Asunto(s)
Inhibidores Enzimáticos/química , Nucleótidos/biosíntesis , Aspergillus/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Deshidrogenasas de Carbohidratos/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Galactosa/análogos & derivados , Galactosa/biosíntesis , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/metabolismo , Azúcares de Nucleósido Difosfato/biosíntesis , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/metabolismo , Pseudomonas aeruginosa/enzimología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/biosíntesisRESUMEN
Galactofuranosyltransferases are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. These enzymes are considered as potential targets for therapeutic action. In addition to the only well-characterised prokaryotic GlfT2 from Mycobacterium tuberculosis, four putative genes in Leishmania major were previously described as potential galactofuranosyltransferases. In this study, we have cloned, over-expressed, purified and fully determined the kinetic parameters of these four eukaryotic enzymes, thus demonstrating their unique potency in catalysing the transfer of the galactofuranosyl moiety into acceptors. Their individual promiscuity revealed to be different, as some of them could efficiently use NDP-pyranoses as donor substrates in addition to the natural UDP-galactofuranose. Such results pave the way for the development of chemoenzymatic synthesis of furanosyl-containing glycoconjugates as well as the design of improved drugs against leishmaniasis.
Asunto(s)
Galactosa/análogos & derivados , Galactosiltransferasas/biosíntesis , Galactosiltransferasas/química , Leishmania major/enzimología , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Uridina Difosfato/análogos & derivados , Biocatálisis , Escherichia coli/genética , Galactosa/metabolismo , Cinética , Especificidad por Sustrato , Uridina Difosfato/metabolismoRESUMEN
O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP-peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol-ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA ( Ki = 1.3 µM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/farmacología , Diseño de Fármacos , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , N-Acetilglucosaminiltransferasas/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal p), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal f). Gal f biosynthesis begins with the conversion of UDP-Gal p to UDP-Gal f catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus Aspergillus fumigatus also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara p), which differs from UDP-Gal p by lacking a -CH2-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara p. This result implicated residues that contact the -CH2-OH of UDP-Gal p in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara p. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path.
Asunto(s)
Aspergillus fumigatus/enzimología , Transferasas Intramoleculares/metabolismo , Aspergilosis/microbiología , Aspergillus fumigatus/química , Aspergillus fumigatus/metabolismo , Cristalografía por Rayos X , Galactosa/análogos & derivados , Galactosa/metabolismo , Humanos , Transferasas Intramoleculares/química , Isomerismo , Cinética , Modelos Moleculares , Conformación Proteica , Especificidad por Sustrato , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , Uridina Difosfato Galactosa/metabolismo , Azúcares de Uridina Difosfato/metabolismoRESUMEN
Cryptococcus neoformans, an AIDS-defining opportunistic pathogen, is the leading cause of fungal meningitis worldwide and is responsible for hundreds of thousands of deaths annually. Cryptococcal glycans are required for fungal survival in the host and for pathogenesis. Most glycans are made in the secretory pathway, although the activated precursors for their synthesis, nucleotide sugars, are made primarily in the cytosol. Nucleotide sugar transporters are membrane proteins that solve this topological problem, by exchanging nucleotide sugars for the corresponding nucleoside phosphates. The major virulence factor of C. neoformans is an anti-phagocytic polysaccharide capsule that is displayed on the cell surface; capsule polysaccharides are also shed from the cell and impede the host immune response. Xylose, a neutral monosaccharide that is absent from model yeast, is a significant capsule component. Here we show that Uxt1 and Uxt2 are both transporters specific for the xylose donor, UDP-xylose, although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Both proteins also transport the galactofuranose donor, UDP-galactofuranose. We further show that Uxt1 and Uxt2 are required for xylose incorporation into capsule and protein; they are also necessary for C. neoformans to cause disease in mice, although surprisingly not for fungal viability in the context of infection. These findings provide a starting point for deciphering the substrate specificity of an important class of transporters, elucidate a synthetic pathway that may be productively targeted for therapy, and contribute to our understanding of fundamental glycobiology.
Asunto(s)
Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Uridina Difosfato Xilosa/metabolismo , Animales , Transporte Biológico , Criptococosis/microbiología , Criptococosis/patología , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/ultraestructura , Femenino , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/ultraestructura , Proteínas Fúngicas/genética , Galactosa/análogos & derivados , Galactosa/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Glicoproteínas/genética , Cinética , Ratones , Microscopía Electrónica de Transmisión , Mutación , Proteínas de Transporte de Nucleótidos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , VirulenciaRESUMEN
Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs.IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs.
Asunto(s)
Antifúngicos/metabolismo , Botrytis/metabolismo , Glucosa/análogos & derivados , Transferasas Intramoleculares/antagonistas & inhibidores , Uridina Difosfato/análogos & derivados , Pared Celular/metabolismo , Cristalografía por Rayos X , Glucosa/metabolismo , Cinética , Redes y Vías Metabólicas , Phaseolus/microbiología , Hojas de la Planta/microbiología , Uridina Difosfato/metabolismo , Azúcares de Uridina Difosfato/metabolismoRESUMEN
Although ancillary pathways of glucose metabolism are critical for synthesizing cellular building blocks and modulating stress responses, how they are regulated remains unclear. In the present study, we used radiometric glycolysis assays, [13C6]-glucose isotope tracing, and extracellular flux analysis to understand how phosphofructokinase (PFK)-mediated changes in glycolysis regulate glucose carbon partitioning into catabolic and anabolic pathways. Expression of kinase-deficient or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat neonatal cardiomyocytes co-ordinately regulated glycolytic rate and lactate production. Nevertheless, in all groups, >40% of glucose consumed by the cells was unaccounted for via catabolism to pyruvate, which suggests entry of glucose carbons into ancillary pathways branching from metabolites formed in the preparatory phase of glycolysis. Analysis of 13C fractional enrichment patterns suggests that PFK activity regulates glucose carbon incorporation directly into the ribose and the glycerol moieties of purines and phospholipids, respectively. Pyrimidines, UDP-N-acetylhexosamine, and the fatty acyl chains of phosphatidylinositol and triglycerides showed lower 13C incorporation under conditions of high PFK activity; the isotopologue 13C enrichment pattern of each metabolite indicated limitations in mitochondria-engendered aspartate, acetyl CoA and fatty acids. Consistent with this notion, high glycolytic rate diminished mitochondrial activity and the coupling of glycolysis to glucose oxidation. These findings suggest that a major portion of intracellular glucose in cardiac myocytes is apportioned for ancillary biosynthetic reactions and that PFK co-ordinates the activities of the pentose phosphate, hexosamine biosynthetic, and glycerolipid synthesis pathways by directly modulating glycolytic intermediate entry into auxiliary glucose metabolism pathways and by indirectly regulating mitochondrial cataplerosis.
Asunto(s)
Glucosa/metabolismo , Glucólisis , Mitocondrias Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Vía de Pentosa Fosfato , Fosfofructoquinasa-1 Tipo Hepático/metabolismo , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Isótopos de Carbono , Células Cultivadas , Medio de Cultivo Libre de Suero , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hexosaminas/metabolismo , Ácido Láctico/metabolismo , Mitocondrias Musculares/enzimología , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fosfofructoquinasa-1 Tipo Hepático/genética , Mutación Puntual , Pirimidinas/metabolismo , Ácido Pirúvico/metabolismo , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismoRESUMEN
Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 µM·gcdw-1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Condroitín/metabolismo , Disacáridos/metabolismo , Escherichia coli/metabolismo , Heparina/metabolismo , Vías Biosintéticas , Sulfatos de Condroitina/metabolismo , Escherichia coli/crecimiento & desarrollo , Microbiología Industrial , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismoRESUMEN
Uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5'-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.
Asunto(s)
Acetilglucosamina/análogos & derivados , Bifidobacterium/enzimología , Enterococcus/enzimología , Escherichia coli/enzimología , Lactobacillus/enzimología , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato/análogos & derivados , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Bifidobacterium/genética , Bifidobacterium/metabolismo , Vías Biosintéticas , Clonación Molecular , Enterococcus/genética , Enterococcus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Lactobacillus/genética , Lactobacillus/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato/genética , Uridina Difosfato/metabolismo , Uridina Difosfato N-Acetilglucosamina/genéticaRESUMEN
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
Uridine 5'-diphosphate-5-fluorogalactopyranose (UDP-5F-Galp, 7) was synthesized, and its effect on UDP-Galp mutase (UGM) was investigated. UGM facilitated the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but no 11 was detected. (19)F NMR and trapping experiments demonstrated that the reaction involves the initial formation of a substrate-cofactor adduct followed by decomposition of the resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate (10). The results support the current mechanistic proposal for UGM and suggest new directions for designing mechanism-based inhibitors.
Asunto(s)
Galactosa/análogos & derivados , Galactosa/química , Transferasas Intramoleculares/química , Uridina Difosfato/análogos & derivados , Uridina Difosfato/química , Pruebas de Enzimas , Flavina-Adenina Dinucleótido/químicaRESUMEN
Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Isomerasas/antagonistas & inhibidores , Uridina Difosfato/análogos & derivados , Biocatálisis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Galactosa/metabolismo , Hidroxilación , Isomerasas/metabolismo , Mycobacterium tuberculosis/enzimología , Compuestos de Selenio/síntesis química , Compuestos de Selenio/química , Compuestos de Selenio/farmacología , Compuestos de Sulfonio/síntesis química , Compuestos de Sulfonio/química , Compuestos de Sulfonio/farmacología , Uridina Difosfato/metabolismoRESUMEN
A series of novel Galf mimics has been synthesized and characterized by IR, (1)H NMR, (13)C NMR, mass spectral and element analysis. All the newly prepared compounds were screened for their antitubercular activities. Bioactivity assays manifested that most of Galf mimics exhibited good antitubercular activities. Especially compound 4d and 4e displayed remarkable antitubercular efficacies, which were comparable to ethambutol.
Asunto(s)
Antituberculosos/síntesis química , Pared Celular/efectos de los fármacos , Diseño de Fármacos , Glicoconjugados/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Pared Celular/metabolismo , Etambutol/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/análogos & derivados , Galactosa/química , Galactosa/genética , Galactosa/metabolismo , Expresión Génica , Glicoconjugados/química , Iminofuranosas/química , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Pruebas de Sensibilidad Microbiana , Imitación Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Transferasas/genética , Transferasas/metabolismo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/química , Uridina Difosfato/genética , Uridina Difosfato/metabolismoRESUMEN
BACKGROUND: Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. METHODS: Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. RESULTS: The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. CONCLUSION: A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.
Asunto(s)
Aspergillus niger/metabolismo , Galactosa/análogos & derivados , Aparato de Golgi/metabolismo , Transferasas/metabolismo , Uridina Difosfato/análogos & derivados , Aspergillus niger/química , Aspergillus niger/genética , Pared Celular/metabolismo , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/metabolismo , Eliminación de Gen , Duplicación de Gen , Homología de Secuencia de Ácido Nucleico , Transferasas/química , Transferasas/genética , Uridina Difosfato/metabolismoRESUMEN
Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.
Asunto(s)
Adenosina Trifosfato/metabolismo , Páncreas Exocrino/metabolismo , Conductos Pancreáticos/metabolismo , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/biosíntesis , Ácidos y Sales Biliares/metabolismo , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , Concentración Osmolar , Páncreas Exocrino/citología , Conductos Pancreáticos/citología , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Estrés Fisiológico , Uridina Difosfato/análogos & derivados , Uridina Difosfato/farmacología , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/farmacologíaRESUMEN
The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50 â values of 2.6×, 7.6×, and 7.6×10(-7) M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery.
Asunto(s)
Glicosiltransferasas/metabolismo , Mangifera/enzimología , Glucosa/análogos & derivados , Glucosa/metabolismo , Glicósidos/química , Glicósidos/metabolismo , Glicosilación , Glicosiltransferasas/química , Mangifera/química , Mangifera/metabolismo , Especificidad por Sustrato , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , Xantonas/metabolismo , Xilosa/análogos & derivados , Xilosa/metabolismoRESUMEN
Unlike the complex glycans decorating the cell surface, the O-linked ß-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate-GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.
Asunto(s)
Acetilglucosamina/química , Mitocondrias/metabolismo , N-Acetilglucosaminiltransferasas/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/biosíntesis , Empalme Alternativo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Células Madre/metabolismo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/químicaRESUMEN
Metabotropic P2Y receptors subfamily consists of eight functional mammalian receptors. Specifically, P2Y1, P2Y6 and P2Y11 receptors have been described in the sensory nervous system, but their participation, at peripheral level, in behavioral pain models is scarcely understood. This study assessed the role of peripheral P2Y1, P2Y6 and P2Y11 receptors in formalin-induced inflammatory pain. Ipsilateral, but not contralateral peripheral pre-treatment with the endogenous P2Y1 (ADP, 100-1000nmol/paw), P2Y6 (UDP, 180-300nmol/paw) and P2Y11 (ATP, 100-1000nmol/paw), or selective P2Y1 (MRS2365, 0.1-10nmol/paw), P2Y6 (PSB0474, 0.1-0.10pmol/paw) and P2Y11 (NF546, 0.3-3nmol/paw) receptor agonists increased 0.5% formalin-induced flinching behavior. Concordantly, peripheral pre-treatment with the selective P2Y1 (MRS2500, 0.01-10pmol/paw), P2Y6 (MRS2578, 3-30nmol/paw) and P2Y11 (NF340, 1-10nmol/paw) receptor antagonists significantly decreased 1% formalin-induced flinching behavior. Furthermore, the pronociceptive effect of ADP (100nmol/paw) or MRS2365 (10nmol/paw), UDP (300nmol/paw) or PSB0474 (10pmol/paw) and ATP (1000nmol/paw) or NF546 (3nmol/paw) was blocked by the selective P2Y1 (MRS2500, 0.01nmol/paw), P2Y6 (MRS2578, 3nmol/paw), and P2Y11 (NF340, 1nmol/paw) receptor antagonists, respectively. Western blot analysis confirmed the presence of P2Y1 (66kDa), P2Y6 (36kDa) and P2Y11 (75kDa) receptors in dorsal root ganglia (DRG) and sciatic nerve. Results suggest that peripheral activation of P2Y1, P2Y6 and P2Y11 receptors plays a pronociceptive role in formalin-induced pain.
Asunto(s)
Dolor Nociceptivo/fisiopatología , Receptores Purinérgicos P2Y1/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Femenino , Formaldehído/toxicidad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Inflamación/fisiopatología , Isotiocianatos/farmacología , Nocicepción/efectos de los fármacos , Nocicepción/fisiología , Dolor Nociceptivo/inducido químicamente , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Agonistas del Receptor Purinérgico P2/farmacología , Antagonistas del Receptor Purinérgico P2/farmacología , Ratas , Ratas Wistar , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiopatología , Tiourea/análogos & derivados , Tiourea/farmacología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/metabolismo , Uridina Difosfato/farmacologíaRESUMEN
UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in k(cat)/K(M) values of 200-1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together.
Asunto(s)
Aspergillus fumigatus/enzimología , Biocatálisis , Proteínas Fúngicas/metabolismo , Transferasas Intramoleculares/metabolismo , Modelos Moleculares , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Sustitución de Aminoácidos , Dominio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosa/análogos & derivados , Galactosa/metabolismo , Transferasas Intramoleculares/química , Transferasas Intramoleculares/genética , Cinética , Ligandos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Conformación Proteica , Estabilidad Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Especificidad por Sustrato , Uridina Difosfato/análogos & derivados , Uridina Difosfato/química , Uridina Difosfato/metabolismo , Uridina Difosfato Galactosa/metabolismoRESUMEN
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.