Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 137
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nature ; 571(7763): 127-131, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243371

RESUMO

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths1. Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival2,3. However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid4. Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial-mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA.


Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/prevenção & controle , Estabilidade de RNA , Fatores de Transcrição da Família Snail/genética , Uridina Difosfato Glucose/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteína Semelhante a ELAV 1/deficiência , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Fosfotirosina/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fatores de Transcrição da Família Snail/biossíntese , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo
2.
Biomed Res ; 40(1): 17-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30787260

RESUMO

This study aimed to clarify relationships among UDP-glucose-6 dehydrogenase (UGDH) expression, clinicopathological factors, and the prognosis of patients, and to determine the role of UGDH in lung adenocarcinoma (AC). Firstly, UGDH expression and localization in 126 lung AC tissues were immunohistochemically studied, and associations with clinicopathological parameters and patients' prognosis were evaluated. Secondly, serum UGDH levels were measured in 267 lung cancer patients and 100 healthy controls. Finally, the effects of UGDH knockdown by siRNA on migration and invasion abilities were analyzed. As a result, nuclear UGDH staining was significantly correlated with poorer differentiation, a larger tumor size, higher p-TNM stage, positive nodal metastasis, positive lymphatic invasion, and positive vascular invasion in lung AC patients. Nuclear UGDH-positive patients showed significantly poorer survival than nuclear UGDH-negative patients. Serum UGDH levels were especially higher in lung AC patients even in stage I than those in healthy controls. In lung AC cell lines, nuclear expression levels of UGDH were higher in LC-2/ad cells than in A549 cells. UGDH siRNA-treated LC-2/ad cells showed significantly decreased migration and invasion abilities, but no significant differences were observed in UGDH siRNA-treated A549 cells. These data indicate that UGDH expression and localization are an early sero-diagnostic marker in addition to a poor prognostic indicator in lung AC patients.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/mortalidade , Biomarcadores Tumorais , Núcleo Celular/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/genética , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Transporte Proteico , RNA Interferente Pequeno/genética , Carga Tumoral , Uridina Difosfato Glucose Desidrogenase/genética
3.
Biochemistry ; 57(50): 6848-6859, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30457329

RESUMO

Human UDP-glucose dehydrogenase (hUGDH) oxidizes UDP-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is regulated by the conformation of a buried allosteric switch (T131 loop/α6 helix). Substrate binding induces the allosteric switch to slowly isomerize from an inactive E* conformation to the active E state, which can be observed as enzyme hysteresis. When the feedback inhibitor UDP-xylose binds, the allosteric switch and surrounding residues in the protein core repack, converting the hexamer into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition is facilitated by large cavities and declivities in the protein core that provide the space required to accommodate the alternate packing arrangements. Here, we have used the A104L substitution to fill a cavity in the E state and sterically prevent repacking of the core into the EΩ state. Steady state analysis shows that hUGDHA104L binds UDP-xylose with lower affinity and that the inhibition is no longer cooperative. This means that the allosteric transition to the high-UDP-xylose affinity EΩ state is blocked by the substitution. The crystal structures of hUGDHA104L show that the allosteric switch still adopts the E and E* states, albeit with a more rigid protein core. However, the progress curves of hUGDHA104L do not show hysteresis, which suggests that the E* and E states are now in rapid equilibrium. Our data suggest that hysteresis in native hUGDH originates from the conformational entropy of the E* state protein core.


Assuntos
Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Regulação Alostérica , Sítio Alostérico , Substituição de Aminoácidos , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Especificidade por Substrato , Uridina Difosfato Glucose Desidrogenase/genética
4.
J Biol Chem ; 293(40): 15725-15732, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30089654

RESUMO

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the K half of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/genética , GTP Fosfo-Hidrolases/química , Regulação Bacteriana da Expressão Gênica , Complexos Multienzimáticos/química , Nucleotidiltransferases/química , Peptídeo Sintases/química , Uridina Difosfato Glucose Desidrogenase/química , Regulação Alostérica , Sítios de Ligação , Ensaios Enzimáticos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Fólico/biossíntese , Ácido Fólico/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Cinética , Simulação de Acoplamento Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ácidos Pteroilpoliglutâmicos/biossíntese , Ácidos Pteroilpoliglutâmicos/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , Termodinâmica , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo
5.
Oncogene ; 37(20): 2615-2629, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29479058

RESUMO

UDP-glucose 6-dehydrogenase (UGDH) produces UDP-α-D-glucuronic acid, the precursors for glycosaminoglycans (GAGs) and proteoglycans of the extracellular matrix. Elevated GAG formation has been implicated in a variety of human diseases, including glioblastoma (GBM). In our previous study, we found that Krüppel-like factor 4 (KLF4) promotes GBM cell migration by binding to methylated DNA, mainly methylated CpGs (mCpG) and transactivating gene expression. We identified UDGH as one of the downstream targets of KLF4-mCpG binding activity. In this study, we show that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4-induced cell migration in vitro. UGDH knockdown decreases GAG abundance in GBM cells, as well as cell proliferation and migration in vitro. In intracranial xenografts, reduced UGDH inhibits tumor growth and migration, accompanied by a decrease in the expression of extracellular matrix proteins such as tenascin C, brevican. Our studies demonstrate a novel DNA methylation-dependent UGDH upregulation by KLF4. Developing UGDH antagonists to decrease the synthesis of extracellular matrix components will be a useful strategy for GBM therapy.


Assuntos
Neoplasias Encefálicas/enzimologia , Glioblastoma/enzimologia , Fatores de Transcrição Kruppel-Like/metabolismo , Regulação para Cima , Uridina Difosfato Glucose Desidrogenase/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glicosaminoglicanos/metabolismo , Humanos , Camundongos , Transplante de Neoplasias , Proteoglicanas/metabolismo , Regulação para Cima/efeitos dos fármacos , Uridina Difosfato Glucose Desidrogenase/antagonistas & inibidores
6.
Adv Exp Med Biol ; 987: 99-107, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28971451

RESUMO

In recent years there has been an increasingly amount of data stored in biomedical Databases due to the breakthroughs in biology and bioinformatics, biomedical information is growing exponentially making efficient information retrieval from scientist more and more challenging. New Scientific fields as Bioinformatics seem to be the tool needed to extract scientifically important data based on experimental results and information provided by papers and journals. In this paper we are going to implement a custom made IT system in order to find connections between genes in the breast cancer pathways such the BRCA1 with the electrical energy in the human brain with UGDH gene via the TP53 tumor gene. The proposed system will be able to identify the appearance of each gene ID and compare the coexistence of two genes in PubMed articles/papers. The final system could become a useful tool against the struggle of scientists and medical professionals in the near future.


Assuntos
Proteína BRCA1/genética , Bibliometria , Neoplasias da Mama/diagnóstico , Eletroencefalografia , Proteína Supressora de Tumor p53/genética , Uridina Difosfato Glucose Desidrogenase/genética , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Feminino , Humanos , Armazenamento e Recuperação da Informação/métodos , Armazenamento e Recuperação da Informação/estatística & dados numéricos , Prognóstico , Transdução de Sinais/genética
7.
Protein Pept Lett ; 24(8): 735-741, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28741460

RESUMO

BACKGROUND: The biocatalytic oxidation of UDP-glucose in the presence of NAD+ is catalyzed by UDP-glucose dehydrogenases. OBJECTIVES: The main objective of this study was the characterization of a UDP-glucose dehydrogenase (AmUGD) from Akkermansia muciniphila, a bacterium originally isolated from human faeces in an anaerobic medium containing gastric mucin as the sole carbon source. METHODS: The biochemical analysis of AmUGD was performed using a plate reader-based assay measuring the reaction by-product NADH. Furthermore, HPLC- and MALDI-ToF-MS- based methods were used for the enzyme characterization. RESULTS: The recombinant form of the protein was expressed in E. coli and the purified enzyme exhibited optimum levels of activity at 37°C and pH 9.0. While the enzyme is active in the absence of metal ions, the presence of Zn2+ ions results in markedly enhanced levels of catalysis. CONCLUSION: This study describes the first characterization of a nucleotide-processing enzyme from A. muciniphila. The ease of expression and purification of this enzyme make it ideal for biotechnological applications such as the enzymatic synthesis of nucleotide sugars, which may in turn be used for the synthesis of complex carbohydrates or glycoconjugates.


Assuntos
Proteínas de Bactérias/metabolismo , NAD/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Uridina Difosfato Glucose/metabolismo , Verrucomicrobia/química , Proteínas de Bactérias/genética , Cátions Bivalentes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , NAD/química , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Uridina Difosfato Glucose/química , Uridina Difosfato Glucose Desidrogenase/genética , Verrucomicrobia/enzimologia , Zinco/química , Zinco/metabolismo
8.
Biochemistry ; 56(1): 202-211, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27966912

RESUMO

Human UDP-glucose dehydrogenase (hUGDH) is regulated by an atypical allosteric mechanism in which the feedback inhibitor UDP-xylose (UDP-Xyl) competes with the substrate for the active site. Binding of UDP-Xyl triggers the T131-loop/α6 allosteric switch, which converts the hexameric structure of hUGDH into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the EΩ structure. Here we use a methionine substitution to prevent the burial of A136 and trap the T131-loop/α6 switch in the active conformation. We show that hUGDHA136M does not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low-UDP-Glc-affinity EΩ state. In addition, the inhibitor affinity of hUGDHA136M is reduced 14-fold, which most likely represents the Ki for competitive inhibition in the absence of the allosteric transition to the higher-affinity EΩ state. hUGDH also displays a lag in progress curves, which is caused by a slow, substrate-induced isomerization that activates the enzyme. Stopped-flow analysis shows that hUGDHA136M does not exhibit hysteresis, which suggests that the T131-loop/α6 switch is the source of the slow isomerization. This interpretation is supported by the 2.05 Å resolution crystal structure of hUGDHA136M, which shows that the A136M substitution has stabilized the active conformation of the T131-loop/α6 allosteric switch. This work shows that the T131-loop/α6 allosteric switch couples allostery and hysteresis in hUGDH.


Assuntos
Regulação Alostérica , Domínio Catalítico , Uridina Difosfato Glucose Desidrogenase/metabolismo , Uridina Difosfato Xilose/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Ligação Competitiva , Biocatálise , Cristalização , Cristalografia por Raios X , Humanos , Cinética , Metionina/química , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação Proteica , Multimerização Proteica , Especificidade por Substrato , Fatores de Tempo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/genética
9.
Enzyme Microb Technol ; 85: 64-70, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26920483

RESUMO

Hyaluronic acid (HA), a vital acid mucopolysaccharide, has immense applied value in foodstuffs, medicaments, and cosmetics among others. UDP-glucose dehydrogenase (UGDH, EC 1.1.1.22) is an essential enzyme for HA synthesis. In this study, a UGDH (PmuHasB, 45.9 kDa) from Pasteurella multocida CVCC 408 was expressed in Escherichia coli BL21 (DE3). It was purified by two chromatographic columns with a specific activity of 6.58 IU/mg. The optimum pH and temperature were determined to be 10.0 and 37°C, respectively. The activity was stable across the pH range 6-10, and had a half-life of about 3 h at 45°C. The estimated apparent Km values for UDP-glucose and NAD(+) were 0.11 and 0.069 mM, respectively. The results indicated that PmuHasB was an alkaline and mesophilic UGDH. PmuHasB and PmuHasA (HA synthase, HAS) were co-expressed in E. coli BW25113 to obtain a HA high-producing strain pBPAB/BW25113. It produced about 2.39 g/L HA in shake flask by using the method of whole-cell catalysis. Investigation of the different UGDHs on HA synthesis revealed that intracellular UGDH activity and HA total yield of pBPAB/BW25113 (0.15 IU/mg and 5.4 g/L) were higher than from pBPASB/BW25113 (0.013 IU/mg and 2.8 g/L) and pBPAEB/BW25113 (0.010 IU/mg and 2.22 g/L). These results indicated that the activity and stability of UGDH plays a significant role in HA production, and should prove useful for further genetic engineering research with a view to construct other glucuronic acid polysaccharide synthesis pathways.


Assuntos
Proteínas de Bactérias/metabolismo , Ácido Hialurônico/biossíntese , Pasteurella multocida/enzimologia , Uridina Difosfato Glucose Desidrogenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Genes Bacterianos , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/genética
10.
Anal Biochem ; 490: 46-51, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26297818

RESUMO

Glycosyltransferases (GTs) are abundant in nature and diverse in their range of substrates. Application of GTs is, however, often complicated by their narrow substrate specificity. GTs with tailored specificities are highly demanded for targeted glycosylation reactions. Engineering of such GTs is, however, restricted by lack of practical and broad-scope assays currently available. Here we present an improvement of an inexpensive and simple assay that relies on the enzymatic detection of inorganic phosphate cleaved from nucleoside phosphate products released in GT reactions. This phosphatase-coupled assay (PCA) is compared with other GT assays: a pH shift assay and a commercially available immunoassay in Escherichia coli cell-free extract (CE). Furthermore, we probe PCA with three GTs with different specificities. Our results demonstrate that PCA is a versatile and apparently general GT assay with a detection limit as low as 1 mU. The detection limit of the pH shift assay is roughly 4 times higher. The immunoassay, by contrast, detected only nucleoside diphosphates (NDPs) but had the lowest detection limit. Compared with these assays, PCA showed superior robustness and, therefore, appears to be a suitable general screening assay for nucleotide sugar-dependent GTs.


Assuntos
Fosfatase Alcalina/metabolismo , Glicosiltransferases/metabolismo , Nucleosídeos/metabolismo , Fosfatos/análise , Adsorção , Óxido de Alumínio/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Livre de Células/enzimologia , Sistema Livre de Células/metabolismo , Centrifugação , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glicosiltransferases/genética , Humanos , Hidrólise , Indicadores e Reagentes/química , Cinética , Limite de Detecção , Fosfatos/química , Fosfatos/isolamento & purificação , Fosfatos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Especificidade por Substrato , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo
11.
Protein Pept Lett ; 22(7): 628-34, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26008638

RESUMO

UDP-glucose dehydrogenases (EC 1.1.1.22) are responsible for the conversion of UDP-glucose to UDP-glucuronic acid, a key precursor in the biosynthesis of glycoconjugates. Herein we report the discovery and characterization of a UDPglucose dehydrogenase (GbUGD) from Granulibacter bethesdensis, a bacterium originally isolated from the lymph nodes of patients with chronic granulomatous disease (CGD). The recombinant form of the protein was expressed in high yield and the purified enzyme showed highest activity at 37°C/pH 9.0 and was strongly inhibited by Zn(2+) ions, sodium dodecyl sulfate (SDS) and urea. UDP-xylose, an allosteric feedback inhibitor, reduced significantly the activity of the enzyme. High activities were observed using the co-substrates UDP-glucose and NAD+, whereas no activity could be detected using other nucleotide sugars or NADP(+) as potential alternative substrates. The high activity combined with the simple purification procedure used make GbUGD a valuable new alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems.


Assuntos
Acetobacteraceae/enzimologia , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Acetobacteraceae/genética , Clonagem Molecular , Detergentes/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Uridina Difosfato Glucose Desidrogenase/antagonistas & inibidores , Uridina Difosfato Glucose Desidrogenase/genética
12.
Prikl Biokhim Mikrobiol ; 51(1): 30-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25842901

RESUMO

Biopolymer Ss of Sphingomonas sanxanigenens strain NX02 is an sphingan that can be extracted using a small quantity of acid, which is a low cost extraction process. A UDP-glucose dehydrogenase gene (ugdG), related to Ss biosynthesis, was cloned from S. sanxanigenens NX02 and expressed in Escherichia coli. It encoded a 454-residue protein of 48.2 kDa. The deduced amino acid sequence had 77% identity with UDP-glucose dehydrogenase (UgdG) from Sphingomonas sp. KC8, and 73% identity with UgdG from Sphingomonas elodea ATCC31461. Purified recombinant UgdG had maximum activity at 35°C and pH 8.0, with Km values of 0.47 and 0.38 mM for UDP-glucose and NAD+, respectively. Overexpression of the ugdG gene in S. sanxanigenens resulted in increased (14.9 ± 0.5)% Ss production and higher fermentation broth viscosity. Furthermore, the weight-average molecular weight of polymer Ss from the recombinant strain was (5.3 ± 0.16)% higher and the viscosity was (74 ± 0.15)% higher than those from the WT strain at a shear rate of 1 rev/min.


Assuntos
Biopolímeros/biossíntese , Sphingomonas/enzimologia , Uridina Difosfato Glucose Desidrogenase/genética , Aminoácidos/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Cinética , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Uridina Difosfato Glucose Desidrogenase/biossíntese , Uridina Difosfato Glucose Desidrogenase/química
13.
Appl Environ Microbiol ; 80(9): 2754-62, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24561591

RESUMO

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.


Assuntos
Arabinose/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides/metabolismo , Engenharia Metabólica , Xilose/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Carboxiliases/genética , Carboxiliases/metabolismo , Flavonoides/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo , Açúcares de Uridina Difosfato/metabolismo
14.
Dev Biol ; 387(2): 154-66, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24462977

RESUMO

Growth factors and morphogens regulate embryonic patterning, cell fate specification, cell migration, and morphogenesis. The activity and behavior of these signaling molecules are regulated in the extracellular space through interactions with proteoglycans (Bernfield et al., 1999; Perrimon and Bernfield 2000; Lander and Selleck 2000; Selleck 2000). Proteoglycans are high molecular-weight proteins consisting of a core protein with covalently linked glycosaminoglycan (GAG) side chains, which are thought to mediate ligand interaction. Drosophila mutant embryos deficient for UDP-glucose dehydrogenase activity (Ugdh, required for GAG synthesis) exhibit abnormal Fgf, Wnt and TGFß signaling and die during gastrulation, indicating a broad and critical role for proteoglycans during early embryonic development (Lin et al., 1999; Lin and Perrimon 2000) (Hacker et al., 1997). Mouse Ugdh mutants also die at gastrulation, however, only Fgf signaling appears disrupted (Garcia-Garcia and Anderson, 2003). These findings suggested a possible divergence in the requirement for proteoglycans during Drosophila and mouse embryogenesis, and that mammals may have evolved alternative means of regulating Wnt and TGFß activity. To further examine the function of proteoglycans in vertebrate development, we have characterized zebrafish mutants devoid of both maternal and zygotic Ugdh/Jekyll activity (MZjekyll). We demonstrate that MZjekyll mutant embryos display abnormal Fgf, Shh, and Wnt signaling activities, with concomitant defects in central nervous system patterning, cardiac ventricular fate specification and axial morphogenesis. Furthermore, we uncover a novel role for proteoglycans in left-right pattern formation. Our findings resolve longstanding questions into the evolutionary conservation of Ugdh function and provide new mechanistic insights into the initiation of left-right asymmetry.


Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Síndrome de Heterotaxia/genética , Proteoglicanas/metabolismo , Uridina Difosfato Glucose Desidrogenase/genética , Zigoto , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicosaminoglicanos/metabolismo , Proteínas Hedgehog/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Camundongos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
15.
J Biol Chem ; 288(49): 35049-57, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24145036

RESUMO

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.


Assuntos
Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Substituição de Aminoácidos , Estabilidade Enzimática , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteólise , Termodinâmica , Uridina Difosfato Glucose Desidrogenase/genética
16.
J Biol Chem ; 288(32): 23064-74, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23792965

RESUMO

UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.


Assuntos
Trifosfato de Adenosina , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli , NAD , Uridina Difosfato Glucose Desidrogenase , Fatores de Virulência , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Escherichia coli K12/genética , Escherichia coli K12/patogenicidade , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , NAD/química , NAD/genética , NAD/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/química , Polissacarídeos/genética , Uridina Difosfato Glucose Desidrogenase/antagonistas & inibidores , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
17.
Microbiology ; 159(Pt 7): 1471-86, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23676433

RESUMO

The genus Edwardsiella comprises a genetically distinct taxon related to other members of the family Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cationic antimicrobial peptides (CAMPs) is a specific property of the genus Edwardsiella. In particular, Edwardsiella ictaluri, an important pathogen of the catfish (Ictalurus punctatus) aquaculture and the causative agent of a fatal systemic infection, is highly resistant to CAMPs. E. ictaluri mechanisms of resistance to CAMPs are unknown. We hypothesized that E. ictaluri lipopolysaccharide (LPS) plays a role in both virulence and resistance to CAMPs. The putative genes related to LPS oligo-polysaccharide (O-PS) synthesis were in-frame deleted. Individual deletions of wibT, gne and ugd eliminated synthesis of the O-PS, causing auto-agglutination, rough colonies, biofilm-like formation and motility defects. Deletion of ugd, the gene that encodes the UDP-glucose dehydrogenase enzyme responsible for synthesis of UDP-glucuronic acid, causes sensitivity to CAMPs, indicating that UDP-glucuronic acid and its derivatives are related to CAMP intrinsic resistance. E. ictaluri OP-S mutants showed different levels of attenuation, colonization of lymphoid tissues and immune protection in zebrafish (Danio rerio) and catfish. Orally inoculated catfish with O-PS mutant strains presented different degrees of gut inflammation and colonization of lymphoid tissues. Here we conclude that intrinsic resistance to CAMPs is mediated by Ugd enzyme, which has a pleiotropic effect in E. ictaluri influencing LPS synthesis, motility, agglutination, fish gut inflammation and virulence.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Peixes-Gato/microbiologia , Farmacorresistência Bacteriana , Edwardsiella ictaluri/efeitos dos fármacos , Edwardsiella ictaluri/patogenicidade , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/microbiologia , Uridina Difosfato Glucose Desidrogenase/metabolismo , Animais , Aquicultura , Edwardsiella ictaluri/enzimologia , Edwardsiella ictaluri/genética , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Inflamação/imunologia , Inflamação/microbiologia , Uridina Difosfato Glucose Desidrogenase/genética , Virulência
18.
Appl Environ Microbiol ; 79(9): 2968-78, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23435893

RESUMO

Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Pseudomonas aeruginosa/enzimologia , Alginatos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácido Glucurônico/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/isolamento & purificação , Ácidos Hexurônicos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão , Deleção de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo
19.
BMC Genomics ; 13: 590, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-23122059

RESUMO

BACKGROUND: We previously localized a quantitative trait locus (QTL) on bovine chromosome 6 affecting milk production traits to a 1.5-Mb region between BMS483 and MNB-209 via genome scanning followed by fine mapping. RESULTS: Totally 15 genes were mapped within such linkage region through bioinformatic analysis of the cattle-human comparative map and bovine genome assembly. Of them, the UDP-glucose dehydrogenase (UGDH) was suggested as a potential positional candidate gene for milk production traits based on its corresponding physiological and biochemical functions and genetic effects. By sequencing all the coding exons and the untranslated regions in UGDH with pooled DNA of 8 sires represented the separated families detected in our previous studies, a total of ten SNPs were identified and genotyped in 1417 Holstein cows of 8 separation families. Individual SNP-based association analysis revealed 4 significant associations of SNP Ex1-1, SNP Int3-1, SNP Int5-1, and SNP Ex12-3 with milk yield (P < 0.05), and 2 significant associations of SNP Ex1-1 and SNP Ex12-3 with protein yield (P < 0.05). Furthermore, our haplotype-based association analyses indicated that haplotypes G-C-C, formed by SNP Ex12-2-SNP Int11-1-SNP Ex11-1, T-G, formed by SNP Int9-3-SNP Int9-2, and C-C, formed by SNP Int5-1-SNP Int3-1, are significantly associated with protein percentage (F=4.15; P=0.0418) and fat percentage (F=5.18~7.25; P=0.0072~0.0231). Finally, by using an in vitro expression assay, we demonstrated that the A allele of SNP Ex1-1 and T allele of SNP Ex11-1of UGDH significantly decreases the expression of UGDH by 68.0% at the RNA, and 50.1% at the protein level, suggesting that SNP Ex1-1 and Ex11-1 represent two functional polymorphisms affecting expression of UGDH and may partly contributed to the observed association of the gene with milk production traits in our samples. CONCLUSIONS: Taken together, our findings strongly indicate that UGDH gene could be involved in genetic variation underlying the QTL for milk production traits.


Assuntos
Leite/metabolismo , Polimorfismo de Nucleotídeo Único , Uridina Difosfato Glucose Desidrogenase/genética , Alelos , Animais , Células COS , Bovinos , China , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Genoma , Genótipo , Haplótipos , Mutagênese , Locos de Características Quantitativas , Transfecção , Uridina Difosfato Glucose Desidrogenase/metabolismo
20.
J Bacteriol ; 194(22): 6154-61, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22961854

RESUMO

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.


Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Óperon/genética , Streptococcus pyogenes/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Conformação Proteica , Streptococcus pyogenes/genética , Uridina Difosfato Glucose Desidrogenase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA