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1.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540748

RESUMO

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Fosfofrutoquinases/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Proteínas de Bactérias/antagonistas & inibidores , Catálise , Indução Enzimática , Retroalimentação Fisiológica , Frutosedifosfatos/biossíntese , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Gluconeogênese , Glicólise , Hexosefosfatos/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Oxigênio/farmacologia , Fosfofrutoquinases/antagonistas & inibidores , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
2.
Biomolecules ; 10(12)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302546

RESUMO

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.


Assuntos
Proteínas Arqueais/química , DNA Polimerase beta/química , DNA Arqueal/química , Hexosefosfatos/química , Nucleotídeos/química , RNA Arqueal/química , Thermococcus/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hexosefosfatos/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , RNA Arqueal/genética , RNA Arqueal/metabolismo , Especificidade por Substrato , Thermococcus/enzimologia
3.
Commun Biol ; 3(1): 423, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32759958

RESUMO

The rare sugar D-tagatose is a safe natural product used as a commercial food ingredient. Here, we show that D-tagatose controls a wide range of plant diseases and focus on downy mildews to analyze its mode of action. It likely acts directly on the pathogen, rather than as a plant defense activator. Synthesis of mannan and related products of D-mannose metabolism are essential for development of fungi and oomycetes; D-tagatose inhibits the first step of mannose metabolism, the phosphorylation of D-fructose to D-fructose 6-phosphate by fructokinase, and also produces D-tagatose 6-phosphate. D-Tagatose 6-phosphate sequentially inhibits phosphomannose isomerase, causing a reduction in D-glucose 6-phosphate and D-fructose 6-phosphate, common substrates for glycolysis, and in D-mannose 6-phosphate, needed to synthesize mannan and related products. These chain-inhibitory effects on metabolic steps are significant enough to block initial infection and structural development needed for reproduction such as conidiophore and conidiospore formation of downy mildew.


Assuntos
Fungos/efeitos dos fármacos , Hexoses/farmacologia , Doenças das Plantas/prevenção & controle , Substâncias Protetoras/farmacologia , Agroquímicos/química , Agroquímicos/farmacologia , Fungos/patogenicidade , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Hexosefosfatos/genética , Hexoses/química , Fosforilação/efeitos dos fármacos , Doenças das Plantas/microbiologia
4.
Neurochem Res ; 45(11): 2529-2552, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32815045

RESUMO

The isoform of glucose-6-phosphatase in liver, G6PC1, has a major role in whole-body glucose homeostasis, whereas G6PC3 is widely distributed among organs but has poorly-understood functions. A recent, elegant analysis of neutrophil dysfunction in G6PC3-deficient patients revealed G6PC3 is a neutrophil metabolite repair enzyme that hydrolyzes 1,5-anhydroglucitol-6-phosphate, a toxic metabolite derived from a glucose analog present in food. These patients exhibit a spectrum of phenotypic characteristics and some have learning disabilities, revealing a potential linkage between cognitive processes and G6PC3 activity. Previously-debated and discounted functions for brain G6PC3 include causing an ATP-consuming futile cycle that interferes with metabolic brain imaging assays and a nutritional role involving astrocyte-neuron glucose-lactate trafficking. Detailed analysis of the anhydroglucitol literature reveals that it competes with glucose for transport into brain, is present in human cerebrospinal fluid, and is phosphorylated by hexokinase. Anhydroglucitol-6-phosphate is present in rodent brain and other organs where its accumulation can inhibit hexokinase by competition with ATP. Calculated hexokinase inhibition indicates that energetics of brain and erythrocytes would be more adversely affected by anhydroglucitol-6-phosphate accumulation than heart. These findings strongly support the paradigm-shifting hypothesis that brain G6PC3 removes a toxic metabolite, thereby maintaining brain glucose metabolism- and ATP-dependent functions, including cognitive processes.


Assuntos
Encéfalo/metabolismo , Glucose-6-Fosfatase/metabolismo , Hexosefosfatos/metabolismo , Neuroproteção/fisiologia , Animais , Desoxiglucose/metabolismo , Inibidores Enzimáticos/metabolismo , Hexoquinase/antagonistas & inibidores , Hexoquinase/metabolismo , Humanos , Fosforilação , Isoformas de Proteínas/metabolismo
5.
Blood ; 136(9): 1033-1043, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32294159

RESUMO

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Glucosídeos/uso terapêutico , Doença de Depósito de Glicogênio Tipo I/complicações , Hexosefosfatos/sangue , Neutropenia/tratamento farmacológico , Neutrófilos/patologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Compostos Benzidrílicos/efeitos adversos , Glicemia/análise , Quimiotaxia de Leucócito/efeitos dos fármacos , Pré-Escolar , Reposicionamento de Medicamentos , Resistência a Medicamentos , Feminino , Glucosídeos/efeitos adversos , Doença de Depósito de Glicogênio Tipo I/sangue , Doença de Depósito de Glicogênio Tipo I/imunologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Granulócitos/química , Humanos , Recém-Nascido , Proteína 2 de Membrana Associada ao Lisossomo/sangue , Masculino , Neutropenia/sangue , Uso Off-Label , Explosão Respiratória/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Adulto Jovem
6.
Mol Microbiol ; 112(4): 1178-1198, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31332863

RESUMO

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell-cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O-antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O-antigen in M. xanthus social behaviors. We identified WbaPMx (MXAN_2922) as the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for priming O-antigen synthesis. In heterologous expression experiments, WbaPMx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O-antigen synthesis, indicating that WbaPMx transfers galactose-1-P to undecaprenyl-phosphate. We also identified WaaLMx (MXAN_2919), as the O-antigen ligase that joins O-antigen to lipid A-core. Our data also support the previous suggestion that WzmMx (MXAN_4622) and WztMx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O-antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaPMx deletion mutant, we revisited the role of LPS O-antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili-dependent motility and required to complete the developmental program leading to the formation of spore-filled fruiting bodies.


Assuntos
Lipopolissacarídeos/biossíntese , Myxococcus xanthus/metabolismo , Antígenos O/biossíntese , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Hexosefosfatos/metabolismo , Ligases/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas Motores Moleculares/metabolismo , Mutação , Myxococcus xanthus/genética , Antígenos O/metabolismo , Fenótipo , Fosfatos de Poli-Isoprenil/metabolismo
7.
Planta ; 249(5): 1319-1336, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30627889

RESUMO

MAIN CONCLUSION: Futile cycling between free sugars and hexose phosphates occurring under phosphate deficiency could be involved in the maintenance of a threshold level of free cellular phosphate to preserve respiratory metabolism. We studied the metabolic response of potato cell cultures growing in Pi sufficient (2.5 mM, +Pi) or deficient (125 µM, -Pi) conditions. Under Pi deficiency, cellular growth was severely affected, however -Pi cells were able to maintain a low but steady level of free Pi. We surveyed the activities of 33 primary metabolic enzymes during the course of a 12 days Pi deficiency period. Our results show that many of these enzymes had higher specific activity in -Pi cells. Among these, we found typical markers of Pi deficiency such as phosphoenolpyruvate phosphatase and phosphoenolpyruvate carboxylase as well as enzymes involved in the biosynthesis of organic acids. Intriguingly, several ATP-consuming enzymes such as hexokinase (HK) and phosphofructokinase also displayed increased activity in -Pi condition. For HK, this was associated with an increase in the steady state of a specific HK polypeptide. Quantification of glycolytic intermediates showed a pronounced decrease in phosphate esters under Pi deficiency. Adenylate levels also decreased in -Pi cells, but the Adenylate Energy Charge was not affected by the treatment. To investigate the significance of HK induction under low Pi, [U-14C]-glucose tracer studies were conducted. We found in vivo evidence of futile cycling between pools of hexose phosphates and free sugars under Pi deficiency. Our study suggests that the futile cycling between hexose phosphates and free sugars which is active under +Pi conditions is sustained under Pi deficiency. The possibility that this process represents a metabolic adaptation to Pi deficiency is discussed with respect to Pi homeostasis in Pi-deficient conditions.


Assuntos
Hexosefosfatos/metabolismo , Fosfatos/deficiência , Solanum tuberosum/metabolismo , Açúcares/metabolismo , Técnicas de Cultura de Células , Hexoquinase/metabolismo , Solanum tuberosum/citologia
8.
J Gen Appl Microbiol ; 64(5): 248-252, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29743459

RESUMO

Sorbitol-6-phosphatase (EC 3.1.3.50) catalyzes sorbitol production from sorbitol-6-phosphate in certain organisms, but has not been identified unequivocally. We screened the activity of the haloacid dehalogenase-like hydrolases (HAD) superfamily and identified four HAD proteins from Escherichia coli as sorbitol-6-phosphatase. Of these proteins, HAD2 (YfbT) exhibited catalytic activity (kcat/Km) that was better than that of the previously reported "preferred" substrate. HAD1 (YniC) and HAD2 exhibited higher sorbitol-6-phosphatase activity than that of HAD12 (YbiV) and HAD13 (YidA). Therefore, genes of HAD may be useful for metabolic engineering of effective sorbitol production.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Cianobactérias/enzimologia , Escherichia coli/enzimologia , Hexosefosfatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cianobactérias/genética , Escherichia coli/genética , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Cinética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sorbitol/metabolismo , Especificidade por Substrato
9.
J Struct Biol ; 203(2): 109-119, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29605571

RESUMO

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Šresolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Erwinia amylovora/enzimologia , Erwinia amylovora/metabolismo , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo , Proteínas de Bactérias/genética , Erwinia amylovora/genética , Hexosefosfatos/metabolismo , Cinética , Rosaceae/microbiologia , Desidrogenase do Álcool de Açúcar/genética , Tomografia Computadorizada por Raios X
10.
Food Microbiol ; 62: 178-187, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27889146

RESUMO

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and ß-galactosidase assay suggest that PTSGal system, PTSLac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway.


Assuntos
Produtos Fermentados do Leite/microbiologia , Galactose/metabolismo , Hexosefosfatos/metabolismo , Leite/química , Animais , Produtos Fermentados do Leite/análise , Galactose/análise , Galactose/biossíntese , Genômica , Hexosefosfatos/genética , Lactose/análise , Lactose/metabolismo , Leite/microbiologia , Especificidade da Espécie , Streptococcus thermophilus/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
11.
Plant Cell Physiol ; 58(1): 145-155, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28011870

RESUMO

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.


Assuntos
Aldeído Redutase/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Prunus domestica/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Frutosedifosfatos/metabolismo , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Dissulfeto de Glutationa/metabolismo , Hexosefosfatos/metabolismo , Hexosefosfatos/farmacologia , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo
12.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 11): 831-839, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27827354

RESUMO

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Šresolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.


Assuntos
Aldeído Liases/química , Fosfato de Di-Hidroxiacetona/química , Proteínas Fúngicas/química , Hexosefosfatos/química , Saccharomycetales/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Fosfato de Di-Hidroxiacetona/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Hexosefosfatos/metabolismo , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
Food Microbiol ; 58: 79-86, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27217362

RESUMO

The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.


Assuntos
Carnobacterium/genética , Galactose/metabolismo , Variação Genética , Genômica , Lactose/metabolismo , Leite/metabolismo , Animais , Carnobacterium/metabolismo , Hexosefosfatos , Filogenia , Análise de Sequência de DNA , Sintenia
14.
Int J Biol Macromol ; 88: 578-85, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27083849

RESUMO

Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile ß-l-fucopyranosyl phosphate (ß-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of ß-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure.


Assuntos
Escherichia coli/química , Lipopolissacarídeos/química , Antígenos O/química , Relação Estrutura-Atividade , Sequência de Carboidratos , Fucose/análogos & derivados , Fucose/química , Hexosefosfatos/química , Lipopolissacarídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Antígenos O/isolamento & purificação
15.
Acta Crystallogr D Struct Biol ; 72(Pt 4): 467-76, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27050126

RESUMO

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to ß-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.


Assuntos
Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Hexosefosfatos/química , Transaldolase/antagonistas & inibidores , Transaldolase/química , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação de Sentido Incorreto , Transaldolase/genética
16.
Appl Environ Microbiol ; 81(24): 8315-29, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26407889

RESUMO

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Biologia Computacional/métodos , Redes e Vias Metabólicas/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Bactérias/genética , Colina/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Hexosefosfatos/metabolismo , Organelas/metabolismo , Propilenoglicóis/metabolismo
17.
J Pharm Biomed Anal ; 115: 402-9, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26279371

RESUMO

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.


Assuntos
Carboidratos/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Nucleotídeos/análise , Soluções Tampão , Fucose/análogos & derivados , Fucose/análise , Hexosefosfatos/análise , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Açúcares de Nucleosídeo Difosfato/análise , Solventes/química , Eletricidade Estática , Fosfatos Açúcares/análise
18.
J Mol Microbiol Biotechnol ; 25(2-3): 106-19, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26159072

RESUMO

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.


Assuntos
Bacillus/metabolismo , Hexoses/química , Hexoses/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Bacillus/enzimologia , Escherichia coli/genética , Frutose/metabolismo , Frutosefosfatos/metabolismo , Hexosefosfatos/metabolismo , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Especificidade por Substrato
19.
Planta ; 240(1): 223-38, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24817585

RESUMO

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP(+) (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.


Assuntos
Simulação de Acoplamento Molecular , Oryza/enzimologia , Processamento de Proteína Pós-Traducional , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Hexosefosfatos/metabolismo , Cinética , Oryza/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/isolamento & purificação
20.
J Biomol Screen ; 19(7): 1014-23, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24717911

RESUMO

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Descoberta de Drogas/métodos , Glucoquinase/química , Trifosfato de Adenosina/química , Regulação Alostérica , Animais , Glicemia/análise , Calorimetria , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fluorescência , Fluorometria , Frutosefosfatos/química , Hepatócitos/metabolismo , Hexosefosfatos/química , Homeostase , Humanos , Hipoglicemia/prevenção & controle , Concentração Inibidora 50 , Luminescência , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ressonância de Plasmônio de Superfície
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