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1.
J Microbiol Biotechnol ; 29(5): 758-764, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30955255

RESUMO

ß-Glucan is a chief structural polymer in the cell wall of yeast. ß-Glucan has attracted intensive attention because of its wide applications in health protection and cosmetic areas. In the present study, the ß-glucan biosynthesis pathway in S. Cerevisiae was engineered to enhance ß-glucan accumulation. A newly identified bacterial ß-1, 6-glucan synthase GsmA from Mycoplasma agalactiae was expressed, and increased ß-glucan content by 43%. In addition, other pathway enzymes were investigated to direct more metabolic flux towards the building of ß-glucan chains. We found that overexpression of Pgm2 (phosphoglucomutase) and Rho1 (a GTPase for activating glucan synthesis) significantly increased ß-glucan accumulation. After further optimization of culture conditions, the ß-glucan content was increased by 53.1%. This study provides a new approach to enhance ß-glucan biosynthesis in Saccharomyces cerevisiae.


Assuntos
Vias Biossintéticas/genética , Glucanos/biossíntese , Glucanos/genética , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Metabolismo dos Carboidratos/genética , Parede Celular/química , Meios de Cultura/química , Glucosiltransferases/genética , Mycoplasma agalactiae/enzimologia , Mycoplasma agalactiae/genética , Fosfoglucomutase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , beta-Glucanas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
2.
PLoS Pathog ; 15(1): e1007537, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668586

RESUMO

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.


Assuntos
Inibidores de Adenilil Ciclases/metabolismo , Adenilil Ciclases/metabolismo , Adenilil Ciclases/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Fosfatos de Dinucleosídeos/antagonistas & inibidores , Fosfatos de Dinucleosídeos/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Óperon/genética , Fosfoglucomutase/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Domínios Proteicos , Espalhamento a Baixo Ângulo , Sistemas do Segundo Mensageiro/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiologia , Difração de Raios X/métodos
3.
Microb Pathog ; 126: 157-164, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30391537

RESUMO

Helicobacter pylori growth requirements is a prerequisite to invade gastric epithelium and the process of injury to gastric cells will eventually lead to gastric cancer. The aim of this study is to investigate the effect of iron challenge on the expression of genes involved in iron homeostasis. The presence of Phosphoglucosamine mutase (glmM), cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) genes and mRNA expression of Iron Regulatory Protein 2 (IRP2), Transferrin Receptor (TFRC) and Ferritin Light Chain (FTL) genes in samples of 28 normal gastric mucosa, 33 chronic gastritis, 29 gastritis with intestinal metaplasia, 29 intestinal type adenocarcinoma patients were examined by real-time PCR. Immunohistochemistry was used to analyze cellular localization and protein levels. In the all H. pylori positive tissues, particularly in the basal regions of foveolar cells, TFRC was overexpressed (P < 0.05), and regardless of the H. pylori infection, FTL was overexpressed in all patient, exclusively in metaplastic glandular cells (P < 0.05). Furthermore, overexpression of IRP2 was associated with H. pylori positive chronic gastritis and intestinal metaplasia (P < 0.05). Our findings confirm the role of transferrin receptor in H. pylori attachment into the gastric mucosa to capture iron. Overexpression of FTL gene in metaplastic cells could be considered as a research background to investigate the role of this gene in the differentiation of gastric cells into intestinal metaplasia. In addition, this gene could be suggested as a diagnostic marker to be included among the other markers routinely performed by clinical diagnostic laboratories.


Assuntos
Apoferritinas/metabolismo , Biomarcadores , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Metaplasia/metabolismo , Receptores da Transferrina/metabolismo , Adenocarcinoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Apoferritinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrite/patologia , Gastrite Atrófica/patologia , Expressão Gênica , Infecções por Helicobacter/patologia , Humanos , Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Masculino , Metaplasia/diagnóstico , Pessoa de Meia-Idade , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , RNA Mensageiro/biossíntese , Neoplasias Gástricas/patologia , Adulto Jovem
4.
Biotechnol J ; 14(3): e1800220, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30076758

RESUMO

Chrysolaminarin, the primary polysaccharide reservoir in some marine algae, has attracted much attention due to its broad health properties. However, its biosynthetic pathway and regulation mechanisms have rarely been reported which hinders the improvement of production efficiency. Therefore, this study aims to identify key metabolic nodes in the chrysolaminarin biosynthetic pathway. A phosphoglucomutase (PGM) in the model microalga Phaeodactylum tricornutum, revealing its critical role in chrysolaminarin biosynthesis is identified. PGM overexpression significantly elevates chrysolaminarin content by 2.54-fold and reaches 25.6% of cell dry weight; while algal growth and photosynthesis are not impaired. Besides, PGM overexpression up- and down-regulates the expression of chrysolaminarin and lipid biosynthetic genes, respectively. Microscopic analysis of aniline blue stained cells reveals that overproduced chrysolaminarin localized predominantly in vacuoles. Lipidomic analyses reveal that PGM overexpression significantly reduces the lipid content. The findings reveal the critical role of PGM in regulating the carbon flux between carbohydrate and lipid biosynthesis in microalgae, and provide a promising candidate for high efficiency production of chrysolaminarin.


Assuntos
Ciclo do Carbono/fisiologia , Carbono/metabolismo , Microalgas/metabolismo , Fosfoglucomutase/metabolismo , Polímeros/metabolismo , Vias Biossintéticas/fisiologia , Regulação para Baixo/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Fotossíntese/fisiologia , Polissacarídeos/metabolismo , Regulação para Cima/fisiologia
5.
Curr Radiopharm ; 12(1): 23-28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30173659

RESUMO

BACKGROUND: Heart injury is one of the most important concerns after exposure to a high dose of radiation in chest cancer radiotherapy or whole body exposure to a radiation disaster. Studies have proposed that increased level of inflammatory and pro-fibrotic cytokines following radiotherapy or radiation events play a key role in the development of several side effects such as cardiovascular disorders. In the current study, we aimed to evaluate the expression of IL-4 and IL-13 cytokines as well as signaling pathways such as IL4Ra1, IL13Ra2, Duox1 and Duox2. In addition, we detected the possible protective effect of curcumin on the expression of these factors and infiltration of inflammatory cells. MATERIALS AND METHODS: Twenty rats were divided into 4 groups including control; curcumin treated; radiation; and radiation plus curcumin. After 10 weeks, rats were sacrificed for evaluation of mentioned parameters. RESULTS: Results showed an increase in the level of IL-4 and all evaluated genes, as well as increased infiltration of lymphocytes and macrophages. Treatment with curcumin could attenuate these changes. CONCLUSION: Curcumin could reduce radiation-induced heart injury markers in rats.


Assuntos
Curcumina/farmacologia , Coração/efeitos dos fármacos , Coração/efeitos da radiação , Protetores contra Radiação/farmacologia , Animais , Oxidases Duais/metabolismo , Ensaio de Imunoadsorção Enzimática , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Fosfoglucomutase/metabolismo , Radiação Ionizante , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
6.
PLoS Biol ; 16(10): e2006483, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30335765

RESUMO

Glycogen metabolism commonly altered in cancer is just beginning to be understood. Phosphoglucomutase 1 (PGM1), the first enzyme in glycogenesis that catalyzes the reversible conversion between glucose 1-phosphate (G-1-P) and glucose 6-phosphate (G-6-P), participates in both the breakdown and synthesis of glycogen. Here, we show that PGM1 is down-regulated in hepatocellular carcinoma (HCC), which is associated with the malignancy and poor prognosis of HCC. Decreased PGM1 expression obstructed glycogenesis pathway, which leads to the increased flow of glucose into glycolysis, thereby promoting tumor cell proliferation and HCC development. The loss of forkhead box protein J2 (FOXJ2), at least partly due to low genomic copy number in HCC, releases cellular nucleic acid-binding protein (CNBP), a nucleic acid chaperon, to bind to and promote G-quadruplex formation in PGM1 promoter and therefore decreases PGM1 expression. In addition, integrated analyses of PGM1 and FOXJ2 expression provide a better prediction for the malignance and prognosis of HCC. This study establishes a tumor-suppressive role of PGM1 by regulating glucose trafficking and uncovers a novel regulatory mechanism of PGM1 expression.


Assuntos
Carcinoma Hepatocelular/metabolismo , Glucose/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfoglucomutase/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosfoglucomutase/deficiência , Fosfoglucomutase/genética , Prognóstico , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
7.
Structure ; 26(10): 1337-1345.e3, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30122451

RESUMO

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, catalyzing the conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, missense variants of this enzyme were identified as causing an inborn error of metabolism, PGM1 deficiency, with features of a glycogen storage disease and a congenital disorder of glycosylation. Previous studies of selected PGM1 variants have revealed various mechanisms for enzyme dysfunction, including regions of structural disorder and side-chain rearrangements within the active site. Here, we examine variants within a substrate-binding loop in domain 4 (D4) of PGM1 that cause extreme impairment of activity. Biochemical, structural, and computational studies demonstrate multiple detrimental impacts resulting from these variants, including loss of conserved ligand-binding interactions and reduced mobility of the D4 loop, due to perturbation of its conformational ensemble. These potentially synergistic effects make this conserved ligand-binding loop a hotspot for disease-related variants in PGM1 and related enzymes.


Assuntos
Mutação de Sentido Incorreto , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Domínio Catalítico , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfoglucomutase/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos
8.
Methods Enzymol ; 607: 241-267, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30149860

RESUMO

Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in the enzyme mechanism, the phosphorylation state of the catalytic phosphoserine has recently been found to have widespread effects on the structural flexibility of enzymes in this superfamily. These effects must be carefully accounted for when assessing other perturbations to these enzymes, such as mutations or ligand binding. In this chapter, we focus on methods for assessing and modulating the phosphorylation state of the catalytic serine, as well as straightforward ways to probe the impacts of this modification on protein structure/flexibility. This knowledge is essential for producing homogeneous and stable samples of these proteins for biophysical studies. The methods described herein should be widely applicable to enzymes across the PHM superfamily and may also be useful in characterizing the effects of posttranslational modifications on other proteins.


Assuntos
Ensaios Enzimáticos/métodos , Fosfoglucomutase/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Ensaios Enzimáticos/instrumentação , Corantes Fluorescentes/química , Modelos Moleculares , Fosfoglucomutase/química , Fosfoglucomutase/genética , Fosfoglucomutase/isolamento & purificação , Fosforilação , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos
9.
BMC Pediatr ; 18(1): 285, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30157810

RESUMO

BACKGROUND: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. CASE PRESENTATION: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. CONCLUSIONS: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.


Assuntos
Síndromes de Imunodeficiência/genética , Mutação , Fosfoglucomutase/genética , Sítios de Splice de RNA/genética , Pré-Escolar , Evolução Fatal , Feminino , Homozigoto , Humanos , Fosfoglucomutase/metabolismo , RNA Mensageiro/metabolismo
10.
Biochem J ; 475(15): 2547-2557, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-29967067

RESUMO

N-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-d-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.


Assuntos
Acetilglucosamina/análogos & derivados , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/química , Glucose-6-Fosfato/análogos & derivados , Fosfoglucomutase/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Catálise , Proteínas Fúngicas/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Fosfoglucomutase/metabolismo
11.
Plant Signal Behav ; 13(5): e1467698, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29944433

RESUMO

Phosphoglucomutases (PGM) (5.4.2.2.) belong to the Phosphohexomutases superfamily and are highly specific in catalyzing the interconversion of Glc-1-P to Glc-6-P. In this study, we characterize the expression and activity of two cytosolic PGMs (cPGM2 and cPGM3) stigmas of ornamental kale during flower development. In stigmas, cPGM expression and activity showed a gradual increase during stigma development with the highest activity around the time of anthesis. Blocking of cPGM activity in the stigmas using a known inhibitor, resulted in breakdown of self-incompatibility in immature S3 and S4 stigmas, but had no effect on the fully mature S5 stigmas. It is likely that cPGMs are required for accumulation of factors necessary for SI response in mature stigmas.


Assuntos
Brassica/enzimologia , Brassica/metabolismo , Flores/enzimologia , Flores/metabolismo , Fosfoglucomutase/metabolismo , Proteínas de Plantas/metabolismo , Brassica/genética , Flores/genética , Fosfoglucomutase/genética , Proteínas de Plantas/genética
12.
Biochemistry ; 57(30): 4504-4517, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-29952545

RESUMO

α-Phosphoglucomutase (αPGM), in its phosphorylated state, catalyzes the interconversion of α-d-glucose 1-phosphate and α-d-glucose 6-phosphate. The αPGM of Lactococcus lactis is a type C2B member of the haloalkanoic acid dehalogenase (HAD) enzyme family and is comprised of a Rossmann-fold catalytic domain and inserted α/ß-fold cap domain. The active site is formed at the domain-domain interface. Herein, we report the results from a kinetic-based study of L. lactis αPGM catalysis, which demonstrate enzyme activation by autocatalyzed phosphorylation of Asp8 with αG1P, the intermediacy of αG1,6bisP in the phospho Ll-αPGM-catalyzed conversion of αG1P to G6P, and the reorientation of the αG1,6bisP intermediate via dissociation to solvent and rebinding. In order to provide insight into the structural determinants of L. lactis αPGM substrate recognition and catalysis, metal cofactor and substrate specificities were determined as were the contributions made by active-site residues toward catalytic efficiency. Lastly, the structure and catalytic mechanism of L. lactis αPGM are compared with those of HAD family phosphomutases L. lactis ß-phosphoglucomutase and eukayotic α-phosphomannomutase to provide insight into the evolution of phosphohexomutases from HAD family phosphatases.


Assuntos
Lactococcus lactis/enzimologia , Fosfoglucomutase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Glucose-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Cinética , Lactococcus lactis/química , Lactococcus lactis/metabolismo , Modelos Moleculares , Fosfoglucomutase/química , Fosforilação , Conformação Proteica , Especificidade por Substrato
13.
Mol Biol Evol ; 35(8): 1968-1981, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29788479

RESUMO

Repeated evolutionary events imply underlying genetic constraints that can make evolutionary mechanisms predictable. Morphological traits are thought to evolve frequently through cis-regulatory changes because these mechanisms bypass constraints in pleiotropic genes that are reused during development. In contrast, the constraints acting on metabolic traits during evolution are less well studied. Here we show how a metabolic bottleneck gene has repeatedly adopted similar cis-regulatory solutions during evolution, likely due to its pleiotropic role integrating flux from multiple metabolic pathways. Specifically, the genes encoding phosphoglucomutase activity (PGM1/PGM2), which connect GALactose catabolism to glycolysis, have gained and lost direct regulation by the transcription factor Gal4 several times during yeast evolution. Through targeted mutations of predicted Gal4-binding sites in yeast genomes, we show this galactose-mediated regulation of PGM1/2 supports vigorous growth on galactose in multiple yeast species, including Saccharomyces uvarum and Lachancea kluyveri. Furthermore, the addition of galactose-inducible PGM1 alone is sufficient to improve the growth on galactose of multiple species that lack this regulation, including Saccharomyces cerevisiae. The strong association between regulation of PGM1/2 by Gal4 even enables remarkably accurate predictions of galactose growth phenotypes between closely related species. This repeated mode of evolution suggests that this specific cis-regulatory connection is a common way that diverse yeasts can govern flux through the pathway, likely due to the constraints imposed by this pleiotropic bottleneck gene. Since metabolic pathways are highly interconnected, we argue that cis-regulatory evolution might be widespread at pleiotropic genes that control metabolic bottlenecks and intersections.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Galactose/metabolismo , Fosfoglucomutase/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Fatores de Transcrição/metabolismo , Fosfoglucomutase/metabolismo , Saccharomycetales/metabolismo
14.
J Agric Food Chem ; 66(18): 4702-4709, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29693394

RESUMO

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.


Assuntos
Agaricales/genética , Fosfoglucomutase/genética , Polissacarídeos/antagonistas & inibidores , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Agaricales/metabolismo , Vias Biossintéticas , Expressão Gênica , Engenharia Metabólica , Fosfoglucomutase/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo
15.
J Bacteriol ; 200(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29507091

RESUMO

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.


Assuntos
Acetilgalactosamina/biossíntese , Proteínas Arqueais/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Fosfoglucomutase/metabolismo , Sulfolobus/enzimologia , Uridina Difosfato N-Acetilglicosamina/biossíntese , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas Arqueais/genética , Vias Biossintéticas , Galactosamina/análogos & derivados , Galactosamina/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Fosfatos/metabolismo , Fosfoglucomutase/genética , Sulfolobus/genética
16.
Appl Environ Microbiol ; 84(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29523547

RESUMO

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2, which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiaePGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects.IMPORTANCESaccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas).


Assuntos
Proteínas Fúngicas/genética , Galactose/metabolismo , Fosfoglucomutase/genética , Probióticos/metabolismo , Saccharomyces boulardii/metabolismo , Proteínas Fúngicas/metabolismo , Mutação , Fosfoglucomutase/metabolismo , Saccharomyces boulardii/enzimologia , Saccharomyces boulardii/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos
17.
Cell Death Dis ; 9(3): 377, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515119

RESUMO

Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumor progression. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Here we report the preclinical evaluation of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N- and O-glycosylation level that cause also a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with the activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice, supporting the advantage of targeting HBP for therapeutic purpose and encouraging further investigation about the use of this small molecule as a promising compound for breast cancer therapy.


Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Hexosaminas/biossíntese , Fosfoglucomutase/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Fosfoglucomutase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Rep ; 8(1): 2520, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29410524

RESUMO

While fatty acids are known to be toxic to dermatophytes, key physiological aspects of the Trichophyton rubrum response to undecanoic acid (UDA), a medium chain saturated fatty acid (C11:0), are not well understood. Thus, we analysed RNA-seq data from T. rubrum exposed to sub-lethal doses of UDA for 3 and 12 h. Three putative pathways were primarily involved in UDA detoxification: lipid metabolism and cellular membrane composition, oxidative stress, and pathogenesis. Biochemical assays showed cell membrane impairment, reductions in ergosterol content, and an increase in keratinolytic activity following UDA exposure. Moreover, we assessed differential exon usage and intron retention following UDA exposure. A key enzyme supplying guanine nucleotides to cells, inosine monophosphate dehydrogenase (IMPDH), showed high levels of intron 2 retention. Additionally, phosphoglucomutase (PGM), which is involved in the glycogen synthesis and degradation as well as cell wall biosynthesis, exhibited a significant difference in exon 4 usage following UDA exposure. Owing to the roles of these enzymes in fungal cells, both have emerged as promising antifungal targets. We showed that intron 2 retention in impdh and exon 4 skipping in pgm might be related to an adaptive strategy to combat fatty acid toxicity. Thus, the general effect of UDA fungal toxicity involves changes to fungal metabolism and mechanisms for regulating pre-mRNA processing events.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Antifúngicos/farmacologia , Ácidos Graxos/farmacologia , Transcriptoma/efeitos dos fármacos , Trichophyton/efeitos dos fármacos , Trichophyton/genética , Membrana Celular/efeitos dos fármacos , IMP Desidrogenase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfoglucomutase/metabolismo , Trichophyton/metabolismo , Trichophyton/patogenicidade
19.
Appl Microbiol Biotechnol ; 102(4): 1911-1922, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29349492

RESUMO

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of ß-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.


Assuntos
Parede Celular/metabolismo , Hifas/crescimento & desenvolvimento , Fosfoglucomutase/metabolismo , Polissacarídeos/metabolismo , Reishi/enzimologia , Reishi/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Inativação Gênica , Hifas/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reishi/citologia , Reishi/genética
20.
Appl Microbiol Biotechnol ; 102(3): 1251-1267, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29308528

RESUMO

Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T7 promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 µM) and 11% (9 mg/L; 22 µM) conversion of chrysin to chrysin 6-C-ß-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-ß-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 µM) and 14 mg/L (17%, 34 µM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 µM) conversion of chrysin to chrysin 6-C-ß-D-glucoside at 48 h upon supplementation of 200 µM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 µM) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and ß-glucosidase enzyme, than O-glucosylated derivative of chrysin.


Assuntos
Escherichia coli/metabolismo , Flavonas/biossíntese , Glucosídeos/biossíntese , Engenharia Metabólica , Técnicas de Cultura Celular por Lotes , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Fermentação , Flavonoides/metabolismo , Gentiana/enzimologia , Glucoquinase/genética , Glucoquinase/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Luteolina/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Soja/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Uridina Difosfato Glucose/metabolismo
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