RESUMO
The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.
Assuntos
Acetilesterase/metabolismo , Parede Celular/metabolismo , Polissacarídeos Bacterianos/metabolismo , Streptococcus/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Glucosamina/análogos & derivados , Glucofosfatos , Histonas , Humanos , Ácido Nitroso , Peptidoglicano/química , Peptidoglicano/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus mutansRESUMO
Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.
Assuntos
Glucose/química , Glucofosfatos/química , Glicogênio Fosforilase/química , Glicogênio/química , Amidas/farmacologia , Animais , Cafeína/farmacologia , Ácido Elágico/farmacologia , Ensaios Enzimáticos , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/isolamento & purificação , Ensaios de Triagem em Larga Escala , Indóis/farmacologia , Cinética , Coelhos , Soluções , Relação Estrutura-AtividadeRESUMO
Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-ß-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 µM, while IAA-Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.
Assuntos
Glucosiltransferases/química , Uridina Difosfato Glucose/química , Zea mays/enzimologia , Ácido 2,4-Diclorofenoxiacético/química , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Sítios de Ligação , Cátions , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Glucose/química , Glucofosfatos/química , Glucosiltransferases/antagonistas & inibidores , Homeostase , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Recombinantes/química , Especificidade por Substrato , Zea mays/efeitos dos fármacosRESUMO
Tagatose is a rare sugar metabolised by a limited number of microorganisms that inhibits a large spectrum of phytopathogens. In particular, tagatose inhibited Phytophthora infestans growth and negatively affected mitochondrial processes. However, the possible effects of tagatose on P. infestans metabolism have not yet been investigated. The aim of this study was to analyse the impact of this rare sugar on the sugar metabolism in P. infestans, in order to better understand its mode of action. Tagatose inhibited the growth of P. infestans with a precise reprogramming of the carbohydrate metabolism that involved a decrease of glucose, glucose-1-phosphate and mannose content and ß-glucosidase activity. The combination of tagatose with common sugars led to three different responses and highlighted antagonistic interactions. In particular, glucose partially attenuated the inhibitory effects of tagatose, while fructose fully impaired tagatose-mediated growth inhibition and metabolite changes. Moreover, sucrose did not attenuate tagatose effects, suggesting that the inhibition of sucrose catabolism and the alteration of glucose-related pathways contributed to the growth inhibition caused by tagatose to P. infestans. The interactions of tagatose with the common sugar metabolism were found to be a key mode of action against P. infestans growth, which may represent the basis for the further development of tagatose as an eco-friendly fungicide.
Assuntos
Metabolismo dos Carboidratos , Hexoses/metabolismo , Phytophthora infestans/crescimento & desenvolvimento , Phytophthora infestans/metabolismo , Fungicidas Industriais/farmacologia , Glucose , Glucofosfatos , Hexoses/farmacologia , Manose/metabolismo , Phytophthora infestans/efeitos dos fármacos , Doenças das Plantas , Sacarose , beta-Glucosidase/metabolismoRESUMO
Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
Assuntos
Peixes , Fosfoglucomutase/metabolismo , Animais , Domínio Catalítico , Glucofosfatos/metabolismo , Fosfoglucomutase/química , Ligação Proteica , Especificidade por SubstratoRESUMO
Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.
Assuntos
Fosfotransferases (Fosfomutases)/metabolismo , Processamento de Proteína Pós-Traducional , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Ensaios Enzimáticos , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glicólise , Isomerismo , Cinética , Conformação Molecular , Fosforilação , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/isolamento & purificação , Fosfotransferases (Fosfomutases)/ultraestrutura , Prolina/química , Domínios Proteicos , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Limited knowledge is currently available on the biochemical basis for the development of dark-cutting beef. The objective of this research was to determine the metabolite profile and mitochondrial content differences between normal-pH and dark-cutting beef. A gas chromatography-mass spectrometer-based nontargeted metabolomic approach indicated downregulation of glycolytic metabolites, including glucose-1- and 6-phosphate and upregulation of tricarboxylic substrates such as malic and fumaric acids occurred in dark-cutting beef when compared to normal-pH beef. Neurotransmitters such as 4-aminobutyric acid and succinate semialdehyde were upregulated in dark-cutting beef than normal-pH beef. Immunohistochemistry indicated a more oxidative fiber type in dark-cutting beef than normal-pH beef. In support, the mitochondrial protein and DNA content were greater in dark-cutting beef. This increased mitochondrial content, in part, could influence oxygen consumption and myoglobin oxygenation/appearance of dark-cutting beef. The current results demonstrate that the more tricarboxylic metabolites and mitochondrial content in dark-cutting beef impact muscle pH and color.
Assuntos
Bovinos/metabolismo , Carne/análise , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Animais , Bovinos/genética , Cor , Fumaratos/análise , Fumaratos/metabolismo , Glucofosfatos/análise , Glucofosfatos/metabolismo , Concentração de Íons de Hidrogênio , Malatos/análise , Malatos/metabolismo , Músculo Esquelético/química , Mioglobina/química , Mioglobina/metabolismo , OxirreduçãoRESUMO
Two glycosyl 1-phosphate polymers containing monoglycosyl 1-phosphate, -6)-α-D-Glcp-(1-P-, and diglycosyl 1-phosphate, -6)-α-D-GalpNAc-(1â6)-α-D-GlcpNAc-(1-P-, in the repeating unit were identified in the cell wall of Glutamicibacter protophormiae VKM Ac-2104T (formerly, Arthrobacter protophormiae). The structures of these polymers were described for the first time in prokaryotes. Teichulosonic acid, the third identified polymer, with 3-deoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the main chain, â6)-ß-D-Glcp-(1â8)-α-Kdn-(2â, has been previously detected in a number of actinobacteria. The structures of these glycopolymers were established based on the results of chemical analysis and one-dimensional 1H, 13C, and 31P NMR spectroscopy using two-dimensional homonuclear (1H,1H COZY, TOCSY, ROESY) and heteronuclear (1H,13C HSQC, HSQC-TOCSY, HMBC, and 1H,31P HMBC) techniques.
Assuntos
Parede Celular/metabolismo , Glucofosfatos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Micrococcaceae/metabolismo , Polímeros/química , Polissacarídeos Bacterianos/metabolismo , Ácidos Teicoicos/metabolismo , Parede Celular/química , Glucofosfatos/química , Polissacarídeos Bacterianos/química , Ácidos Teicoicos/químicaRESUMO
Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.
Assuntos
Nucleotídeos de Adenina , Agrobacterium , Glucofosfatos , Glicogênio , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Agrobacterium/química , Agrobacterium/metabolismo , Ésteres/química , Ésteres/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Glicogênio/química , Glicogênio/metabolismo , Proteínas Periplásmicas de Ligação/metabolismoRESUMO
Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-D-phosphohexomutases, a large enzyme superfamily with members in all three domains of life. PGM1 catalyzes the bi-directional interconversion between α-D-glucose 1-phosphate (G1P) and α-D-glucose 6-phosphate (G6P), a reaction that is essential for normal carbohydrate metabolism and also important in the cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. Clinical studies have shown that mutations in the PGM1 gene may cause PGM1 deficiency, an inborn error of metabolism previously classified as a glycogen storage disease, and PGM1 deficiency was recently also shown to be a congenital disorder of glycosylation. Here we present three crystal structures of the isoform 2 variant of PGM1, both as a free enzyme and in complex with its substrate and product. The structures show the longer N-terminal of this PGM1 variant, and the ligand complex structures reveal for the first time the detailed structural basis for both G1P substrate and G6P product recognition by human PGM1. We also show that PGM1 and the paralogous gene PGM5 are the results of a gene duplication event in a common ancestor of jawed vertebrates, and, importantly, that both PGM1 isoforms are conserved and of functional significance in all vertebrates. Our finding that PGM1 encodes two equally conserved and functionally important isoforms in the human organism should be taken into account in the evaluation of disease-related missense mutations in patients in the future.
Assuntos
Fosfoglucomutase/genética , Fosfotransferases (Fosfomutases)/genética , Isoformas de Proteínas/genética , Animais , Domínio Catalítico/genética , Citoplasma/genética , Glucose-6-Fosfato/genética , Glucofosfatos/genética , Doença de Depósito de Glicogênio/genética , Glicosilação , Humanos , Ligantes , Mutação de Sentido Incorreto/genética , Vertebrados/genéticaRESUMO
Condylomata acuminata (CA) is caused by human papillomavirus (HPV) infections of keratinocytes and is a common sexually transmitted disease. The main clinical feature and risk of CA is the high recurrence of genital warts formed by infected keratinocytes. Metabolic reprogramming of most types of mammalian cells including keratinocytes can provide energy and intermediates essential for their survival. Here, we report that HPV infection develops a hypoxic microenvironment in CA warts by inducing the accumulation of glycogen and increased glycogen metabolism in the infected keratinocytes in a hypoxia-inducible factor 1α (HIF-1α) -dependent pathway. Our in vitro studies show that the increased glycogen metabolism is essential for the survival and proliferation of keratinocytes. Regarding its mechanism of action, glycogenolysis generates glucose-1-phosphate that fluxes into the pentose phosphate pathway and, then, generates abundant nicotinamide adenine dinucleotide phosphate, thereby ensuring high levels of glutathione in keratinocytes under hypoxia. The abrogation of glycogen synthesis and glycogenolysis decreases the ratio of glutathione and glutathione disulfide and increases the level of ROS, further resulting in the impairment of keratinocyte survival. Collectively, our work offers an insight into the metabolic reprogramming in the development of CA and implies that the intervention of glycogen metabolism would be a promising therapeutic target for CA.
Assuntos
Condiloma Acuminado/patologia , Glicogênio/metabolismo , Glicogenólise , Queratinócitos/metabolismo , Papillomaviridae/patogenicidade , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Condiloma Acuminado/virologia , Glucofosfatos/metabolismo , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , Masculino , Via de Pentose FosfatoRESUMO
Trehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM ß-glucose 1-phosphate (ß-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of ß-phosphoglucomutase to this reaction produced Glc6P from ß-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained.
Assuntos
Glucosiltransferases/metabolismo , Lactococcus lactis/enzimologia , Fosfatos Açúcares/biossíntese , Trealose/análogos & derivados , Leveduras/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Clonagem Molecular , Glucose-6-Fosfatase/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferases/genética , Lactococcus lactis/genética , Fosfatos/metabolismo , Trealose/biossíntese , Leveduras/genéticaRESUMO
Primary carbohydrate metabolism in plants includes several sugar and sugar-derivative transport processes. Over recent years, evidences have shown that in starch-related transport processes, in addition to glucose 6-phosphate, maltose, glucose and triose-phosphates, glucose 1-phosphate also plays a role and thereby increases the possible fluxes of sugar metabolites in planta. In this study, we report the characterization of two highly similar transporters, At1g34020 and At4g09810, in Arabidopsis thaliana, which allow the import of glucose 1-phosphate through the plasma membrane. Both transporters were expressed in yeast and were biochemically analyzed to reveal an antiport of glucose 1-phosphate/phosphate. Furthermore, we showed that the apoplast of Arabidopsis leaves contained glucose 1-phosphate and that the corresponding mutant of these transporters had higher glucose 1-phosphate amounts in the apoplast and alterations in starch and starch-related metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Glucofosfatos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/fisiologia , Metabolismo dos Carboidratos , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Mutação , Folhas de Planta/metabolismo , Protoplastos , Amido/metabolismo , TranscriptomaRESUMO
Cellodextrins are linear ß-1,4-gluco-oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP-controlled, bottom-up synthesis from expedient substrates is desired for their bulk production. Here, a three-enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short-chain (soluble) cellodextrins (DP range 3-6). The cascade reaction involves iterative ß-1,4-glucosylation of glucose from α-glucose 1-phosphate (αGlc1-P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1-P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L-1 is obtained under near-complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)-1 . Through a simple two-step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.
Assuntos
Celulose/análogos & derivados , Dextrinas/metabolismo , Fosforilases/metabolismo , Cellulomonas/enzimologia , Celulose/metabolismo , Glucose/metabolismo , Glucofosfatos/metabolismo , Fosfatos/metabolismo , Fosforilases/genética , Sacarose/metabolismoRESUMO
Uridine-5'-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism-Rhodococcus opacus 1CP-were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the Vmax was determined to be 177 U mg-1 (uridine-5'-triphosphate (UTP)) and Km to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Rhodococcus/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Sequência de Aminoácidos , Produtos Biológicos/metabolismo , Glucofosfatos/metabolismo , Alinhamento de Sequência , Trealose/metabolismo , Uridina Difosfato Glucose/metabolismoRESUMO
Because polysaccharides have very complicated chemical structures constructed by a great diversity of monosaccharide residues and glycosidic linkages, enzymatic approaches have been identified as powerful tools to precisely synthesize polysaccharides as the reactions progress in highly controlled regio- and stereoarrangements. α-Glucan phosphorylase (GP) is one of the enzymes that have acted as catalysts for the practical production of well-defined polysaccharides. GP can catalyze enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) as a monomer from a maltooligosaccharide primer to produce a pure amylose with well-defined structure via the formation of α(1â4)-glycosidic linkages. Here, the author presents methods which achieve the enzymatic synthesis of functional amylosic materials and amylose analog polysaccharides by GP-catalyzed enzymatic polymerization approaches. As the polymerization progresses at the non-reducing end of the primer, it can be conducted using polymeric primers that are modified at the reducing end and covalently attached on suitable polymeric chains. By using such polymeric primers, various amylose-grafted functional materials can be enzymatically synthesized. For example, the detailed protocol for the synthesis of amylose-grafted poly(γ-glutamic acid) is described. GP shows loose specificity for the recognition of substrates, which allows to recognize some monosaccharide 1-phosphates as analog substrates of Glc-1-P. Representatively, the experimental procedure of the GP-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate as the analog substrate is presented to synthesize an α(1â4)-linked glucosamine polymer, that is called amylosamine. By means of a similar approach catalyzed by GP, several amylose analog polysaccharides have been obtained.
Assuntos
Amilose/biossíntese , Biocatálise , Glucosamina/análogos & derivados , Glucofosfatos/metabolismo , Amilose/análogos & derivados , Glucosamina/metabolismo , Polimerização , Polissacarídeos/biossínteseRESUMO
Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10-18 mol) to fmol (10-15 mol), providing the foundation of metabolite quantitation of single cells. We applied this method to investigate the changes of metabolites including glucose-phosphate, 2-deoxy-d-glucose-phosphate, and ribose-phosphate in single K562 cells stimulated by 2-deoxy-d-glucose. With the robust quantitative capabilities, the developed method holds great potential for studying a drugs' mechanism of action and resistance at single cell level.
Assuntos
Glucofosfatos/análise , Glucofosfatos/isolamento & purificação , Dispositivos Lab-On-A-Chip , Microextração em Fase Líquida/instrumentação , Espectrometria de Massas/instrumentação , Análise de Célula Única/métodos , Humanos , Células K562RESUMO
The first non-natural derivative of the rare d-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.
Assuntos
Agrobacterium/química , Proteínas de Bactérias/metabolismo , Ésteres/síntese química , Glucofosfatos/síntese química , Proteínas Periplásmicas de Ligação/metabolismo , Agrobacterium/crescimento & desenvolvimento , Cristalografia por Raios X , Ésteres/química , Ésteres/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-ß-CD: G4- ß-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicogênio/metabolismo , Ciclodextrinas/metabolismo , Dextrinas/antagonistas & inibidores , Dextrinas/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Glucose/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferases/metabolismo , Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicogênio Fosforilase/metabolismo , Glicosilação , Redes e Vias Metabólicas , Família MultigênicaRESUMO
Online detection and quantification of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate was achieved by coupling sheath-flow surface enhanced Raman spectroscopy (SERS) to liquid chromatography. The presence of an alkanethiol (hexanethiol) self-assembled monolayer adsorbed to a silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The quantification of each analyte was achieved using partial least-squares (PLS) regression analysis and acetonitrile in the mobile phases as an internal standard. These results illustrate the utility of sheath-flow SERS for molecular specific detection in complex biological samples appropriate for metabolomics and other applications.