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1.
J Med Chem ; 63(8): 4227-4255, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32216347

RESUMO

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing ß cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or ß) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.


Assuntos
Ativação Enzimática/fisiologia , Glucuronidase/metabolismo , Glicosídeos/metabolismo , Heparitina Sulfato/metabolismo , Oligossacarídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glicosídeos/síntese química , Heparina/farmacologia , Heparitina Sulfato/antagonistas & inibidores , Humanos , Camundongos , Simulação de Acoplamento Molecular/métodos , Oligossacarídeos/síntese química
2.
Food Chem ; 317: 126406, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097823

RESUMO

In this study, 50 tomato landraces grown in Turkey were investigated in terms of their secondary metabolite profiles. Each accession was planted in 2016 and 2017 in 3 replicates in an open field. In this study, color, pH and brix of the fruit samples were measured and an unbiased LCMS-based metabolomics approach was applied. Based on Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) of the relative abundance levels of >250 metabolites, it could be concluded that fruit size was the most influential to the biochemical composition, rather than the geographical origin of accessions. Results indicated substantial biodiversity in various metabolites generally regarded as key to fruit quality aspects, including sugars; phenolic compounds like phenylpropanoids and flavonoids; alkaloids and glycosides of flavour-related volatile compounds. The phytochemical data provides insight into which Turkish accessions might be most promising as starting materials for the tomato processing and breeding industries.


Assuntos
Frutas/metabolismo , Lycopersicon esculentum/metabolismo , Alcaloides/análise , Alcaloides/metabolismo , Cromatografia Líquida , Análise por Conglomerados , Flavonoides/análise , Flavonoides/metabolismo , Frutas/química , Glicosídeos/análise , Glicosídeos/metabolismo , Concentração de Íons de Hidrogênio , Lycopersicon esculentum/química , Espectrometria de Massas , Metabolômica/métodos , Metabolômica/estatística & dados numéricos , Fenóis/análise , Fenóis/metabolismo , Análise de Componente Principal , Metabolismo Secundário , Turquia
3.
Appl Microbiol Biotechnol ; 104(5): 1883-1890, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932892

RESUMO

C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C-C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C-C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a "C-glycosidase" directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C-C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/metabolismo , Intestinos/microbiologia , Animais , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Biotransformação , Glicosídeo Hidrolases/genética , Glicosídeos/química , Glicosilação , Humanos , Estrutura Molecular
4.
J Agric Food Chem ; 68(5): 1419-1426, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31888328

RESUMO

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.


Assuntos
Bebidas Alcoólicas/microbiologia , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeos/metabolismo , alfa-Glucosidases/metabolismo , Bebidas Alcoólicas/análise , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Etanol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Glicosídeos/química , Glicosilação , Maltose/metabolismo , Oryza/metabolismo , Oryza/microbiologia , alfa-Glucosidases/genética
5.
J Agric Food Chem ; 68(5): 1494-1504, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31917553

RESUMO

Flavonoids are widely distributed in mulberry leaves and have been recognized for their beneficial physiological effects on the human health. Here, we analyzed variations in 44 flavonoid compounds among 91 mulberry resources. Metabolic profiling revealed that O-rhamnosylated flavonols and malonylated flavonol glycosides, including rutin and quercetin 3-O-(6″-O-malonylglucoside) (Q3MG), were absent from Morus notabilis and multiple mulberry (Morus alba L.) resources. Transcriptome and phylogenetic analyses of flavonoid-related UDP-glycosyltransferases (UGTs) suggested that the flavonol 3-O-glucoside-O-rhamnosyltransferase (FGRT) KT324624 is a key enzyme involved in rutin synthesis. A recombinant FGRT protein was able to convert kaempferol/quercetin 3-O-glucoside to kaempferol 3-O-rutinoside (K3G6″Rha) and rutin. The recombinant FGRT was able to use 3-O-glucosylated flavonols but not flavonoid aglycones or 7-O-glycosylated flavonoids as substrates. The enzyme preferentially used UDP-rhamnose as the sugar donor, indicating that it was a flavonol 3-O-glucoside: 6″-O-rhamnosyltransferase. This study provided insights into the biosynthesis of rutin in mulberry.


Assuntos
Flavonoides/biossíntese , Morus/metabolismo , Extratos Vegetais/biossíntese , Proteínas de Plantas/genética , Flavonoides/química , Perfilação da Expressão Gênica , Glicosídeos/química , Glicosídeos/metabolismo , Metabolômica , Morus/química , Morus/genética , Filogenia , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Rutina/metabolismo
6.
J Biol Chem ; 295(8): 2227-2238, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31932305

RESUMO

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4-7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.


Assuntos
Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicosídeos/metabolismo , Glicosilação , Lectinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Receptores da Transferrina/metabolismo , Especificidade por Substrato
7.
Biochem Biophys Res Commun ; 523(2): 487-492, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31889533

RESUMO

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5-7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.


Assuntos
Glicosídeos/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Sphingobacterium/enzimologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Cricetulus , Concentração de Íons de Hidrogênio , Hidrólise , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Ácidos Neuramínicos , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/genética , Sphingobacterium/genética , Especificidade por Substrato , Temperatura
8.
Food Chem ; 311: 125895, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31780220

RESUMO

Pile-fermentation is the most important process of producing ripened pu-erh tea. To study the chemical changes of tea leaves during pile-fermentation (PF), liquid chromatography coupled with tandem mass spectrometry (LC-MS) was used. Untargeted metabolomics analysis revealed that the first stage of PF is crucial in transforming the original secondary metabolites, whereas the contents of flavan-3-ols and gallic acid were decreased after long-term PF. Targeted metabolomics analysis indicated that the levels of puerins (N-ethyl-2-pyrrolidinone substituted gallocatechin and catechin) were significant increased after the first stage of PF, but after long-term PF the levels of flavonol glycosides, procyanidins and galloylated flavan-3-ols were significantly decreased. Accordingly, long-term PF also decreased the inhibition of α-amylase and α-glucosidase activities of the extracts. As a conclusion, pile-fermentation is an important step of changing the polyphenols and bioactivities of pu-erh tea.


Assuntos
Camellia/metabolismo , Metabolômica , Amilases/antagonistas & inibidores , Amilases/metabolismo , Camellia/química , Análise por Conglomerados , Fermentação , Flavonóis/metabolismo , Ácido Gálico/metabolismo , Glicosídeos/metabolismo , Análise dos Mínimos Quadrados , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Análise de Componente Principal , Proantocianidinas/metabolismo
9.
J Mass Spectrom ; 55(1): e4484, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786817

RESUMO

Qixianqingming granules (QXQM) comprise a traditional Chinese medicine (TCM) formula that was developed based on the combination of TCM theory and clinical practice. This formula has been proven to effectively treat asthma. In this study, an analytical procedure using ultraperformance liquid chromatography, coupled with electrospray ionization quadrupole time-of-flight mass spectrometry, was established for the rapid separation and sensitive identification of the chemical components in QXQM and its metabolites in serum of rats. Seventy-two compounds were systematically identified in QXQM, including flavonoids, terpenoids, anthraquinones, phenylethanoid glycosides, stilbenes, alkaloids, and organic acids. Thirteen prototype compounds and 29 metabolites were detected in the serum of rats. The results provided fundamental information for further studying the mechanisms and clinical application of QXQM.


Assuntos
Medicamentos de Ervas Chinesas/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Alcaloides/análise , Alcaloides/metabolismo , Animais , Antraquinonas/análise , Antraquinonas/metabolismo , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/metabolismo , Flavonoides/análise , Flavonoides/metabolismo , Glicosídeos/análise , Glicosídeos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Estilbenos/análise , Estilbenos/metabolismo , Terpenos/análise , Terpenos/metabolismo
10.
Insect Biochem Mol Biol ; 116: 103259, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31698083

RESUMO

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.


Assuntos
Borboletas/metabolismo , Glicosídeos/biossíntese , Glicosídeos/metabolismo , Animais , Coevolução Biológica , Borboletas/química , Borboletas/enzimologia , Borboletas/crescimento & desenvolvimento , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Perfilação da Expressão Gênica , Glucosídeos/metabolismo , Herbivoria , Larva/enzimologia , Larva/metabolismo , Estágios do Ciclo de Vida/fisiologia , Nitrilos/metabolismo , Passiflora/química
11.
Protein Expr Purif ; 166: 105502, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31546007

RESUMO

Cellouronate is a (1,4)-ß-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (ß-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized ß-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.


Assuntos
Caulobacteraceae/genética , Polissacarídeo-Liase/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Sequência de Bases , Caulobacteraceae/enzimologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Glicosídeos/química , Glicosídeos/metabolismo , Oxirredução , Polissacarídeo-Liase/química , Polissacarídeo-Liase/classificação , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação
12.
BMC Complement Altern Med ; 19(1): 346, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791311

RESUMO

BACKGROUND: Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS: MDCK cells were exposed to Q3R and 100CCID50/100 µl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS: The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of - 10.81, - 10.47, - 9.52, - 9.24 and - 8.78 Kcal/mol, respectively. CONCLUSIONS: Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.


Assuntos
Antivirais , Glicosídeos , Vírus da Influenza A Subtipo H1N1 , Myrsine/química , Compostos Fitoquímicos , Quercetina/análogos & derivados , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Cães , Glicosídeos/química , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Compostos Fitoquímicos/farmacologia , Quercetina/química , Quercetina/metabolismo , Quercetina/farmacologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
13.
Int J Mol Sci ; 20(24)2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817903

RESUMO

Fungal ß-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The ß-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model ß-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-ß-d-glucosaminide or 4-nitrophenyl N-acetyl-ß-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the ß(1-4) position, the galacto-acceptor afforded a ß(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on ß-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.


Assuntos
Glicosídeos/metabolismo , Oligossacarídeos/metabolismo , Talaromyces/enzimologia , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Cinética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
14.
Int J Mol Sci ; 20(23)2019 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-31771257

RESUMO

Flavonols, the second most abundant flavonoids in green tea, exist mainly in the form of glycosides. Flavonols are known to have a variety of beneficial health effects; however, limited information is available on their fate in the digestive system. We investigated the digestive stability of flavonol aglycones and glycosides from green tea under simulated digestion and anaerobic human fecal fermentation. Green tea fractions rich in flavonol glycosides and aglycones, termed flavonol-glycoside-rich fraction (FLG) and flavonol-aglycone-rich fraction (FLA) hereafter, were obtained after treatment with cellulase and tannase, respectively. Kaempferol and its glycosides were found to be more stable in simulated gastric and intestinal fluids than the derivatives of quercetin and myricetin. Anaerobic human fecal fermentation with FLG and FLA increased the populations of Lactobacilli spp. and Bifidobacteria spp. and generated various organic acids, such as acetate, butyrate, propionate, and lactate, among which butyrate was produced in the highest amount. Our findings indicate that some stable polyphenols have higher bioaccessibilities in the gastrointestinal tract and that their health-modulating effects result from their interactions with microbes in the gut.


Assuntos
Fezes/microbiologia , Flavonóis/metabolismo , Chá/química , Técnicas de Cultura Celular por Lotes , Bifidobacterium/isolamento & purificação , Hidrolases de Éster Carboxílico/metabolismo , Celulase/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Flavonóis/química , Glicosídeos/química , Glicosídeos/metabolismo , Humanos , Quempferóis/química , Quempferóis/metabolismo , Lactobacillus/isolamento & purificação , Quercetina/química , Quercetina/metabolismo , Chá/metabolismo
15.
Appl Microbiol Biotechnol ; 103(23-24): 9423-9432, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713673

RESUMO

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeos/metabolismo , alfa-Glucosidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Catálise , Expressão Gênica , Glucose/metabolismo , Glicosídeos/química , Glicosilação , Hidrólise , Cinética , Maltose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Água/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/isolamento & purificação
16.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683666

RESUMO

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeos/metabolismo , Solventes/química , Thermotoga maritima/enzimologia , alfa-Amilases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Colina/química , Dicroísmo Circular , Estabilidade Enzimática , Temperatura Alta , Ligação de Hidrogênio , Hidrólise , Metanol/química , Conformação Proteica , Ureia/química , alfa-Amilases/química
17.
Nutrients ; 11(11)2019 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-31684148

RESUMO

Diets rich in (poly)phenols are associated with a reduced reduction in the incidence of cardiovascular disorders. While the absorption and metabolism of (poly)phenols has been described, it is not clear how their metabolic fate is affected under pathological conditions. This study evaluated the metabolic fate of berry (poly)phenols in an in vivo model of hypertension as well as the associated microbiota response. Dahl salt-sensitive rats were fed either a low-salt diet (0.26% NaCl) or a high-salt diet (8% NaCl), with or without a berry mixture (blueberries, blackberries, raspberries, Portuguese crowberry and strawberry tree fruit) for 9 weeks. The salt-enriched diet promoted an increase in the urinary excretion of berry (poly)phenol metabolites, while the abundance of these metabolites decreased in faeces, as revealed by UPLC-MS/MS. Moreover, salt and berries modulated gut microbiota composition as demonstrated by 16S rRNA analysis. Some changes in the microbiota composition were associated with the high-salt diet and revealed an expansion of the families Proteobacteria and Erysipelotrichaceae. However, this effect was mitigated by the dietary supplementation with berries. Alterations in the metabolic fate of (poly)phenols occur in parallel with the modulation of gut microbiota in hypertensive rats. Thus, beneficial effects of (poly)phenols could be related with these interlinked modifications, between metabolites and microbiota environments.


Assuntos
Frutas , Microbioma Gastrointestinal/fisiologia , Fenóis/metabolismo , Animais , Dieta , Disbiose/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Glicosídeos/metabolismo , Masculino , Fenóis/análise , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/metabolismo , Ratos , Ratos Endogâmicos Dahl , Sódio na Dieta
18.
J Agric Food Chem ; 67(46): 12824-12832, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31650839

RESUMO

Transglycosylation of amylosucrase from Deinococcus geothermalis (DGAS) was performed using daidzin (daidzein-7-O-glucoside). Unlike cyclodextrin glucanotransferase, DGAS led to the production of new daidzin glucosides with high conversion yields (89%). Structures of these daidzin glucosides (i.e., DA2 and DA3) were daidzein-7-O-α-d-glucopyranosyl-(4 → 1)-O-ß-d-glucopyranoside (daidzin-4″-O-α-d-glucopyranoside) and daidzein-4'-O-α-d-glucopyranosyl-7-O-α-d-glucopyranosyl-(1 → 4)-O-ß-d-glucopyranoside (daidzin-4',4″-O-α-d-diglucopyranoside), respectively. DA2 and DA3 showed increased solubility of 15.4 mM (127-fold) and 203.3 mM (1686-fold) compared with daidzin, respectively. Kinetic studies revealed Vmax of 1.0 µM/min and K'm of 175 µM for DA3 production based on nonlinear regression. DGAS exhibited substrate inhibition behavior at high sucrose concentrations (700-1500 mM). Taken together, these findings indicate that DGAS can attach a glucose unit to a free C4'-OH via an α-linkage and then produce highly water-soluble isoflavone glycosides with a simple donor, moderate reaction conditions, less waste production, and high yield compared with that observed using the existing processes and enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/enzimologia , Glucosiltransferases/química , Glicosídeos/química , Isoflavonas/química , Proteínas de Bactérias/química , Biocatálise , Deinococcus/química , Glucosiltransferases/metabolismo , Glicosídeos/metabolismo , Isoflavonas/metabolismo , Cinética
19.
BMC Plant Biol ; 19(1): 436, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638900

RESUMO

BACKGROUND: Stevia rebaudiana (Asteraceae), native from Paraguay, accumulates steviol glycosides (SGs) into its leaves. These compounds exhibit acaloric intense sweet taste which answers to consumer demands for reducing daily sugar intake. Despite the developpement of S. rebaudiana cultivation all over the world, the development of new cultivars is very recent, in particular due to a colossal lack of (1) germplasm collection and breeding, (2) studies on genetic diversity and its structuring, (3) genomic tools. RESULTS: In this study, we developped 18 EST-SSR from 150,258 EST from The Compositae Genome Project of UC Davis ( http://compgenomics.ucdavis.edu/data/ ). We genotyped 145 S. rebaudiana individuals, issued from thirty-one cultivars and thirty-one landraces of various origins worldwide. Markers polymorphic information content (PIC) ranged between 0.60 and 0.84. An average of 12 alleles per locus and a high observed heterozygoty of 0.69 could be observed. The landraces revealed twice as many private alleles as cultivars. The genotypes could be clustered into 3 genetic populations. The landraces were grouped in the same cluster in which the oldest cultivars "Eirete" and "MoritaIII" type are also found. The other two clusters only include cultivated genotypes. One of them revealed an original genetic variability. SG phenotypes could not discriminate the three genetic clusters but phenotyping showed a wide range of composition in terms of bitter to sweet SGs. CONCLUSION: This is the first study of genetic diversity in Stevia rebaudiana involving 145 genotypes, including known cultivars as well as landrace populations of different origin. This study pointed out the structuration of S. rebaudiana germplasm and the resource of the landrace populations for genetic improvement, even on the trait of SG's composition.


Assuntos
Diterpenos de Caurano/metabolismo , Variação Genética , Glucosídeos/metabolismo , Glicosídeos/metabolismo , Stevia/genética , Alelos , Genética Populacional , Genótipo , Melhoramento Vegetal , Folhas de Planta/química , Folhas de Planta/genética , Stevia/química , Paladar
20.
G3 (Bethesda) ; 9(12): 4029-4043, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31604825

RESUMO

In eukaryotes, Polycomb group (PcG) and trithorax group (trxG) factors oppositely regulate gene transcription during development through histone modifications, with PcG factors repressing and trxG factors activating the expression of their target genes. Although plant trxG factors regulate many developmental and physiological processes, their downstream targets are poorly characterized. Here we use transcriptomics to identify genome-wide targets of the Arabidopsis thaliana trxG factor ULTRAPETALA1 (ULT1) during vegetative and reproductive development and compare them with those of the PcG factor CURLY LEAF (CLF). We find that genes involved in development and transcription regulation are over-represented among ULT1 target genes. In addition, stress response genes and defense response genes such as those in glucosinolate metabolic pathways are enriched, revealing a previously unknown role for ULT1 in controlling biotic and abiotic response pathways. Finally, we show that many ULT1 target genes can be oppositely regulated by CLF, suggesting that ULT1 and CLF may have antagonistic effects on plant growth and development in response to various endogenous and environmental cues.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Desenvolvimento Vegetal/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Ontologia Genética , Glucosinolatos/metabolismo , Glicosídeos/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/genética
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