RESUMEN
Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Galf Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.
Asunto(s)
Actinobacteria/metabolismo , Aminoglicósidos/biosíntesis , Proteínas Bacterianas/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Transferasas Intramoleculares/metabolismo , Actinobacteria/genética , Aminoglicósidos/genética , Proteínas Bacterianas/genética , Azúcares de Guanosina Difosfato/genética , Transferasas Intramoleculares/genéticaRESUMEN
The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.
Asunto(s)
Guanosina Difosfato Fucosa/metabolismo , Guanosina Difosfato Manosa/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Plasmodium falciparum/metabolismo , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Cromatografía Liquida , Eritrocitos/parasitología , Gametogénesis/fisiología , Guanosina Difosfato Fucosa/análisis , Guanosina Difosfato Manosa/análisis , Azúcares de Guanosina Difosfato/análisis , Humanos , Estadios del Ciclo de Vida/fisiología , Plasmodium falciparum/crecimiento & desarrollo , Espectrometría de Masas en Tándem , Uridina Difosfato Galactosa/análisis , Uridina Difosfato Glucosa/análisis , Uridina Difosfato N-Acetilgalactosamina/análisisRESUMEN
Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.
Asunto(s)
Azúcares de Guanosina Difosfato/metabolismo , Streptomyces/metabolismo , Fosfatos de Azúcar/biosíntesis , Trehalosa/análogos & derivados , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosa/genética , Galactosa/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Azúcares de Guanosina Difosfato/genética , Streptomyces/genética , Fosfatos de Azúcar/genética , Trehalosa/biosíntesis , Trehalosa/genéticaRESUMEN
Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galß1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Desoxiazúcares/química , Desoxiazúcares/genética , Desoxiazúcares/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Glucosiltransferasas/genética , Azúcares de Guanosina Difosfato/química , Azúcares de Guanosina Difosfato/genética , Azúcares de Guanosina Difosfato/metabolismoRESUMEN
Lincomycin A is a clinically useful antibiotic isolated from Streptomyces lincolnensis. It contains an unusual methylmercapto-substituted octose, methylthiolincosamide (MTL). While it has been demonstrated that the C8 backbone of MTL moiety is derived from D-fructose 6-phosphate and D-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-D-erythro-α-D-gluco-octose (GDP-D-α-D-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-D-α-D-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL.
Asunto(s)
Azúcares de Guanosina Difosfato/metabolismo , Lincomicina/biosíntesis , Monosacáridos/metabolismo , Nucleotidiltransferasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Streptomyces/enzimologíaRESUMEN
Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two ß-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.
Asunto(s)
Carbohidrato Epimerasas/química , Cetona Oxidorreductasas/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Guanosina Difosfato Manosa/análogos & derivados , Guanosina Difosfato Manosa/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Humanos , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/metabolismo , Modelos Moleculares , NADP/química , NADP/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicoesfingolípidos/metabolismo , Manosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ácido Salicílico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Pared Celular/genética , Pared Celular/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Guanosina Difosfato Fucosa/metabolismo , Guanosina Difosfato Manosa/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Immunoblotting , Proteínas de Transporte de Membrana/genética , Microscopía Fluorescente , MutaciónRESUMEN
Gram negative bacteria have lipopolysaccharides (LPS) that are critical for their survival. LPS molecules are composed of antigenic exopolysaccharide chains (O antigens). We are interested in discovering the enzymes involved in the biosynthesis of O antigens in Pseudomonas aeruginosa. The common polysaccharide antigen contains α-linked D-rhamnose residues. We have now synthesized GDP-D-rhamnose by a convenient synthesis in aqueous solution, and have shown that it can be used without extensive purification as the donor substrate for D-rhamnosyltransferase (WbpZ) from the P. aeruginosa strain PAO1. The availability of this nucleotide sugar preparation allows for characterization of D-rhamnosyltransferases.
Asunto(s)
Azúcares de Guanosina Difosfato/síntesis química , Hexosiltransferasas/metabolismo , Pseudomonas aeruginosa/enzimología , Azúcares de Guanosina Difosfato/química , Azúcares de Guanosina Difosfato/metabolismo , Pseudomonas aeruginosa/metabolismo , Especificidad por SustratoRESUMEN
It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 µM) and by ryanodine (50 µm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 µM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Adenosina Trifosfato/metabolismo , ADP-Ribosa Cíclica/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Liso/metabolismo , ADP-Ribosil Ciclasa 1/genética , Adenosina Trifosfato/agonistas , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , ADP-Ribosa Cíclica/análogos & derivados , ADP-Ribosa Cíclica/antagonistas & inhibidores , ADP-Ribosa Cíclica/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Nucleótidos de Guanina/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , NAD/análogos & derivados , NAD/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Rianodina/farmacología , Espectrometría de Fluorescencia , Vejiga UrinariaRESUMEN
Nucleotide sugars are the universal sugar donors for the formation of polysaccharides, glycoproteins, proteoglycans, glycolipids, and glycosylated secondary metabolites. At least 100 genes encode proteins involved in the formation of nucleotide sugars. These nucleotide sugars are formed using the carbohydrate derived from photosynthesis, the sugar generated by hydrolyzing translocated sucrose, the sugars released from storage carbohydrates, the salvage of sugars from glycoproteins and glycolipids, the recycling of sugars released during primary and secondary cell wall restructuring, and the sugar generated during plant-microbe interactions. Here we emphasize the importance of the salvage of sugars released from glycans for the formation of nucleotide sugars. We also outline how recent studies combining biochemical, genetic, molecular and cellular approaches have led to an increased appreciation of the role nucleotide sugars in all aspects of plant growth and development. Nevertheless, our understanding of these pathways at the single cell level is far from complete.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas de Plantas/fisiología , Plantas/metabolismo , Polisacáridos/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Azúcares de Guanosina Difosfato/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/fisiología , Proteínas de Plantas/metabolismo , Plantas/química , Polisacáridos/biosíntesis , Polisacáridos/química , Azúcares de Uridina Difosfato/metabolismoRESUMEN
Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K(m). Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium.
Asunto(s)
Actinobacteria/genética , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfatos de Azúcar/biosíntesis , Trehalosa/análogos & derivados , Actinobacteria/enzimología , Proteínas Bacterianas/genética , Composición de Base , Clonación Molecular , ADN Bacteriano/genética , Expresión Génica , Genes Bacterianos , Glucosiltransferasas/genética , Azúcares de Guanosina Difosfato/metabolismo , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Especificidad por Sustrato , Trehalosa/biosíntesisRESUMEN
Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.
Asunto(s)
Proteínas Bacterianas/química , Carbohidrato Epimerasas/química , Azúcares de Guanosina Difosfato/química , Transaminasas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Dominio Catalítico/genética , Caulobacter crescentus/enzimología , Cristalografía por Rayos X , Azúcares de Guanosina Difosfato/metabolismo , Cinética , Manosa/análogos & derivados , Manosa/química , Manosa/metabolismo , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Ácido Ascórbico/biosíntesis , Azúcares de Guanosina Difosfato/metabolismo , Nucleotidiltransferasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Actinidia/enzimología , Actinidia/genética , Secuencias de Aminoácidos/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ácido Ascórbico/genética , Galactosafosfatos/genética , Galactosafosfatos/metabolismo , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Azúcares de Guanosina Difosfato/genética , Mutación , Nucleotidiltransferasas/genética , Monoéster Fosfórico Hidrolasas/genética , Especificidad por Sustrato/genéticaRESUMEN
Diversity in the polysaccharide component of lipopolysaccharide (LPS) contributes to the persistence and pathogenesis of Gram-negative bacteria. The Nudix hydrolase GDP-mannose mannosyl hydrolase (Gmm) contributes to this diversity by regulating the concentration of mannose in LPS biosynthetic pathways. Here, we present seven high-resolution crystal structures of Gmm from the enteropathogenic E. coli strain O128: the structure of the apo enzyme, the cocrystal structure of Gmm bound to the product Mg2+-GDP, two cocrystal structures of precatalytic and turnover complexes of Gmm-Ca2+-GDP-alpha-d-mannose, and three cocrystal structures of an inactive mutant (His-124 --> Leu) Gmm bound to substrates GDP-alpha-d-mannose, GDP-alpha-d-glucose, and GDP-beta-l-fucose. These crystal structures help explain the molecular basis for substrate specificity and promiscuity and provide a structural framework for reconciling previously determined kinetic parameters. Unexpectedly, these structures reveal concerted changes in the enzyme structure that result in the formation of a catalytically competent active site only in the presence of the substrate/product. These structural views of the enzyme may provide a rationale for the design of inhibitors that target the biosynthesis of LPS by pathogenic bacteria.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Guanosina Difosfato Manosa/biosíntesis , Guanosina Difosfato Manosa/metabolismo , Lipopolisacáridos/química , Sustitución de Aminoácidos , Apoenzimas/química , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Guanosina Difosfato Fucosa/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Holoenzimas/química , Enlace de Hidrógeno , Cinética , Leucina/metabolismo , Modelos Químicos , Conformación Proteica , Especificidad por SustratoRESUMEN
PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.
Asunto(s)
ADP-Ribosil Ciclasa/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , ADP-Ribosa Cíclica/fisiología , Segmento Externo de la Célula en Bastón/enzimología , Animales , Western Blotting , Canales de Calcio/metabolismo , Bovinos , Electroforesis en Gel de Poliacrilamida , Azúcares de Guanosina Difosfato/metabolismo , Nucleótidos de Inosina/metabolismo , Microscopía Confocal , NAD+ Nucleosidasa/metabolismo , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/ultraestructura , Espectrofotometría Atómica , Visión OcularRESUMEN
The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from d-3-phosphoglycerate, with maximal activity at 90 degrees C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Glucósidos/biosíntesis , Ácidos Glicéricos/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Catálisis , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , FilogeniaRESUMEN
The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD(+)) has been proposed to go through an oxocarbenium ion-like transition state. Because of the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD(+) to synthesize two calcium-mobilizing molecules. By using NAD(+) and a surrogate substrate, NGD(+), we captured and determined crystal structures of the enzyme complexed with an intermediate, a substrate, and a product along the reaction pathway. Our results showed that the intermediate is stabilized by polar interactions with the catalytic residue Glu(226) rather than by a covalent linkage. The polar interactions between Glu(226) and the substrate 2',3'-OH groups are essential for initiating catalysis. Ser(193) was demonstrated to have a regulative role during catalysis and is likely to be involved in intermediate stabilization. In addition, a product inhibition effect by ADP-ribose (through the reorientation of the product) or GDP-ribose (through the formation of a covalently linked GDP-ribose dimer) was observed. These structural data provide insights into the understanding of multiple catalysis and clues for drug design.
Asunto(s)
ADP-Ribosil Ciclasa 1/química , ADP-Ribosil Ciclasa 1/metabolismo , NAD+ Nucleosidasa/metabolismo , ADP-Ribosil Ciclasa 1/aislamiento & purificación , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Catálisis , Frío , Cristalografía por Rayos X , ADP-Ribosa Cíclica/metabolismo , Dimerización , Estabilidad de Enzimas , Ácido Glutámico/metabolismo , Azúcares de Guanosina Difosfato/metabolismo , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Homología de Secuencia de Ácido Nucleico , Serina/metabolismo , Electricidad Estática , Especificidad por Sustrato , Agua/químicaRESUMEN
The enzymatic characterization of GDP-d-mannose 3'',5''-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.
Asunto(s)
Arabidopsis/metabolismo , Ácido Ascórbico/biosíntesis , Carbohidrato Epimerasas/metabolismo , Genes de Plantas , Oryza/enzimología , Secuencia de Bases , Sitios de Unión , Carbohidrato Epimerasas/genética , Catálisis , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/genética , Galactosa/metabolismo , Regulación Enzimológica de la Expresión Génica , Azúcares de Guanosina Difosfato/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein alpha to the keto function of a carbohydrate identifies key common features.
Asunto(s)
Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Guanosina Difosfato Manosa/química , Guanosina Difosfato Manosa/metabolismo , Azúcares de Guanosina Difosfato/química , Azúcares de Guanosina Difosfato/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/química , NAD/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis.