RESUMO
BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.
Assuntos
Proteínas de Bactérias/genética , Glucosamina/metabolismo , Rhodococcus/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Proteínas de Bactérias/metabolismo , Duplicação Gênica , Genes Bacterianos , Redes e Vias Metabólicas , Rhodococcus/enzimologia , Rhodococcus/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.
Assuntos
Metabolismo dos Carboidratos , Euglena gracilis/enzimologia , Glucanos/química , UTP-Glucose-1-Fosfato Uridililtransferase/química , Catálise , Glucanos/metabolismo , Cinética , Domínios Proteicos , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalUSpn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalUSpn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.
Assuntos
Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.
Assuntos
Polissacarídeos Bacterianos/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Sinorhizobium meliloti/enzimologia , Sinorhizobium meliloti/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Medicago sativa/microbiologia , Nodulação , Raízes de Plantas/microbiologiaRESUMO
BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.
Assuntos
Glucofosfatos/metabolismo , Glicogênio/biossíntese , Redes e Vias Metabólicas , Mycobacterium tuberculosis/metabolismo , Trealose/biossíntese , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Cinética , Modelos Biológicos , Mycobacterium tuberculosis/enzimologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
Assuntos
Galactosefosfatos/metabolismo , Giardia lamblia/enzimologia , Proteínas de Protozoários/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Giardia lamblia/genética , Glucofosfatos/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxinas/metabolismo , Fatores de Tempo , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
Assuntos
Membrana Celular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/biossíntese , Caules de Planta/enzimologia , Saccharum/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , Membrana Celular/química , Modelos Moleculares , Fosforilação/fisiologia , Proteínas de Plantas/química , Caules de Planta/química , Estrutura Terciária de Proteína , UTP-Glucose-1-Fosfato Uridililtransferase/química , Uridina Difosfato Glucose/biossíntese , Uridina Difosfato Glucose/químicaRESUMO
The function of the UDP-glucose pyrophosphorylase 2 gene (UGP2) in pig is not clear. In the present study, we used RNA isolated from Large White pigs and Chinese indigenous MeiShan pigs to examine the temporal coordination of changes in gene expression within muscle tissues. We cloned both the complete genomic DNA sequence and 2077-bp 5ê-flanking sequence of porcine UGP2, to determine the genomic sequence. Real-time RT-PCR revealed that UGP2 was highly expressed in liver and skeletal muscle of MeiShan pigs. Among different types of muscle fibers, the UGP2 had the highest expression in both soleus muscle and longissimus dorsi in Large White pigs. In the progression of muscle fibers at different growth stages, UGP2 plays a role in the early days after birth in Large White pigs, while in MeiShan pigs it is important later. Furthermore, the 5ê-flanking sequence we cloned exhibited the promoter activity of UGP2, and the sequence 588 bp upstream from the transcriptional site had the greatest activity.
Assuntos
Fígado/metabolismo , Músculo Esquelético/metabolismo , Suínos/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Animais , Linhagem Celular , Clonagem Molecular , Feminino , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Suínos/classificação , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
Assuntos
Escherichia coli/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus mutans/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/genética , Glucofosfatos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Streptococcus mutans/química , Streptococcus mutans/genética , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor making the bacterium resistant to phagocytosis. The galU gene of S. pneumoniae encodes a UDP-glucose pyrophosphorylase absolutely required for capsule biosynthesis. In silico analyses indicated that the galU gene is co-transcribed with the gpdA gene, and four putative promoter regions located upstream of gpdA were predicted. One of them behaved as a functional promoter in a promoter reporter system. It is conceivable that the sequence responsible for initiating transcription of gpdA-galU operon is an extended -10 site TATGATA(T/G)AAT. Semi-quantitative real-time reverse transcription PCR experiments indicated that galU was expressed mainly in the exponential phase of growth.