provide UDP-Galf with excellent reproducibility and in a yield approximately twice as high as those reported previously.
Asunto(s)
Galactosa/síntesis química , Uridina Difosfato/síntesis química , Galactosa/análogos & derivados , Glicosiltransferasas/química , Espectroscopía de Resonancia Magnética , Uridina Difosfato/análogos & derivados , Uridina Difosfato/química , Uridina Monofosfato/químicaRESUMEN
The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.
Asunto(s)
Proteínas de Escherichia coli , Galactosa/análisis , Transferasas Intramoleculares/metabolismo , Uridina Difosfato/análisis , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Galactosa/análogos & derivados , Galactosa/síntesis química , Galactosa/metabolismo , Hidrocarburos Fluorados/análisis , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/metabolismo , Transferasas Intramoleculares/antagonistas & inhibidores , Oxidación-Reducción , Uridina Difosfato/análogos & derivados , Uridina Difosfato/síntesis química , Uridina Difosfato/metabolismoAsunto(s)
Galactosa/síntesis química , Glicosiltransferasas/química , Níquel/química , Ácido Nitrilotriacético/química , Oligosacáridos/síntesis química , Uridina Difosfato/síntesis química , Animales , Escherichia coli/enzimología , Galactosa/análogos & derivados , Galactosa/biosíntesis , Glicosiltransferasas/genética , Rechazo de Injerto/prevención & control , Humanos , Inmovilización , Oligosacáridos/biosíntesis , Porcinos , Trasplante Heterólogo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/biosíntesisRESUMEN
Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.