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2.
Vet Microbiol ; 258: 109102, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33991786

RESUMO

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious economic losses in the pig industry. Phosphorylation is an important mechanism of protein modification. Recent studies have reported that the serine/threonine kinase (STK) gene contributes to the growth and virulence of SS2. However, the mechanism underlying the regulatory functions of STK in SS2 has not been thoroughly elucidated to date. In this study, phosphoproteomic analysis was performed to determine substrates of the STK protein. Twenty-two proteins with different cell functions were identified as potential substrates of STK. Phosphoglucosamine mutase (GlmM) was selected for further investigation among them. In vitro phosphorylation assay and immunoprecipitation assay indicated that GlmM was phosphorylated by STK at the Ser-101 site and the phosphorylation level of GlmM can be affected. We observed that compared to the wild-type strain ZY05719, the glmM-deficient strain (ΔglmM) and the glmM S101A point mutation strain (CΔglmM S101A) showed aberrant cell morphology and attenuated virulence, including enlarged cell volume, absent capsule, decreased resistance, lower survival caused by unusual peptidoglycan synthesis, and significantly attenuated pathogenicity in a mouse infection model. Additionally, compared to ZY05719 and CΔglmM, GlmM enzyme acivities and peptidoglycan concentrations of the stk-deficient strain (Δstk), CΔglmM S101A decreased significantly. These experiments revealed that STK phosphorylates GlmM at the Ser-101 site to impact GlmM enzyme activity and control cell wall peptidoglycan synthesis to affect SS2 pathogenicity.


Assuntos
Parede Celular/metabolismo , Fosfoglucomutase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Streptococcus suis/metabolismo , Animais , Biologia Computacional , Feminino , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Fosfoglucomutase/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Distribuição Aleatória , Infecções Estreptocócicas/microbiologia , Streptococcus suis/classificação , Streptococcus suis/patogenicidade , Virulência
3.
J Bacteriol ; 203(13): e0060220, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33875543

RESUMO

Capsular polysaccharide (CPS) is a major virulence determinant for many human-pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence have been established, knowledge of how CPS production is regulated remains limited. Streptococcus pneumoniae (pneumococcus) CPS expression levels and overall thickness change in response to available oxygen and carbohydrate. These nutrients in addition to transition metal ions can vary significantly between host environmental niches and infection stage. Since the pneumococcus must modulate CPS expression among various host niches during disease progression, we examined the impact of the nutritional transition metal availability of manganese (Mn) and zinc (Zn) on CPS production. We demonstrate that increased Mn/Zn ratios increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway in a transcription-independent manner. Furthermore, we find that the downstream CPS protein CpsB, an Mn-dependent phosphatase, does not promote aberrant dephosphorylation of its target capsule-tyrosine kinase CpsD during Mn stress. Together, these data reveal a direct role for cellular Mn/Zn ratios in the regulation of CPS biosynthesis via the direct activation of Pgm. We propose a multilayer mechanism used by the pneumococcus in regulating CPS levels across various host niches. IMPORTANCE Evolving evidence strongly indicates that maintenance of metal homeostasis is essential for establishing colonization and continued growth of bacterial pathogens in the vertebrate host. In this study, we demonstrate the impact of cellular manganese/zinc (Mn/Zn) ratios on bacterial capsular polysaccharide (CPS) production, an important virulence determinant of many human-pathogenic bacteria, including Streptococcus pneumoniae. We show that higher Mn/Zn ratios increase CPS production via the Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway. The findings provide a direct role for Mn/Zn homeostasis in the regulation of CPS expression levels and further support the ability of metal cations to act as important cellular signaling mediators in bacteria.


Assuntos
Cápsulas Bacterianas/metabolismo , Manganês/metabolismo , Fosfoglucomutase/metabolismo , Streptococcus pneumoniae/metabolismo , Zinco/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicólise , Homeostase , Humanos , Íons/metabolismo , Mutação , Fosfoglucomutase/genética , Fosforilação , Infecções Pneumocócicas/microbiologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/genética , Fatores de Virulência
4.
Biochimie ; 183: 44-48, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32898648

RESUMO

Once experimentally prohibitive, structural studies of individual missense variants in proteins are increasingly feasible, and can provide a new level of insight into human genetic disease. One example of this is the recently identified inborn error of metabolism known as phosphoglucomutase-1 (PGM1) deficiency. Just as different variants of a protein can produce different patient phenotypes, they may also produce distinct biochemical phenotypes, affecting properties such as catalytic activity, protein stability, or 3D structure/dynamics. Experimental studies of missense variants, and particularly structural characterization, can reveal details of the underlying biochemical pathomechanisms of missense variants. Here, we review four examples of enzyme dysfunction observed in disease-related variants of PGM1. These studies are based on 11 crystal structures of wild-type (WT) and mutant enzymes, and multiple biochemical assays. Lessons learned include the value of comparing mutant and WT structures, synergy between structural and biochemical studies, and the rich understanding of molecular pathomechanism provided by experimental characterization relative to the use of predictive algorithms. We further note functional insights into the WT enzyme that can be gained from the study of pathogenic variants.


Assuntos
Doença de Depósito de Glicogênio , Fosfoglucomutase , Cristalografia por Raios X , Doença de Depósito de Glicogênio/enzimologia , Doença de Depósito de Glicogênio/genética , Humanos , Mutação , Fosfoglucomutase/química , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Domínios Proteicos
5.
Biotechnol Lett ; 43(1): 177-192, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32757151

RESUMO

The purification and characterization of PGM (Phosphoglucomutase) from Cordyceps militaris (C. militaris) was investigated. PGM was purified using a combination of ultrafiltration, salting-out and ion exchange chromatography resulting in 4.23-fold enhancement of activity with a recovery of 20.01%. Molecular mass was 50.01 kDa by SDS-PAGE. The optimal activity was achieved at pH 7.5 and 30 °C with NADPH as substrate. The results showed that SDS, DTT Li+, Cu2+, Na+, Mn2+ and Al3+ were effective PGM inhibitors; whereas glycerol, Zn2+, Mg2+, Ca2+, Fe2+ and Fe3+ could enhance the activity of PGM, and the Km and Vmax values were 11.62 mmol/L and 416.67 U/mL, respectively. At the same time, qRT-PCR was used to test the changes of mRNA transcription level of PGM gene encoding under two fermentation conditions: basic medium and optimized medium. The relative quantitative results of PGM target genes resulting in 2.60-fold enhancement than the control group.


Assuntos
Cordyceps , Proteínas Fúngicas , Fosfoglucomutase , Cromatografia por Troca Iônica , Cordyceps/enzimologia , Cordyceps/genética , Cordyceps/metabolismo , Filtração , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Expressão Gênica , Fosfoglucomutase/química , Fosfoglucomutase/genética , Fosfoglucomutase/isolamento & purificação , Fosfoglucomutase/metabolismo
6.
Methods Mol Biol ; 2167: 13-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712912

RESUMO

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaios Enzimáticos/métodos , Faecalibacterium prausnitzii/metabolismo , Magnésio/metabolismo , RNA Catalítico/metabolismo , Transcrição Genética , Proteínas Virais/metabolismo , Proteínas de Bactérias/genética , Catálise , Eletroforese em Gel de Poliacrilamida , Faecalibacterium prausnitzii/enzimologia , Faecalibacterium prausnitzii/genética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Íons/química , Cinética , Ligantes , Magnésio/química , Fosfoglucomutase/metabolismo , RNA Catalítico/genética
7.
Biomolecules ; 10(12)2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287293

RESUMO

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.


Assuntos
Peixes , Fosfoglucomutase/metabolismo , Animais , Domínio Catalítico , Glucofosfatos/metabolismo , Fosfoglucomutase/química , Ligação Proteica , Especificidade por Substrato
8.
PLoS One ; 15(12): e0243867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338036

RESUMO

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Proteômica , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Bacterianos , Anotação de Sequência Molecular , Fosfoglucomutase/metabolismo , Reprodutibilidade dos Testes , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
9.
Vet Res ; 51(1): 97, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32736655

RESUMO

Lipooligosaccharides (LOSs) are virulence determinants of Glaesserella parasuis, a pathogen of the respiratory tract of pigs. We previously reported that disruption of the galU or galE gene in G. parasuis results in increased sensitivity to porcine serum, indicating that the galactose catabolism pathway is required for polysaccharide formation in G. parasuis. Here, we evaluated the role of the HAPS_0849 gene in LOS synthesis. The G. parasuis SC096 HAPS_0849 mutant produced a highly truncated LOS molecule, although a small fraction of intact LOS was still observed, and this mutant was found to be more sensitive to serum than the parental strain. HAPS_0849 was overexpressed and purified for biochemical assays, and this protein exhibited phosphoglucomutase (PGM) activity. Heterologous expression of a pgm gene from Escherichia coli in the HAPS_0849 mutant led to restoration of the wild-type LOS glycoform, further demonstrating the PGM function of HAPS_0849 in G. parasuis. The autoagglutination and biofilm formation ability of this strain were also investigated. Disruption of HAPS_0849 led to an increased tendency to autoagglutinate and form more biofilms, and these enhanced phenotypes were observed in the absence of glucose. In addition, LOSs from HAPS_0849, galU and lgtB mutants had similar truncated glycoforms, while LOSs from the galE and lex-1 mutants exhibited another type of defective LOS pattern. These findings imply that HAPS_0849 may function upstream of GalU in the generation of glucose 1-phosphate. In conclusion, our results preliminarily described the functions of HAPS_0849 in G. parasuis, and this gene was partially required for LOS synthesis.


Assuntos
Proteínas de Bactérias/genética , Haemophilus parasuis/genética , Lipopolissacarídeos/biossíntese , Fosfoglucomutase/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Haemophilus parasuis/enzimologia , Microrganismos Geneticamente Modificados/genética , Fosfoglucomutase/metabolismo
10.
Int J Mol Sci ; 21(16)2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32785068

RESUMO

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of PGM2 mRNA in yeast.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Antimaníacos/farmacologia , Cloreto de Lítio/farmacologia , Biossíntese de Proteínas/genética , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regiões 5' não Traduzidas , Aminoacil-tRNA Sintetases/genética , Antimaníacos/uso terapêutico , Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Cloreto de Lítio/uso terapêutico , Organismos Geneticamente Modificados , Fosfoglucomutase/antagonistas & inibidores , Fosfoglucomutase/metabolismo , RNA Helicases/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
11.
Environ Microbiol ; 22(7): 2771-2791, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32250026

RESUMO

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.


Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/biossíntese , Listeria monocytogenes/fisiologia , Fosfoglucomutase/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Homeostase , Listeria monocytogenes/enzimologia , Pressão Osmótica/fisiologia
12.
Biosci Rep ; 40(4)2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32239183

RESUMO

Cells lining the uterus are responsible for storage and secretion of carbohydrates to support early embryonic development. Histotrophic secretions contain glycogen and glycolytic products such as lactate and pyruvate. Insufficient carbohydrate storage as glycogen has been correlated with infertility in women. While it is clear that changes in estrogen (17-ß-estradiol (E2)) and progesterone (P4) in vivo affect the distribution of glucose in the uterine cells and secretions, the biochemical mechanism(s) by which they affect this crucial allocation is not well understood. Furthermore, in cultured uterine cells, neither E2 nor P4 affect glycogen storage without insulin present. We hypothesized that P4 and E2 alone affect the activity of glycolytic enzymes, glucose and glycolytic flux to increase glycogen storage (E2) and catabolism (P4) and increase pyruvate and lactate levels in culture. We measured the rate of glucose uptake and glycolysis in a mink immortalized epithelial cell line (GMMe) after 24-h exposure to 10 µM P4 and 10 nM E2 (pharmacologic levels) at 5 mM glucose and determined the kinetic parameters (Vmax, Km) of all enzymes. While the activities of many glycolytic enzymes in GMMe cells were shown to be decreased by E2 treatment, in contrast, glucose uptake, glycolytic flux and metabolites levels were not affected by the treatments. The cellular rationale for P4- and E2-induced decreases in the activity of enzymes may be to prime the system for other regulators such as insulin. In vivo, E2 and P4 may be necessary but not sufficient signals for uterine cycle carbohydrate allocation.


Assuntos
Estradiol/metabolismo , Ciclo Estral/metabolismo , Glucose/metabolismo , Progesterona/metabolismo , Útero/metabolismo , Animais , Linhagem Celular , Ensaios Enzimáticos , Células Epiteliais , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glicogênio/metabolismo , Glicólise/fisiologia , Cinética , Vison , Modelos Animais , Fosfoglucomutase/metabolismo
13.
Cancer Lett ; 478: 82-92, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32171858

RESUMO

Cancer cells undergo metabolic reprogramming to sustain their own survival under an environment of increased energy demand; however, the mechanism by which cancer cells ensure survival under glucose deprivation stressed conditions remains elusive. Here, we show that deprivation of glucose, dramatically activated the glycogen pathway, accompanied by elevated phosphoglucomutase 1 (PGM1) expression. We further identified that AMP-activated protein kinase (AMPK) stimulated PGM1 expression by inducing histone deacetylase 8 (HDAC8) phosphorylation. Moreover, we demonstrated that glucose deprivation-induced AMPK activation stimulated the translocation of HDAC8 from the nucleus to the cytoplasm, consequently disrupting the binding between HDAC8 and histone 3. PGM1 expression was also found to be critical for lung cancer glycolysis, the oxidative pentose phosphate pathway, and oxidative phosphorylation under glucose deprivation conditions, and further led to the aberrant expression of metabolic enzymes involved in glucose metabolism mediated by ERK1/2. Finally, PGM1 was found to be highly expressed in lung cancer tissues from patients, which correlated with a poor prognosis. Taken together, these results revealed that AMPK activation by glucose deprivation leads to enhanced PGM1 expression, an essential component of the metabolic switch, to facilitate cancer progression, suggesting PGM1 as promising anti-cancer treatment targets.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Histona Desacetilases/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Proteínas Repressoras/metabolismo , Células A549 , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicólise , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Fosforilação , Transdução de Sinais , Regulação para Cima
14.
ACS Synth Biol ; 9(2): 449-456, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31940436

RESUMO

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Sesquiterpenos Policíclicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Técnicas de Cultura Celular por Lotes , Clausena/enzimologia , Proteínas de Ligação a DNA/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Galactose/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Sesquiterpenos Policíclicos/análise , Sesquiterpenos Policíclicos/química , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Santalum/enzimologia , Fatores de Transcrição/genética
15.
BMC Res Notes ; 12(1): 682, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640766

RESUMO

OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.


Assuntos
Metabolismo dos Carboidratos , Ensaios Enzimáticos/métodos , Glucose/metabolismo , Glicólise , Animais , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Estudos de Viabilidade , Frutose-Bifosfato Aldolase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Células HEK293 , Células Hep G2 , Hexoquinase/metabolismo , Humanos , Camundongos , Fenótipo , Fosfofrutoquinases/metabolismo , Fosfoglucomutase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Projetos Piloto
16.
PLoS Genet ; 15(10): e1008434, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589605

RESUMO

Phosphohexomutase superfamily enzymes catalyze the reversible intramolecular transfer of a phosphoryl moiety on hexose sugars. Bacillus subtilis phosphoglucomutase PgcA catalyzes the reversible interconversion of glucose 6-phosphate (Glc-6-P) and glucose 1-phosphate (Glc-1-P), a precursor of UDP-glucose (UDP-Glc). B. subtilis phosphoglucosamine mutase (GlmM) is a member of the same enzyme superfamily that converts glucosamine 6-phosphate (GlcN-6-P) to glucosamine 1-phosphate (GlcN-1-P), a precursor of the amino sugar moiety of peptidoglycan. Here, we present evidence that B. subtilis PgcA possesses activity as a phosphoglucosamine mutase that contributes to peptidoglycan biosynthesis. This activity was made genetically apparent by the synthetic lethality of pgcA with glmR, a positive regulator of amino sugar biosynthesis, which can be specifically suppressed by overproduction of GlmM. A gain-of-function mutation in a substrate binding loop (PgcA G47S) increases this secondary activity and suppresses a glmR mutant. Our results demonstrate that bacterial phosphoglucomutases may possess secondary phosphoglucosamine mutase activity, and that this dual activity may provide some level of functional redundancy for the essential peptidoglycan biosynthesis pathway.


Assuntos
Bacillus subtilis/enzimologia , Peptidoglicano/biossíntese , Fosfoglucomutase/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Mutação com Ganho de Função , Fosfoglucomutase/genética , Mutações Sintéticas Letais
17.
J Org Chem ; 84(15): 9627-9636, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31264865

RESUMO

α-Phosphomannomutase/phosphoglucomutase (αPMM/PGM) from P. aeruginosa is involved in bacterial cell wall assembly and is implicated in P. aeruginosa virulence, yet few studies have addressed αPMM/PGM inhibition from this important Gram-negative bacterial human pathogen. Four structurally different α-d-glucopyranose 1-phosphate (αG1P) derivatives including 1-C-fluoromethylated analogues (1-3), 1,2-cyclic phosph(on)ate analogues (4-6), isosteric methylene phosphono analogues (7 and 8), and 6-fluoro-αG1P (9), were synthesized and assessed as potential time-dependent or reversible αPMM/PGM inhibitors. The resulting kinetic data were consistent with the crystallographic structures of the highly homologous Xanthomonas citri αPGM with inhibitors 3 and 7-9 binding to the enzyme active site (1.65-1.9 Å). These structural and kinetic insights will enhance the design of future αPMM/PGM inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Fosfoglucomutase/antagonistas & inibidores , Fosfotransferases (Fosfomutases)/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Fosfatos Açúcares/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares , Estrutura Molecular , Fosfoglucomutase/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Pseudomonas aeruginosa/enzimologia , Fosfatos Açúcares/síntese química , Fosfatos Açúcares/química
18.
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
19.
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
20.
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
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