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1.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960931

RESUMO

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridium difficile , Enterocolite Pseudomembranosa , Glucosiltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/enzimologia , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Feminino , Deleção de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Masculino , Camundongos
2.
Proc Natl Acad Sci U S A ; 117(33): 20316-20324, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737163

RESUMO

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucanos/biossíntese , Glucosiltransferases/metabolismo , Células Vegetais/metabolismo , Xilanos/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Mutação , Filogenia
3.
Nat Commun ; 11(1): 2629, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457405

RESUMO

Grain size is an important component trait of grain yield, which is frequently threatened by abiotic stress. However, little is known about how grain yield and abiotic stress tolerance are regulated. Here, we characterize GSA1, a quantitative trait locus (QTL) regulating grain size and abiotic stress tolerance associated with metabolic flux redirection. GSA1 encodes a UDP-glucosyltransferase, which exhibits glucosyltransferase activity toward flavonoids and monolignols. GSA1 regulates grain size by modulating cell proliferation and expansion, which are regulated by flavonoid-mediated auxin levels and related gene expression. GSA1 is required for the redirection of metabolic flux from lignin biosynthesis to flavonoid biosynthesis under abiotic stress and the accumulation of flavonoid glycosides, which protect rice against abiotic stress. GSA1 overexpression results in larger grains and enhanced abiotic stress tolerance. Our findings provide insights into the regulation of grain size and abiotic stress tolerance associated with metabolic flux redirection and a potential means to improve crops.


Assuntos
Adaptação Fisiológica , Grão Comestível/metabolismo , Glucosiltransferases/metabolismo , Oryza/metabolismo , Crescimento Celular , Proliferação de Células , Grão Comestível/citologia , Grão Comestível/genética , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Redes e Vias Metabólicas , Oryza/citologia , Oryza/genética , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas
4.
PLoS One ; 15(5): e0233779, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470059

RESUMO

Trehalose metabolism in yeast has been linked to a variety of phenotypes, including heat resistance, desiccation tolerance, carbon-source utilization, and sporulation. The relationships among the several phenotypes of mutants unable to synthesize trehalose are not understood, even though the pathway is highly conserved. One of these phenotypes is that tps1Δ strains cannot reportedly grow on media containing glucose or fructose, even when another carbon source they can use (e.g. galactose) is present. Here we corroborate the recent observation that a small fraction of yeast tps1Δ cells do grow on glucose, unlike the majority of the population. This is not due to a genetic alteration, but instead resembles the persister phenotype documented in many microorganisms and cancer cells undergoing lethal stress. We extend these observations to show that this phenomenon is glucose-specific, as it does not occur on another highly fermented carbon source, fructose. We further demonstrate that this phenomenon appears to be related to mitochondrial complex III function, but unrelated to inorganic phosphate levels in the cell, as had previously been suggested. Finally, we found that this phenomenon is specific to S288C-derived strains, and is the consequence of a variant in the MKT1 gene.


Assuntos
Glucose/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fermentação , Frutose/metabolismo , Glucosiltransferases/genética , Mutação com Perda de Função , Trealose/biossíntese
5.
Nat Chem Biol ; 16(7): 740-748, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424305

RESUMO

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.


Assuntos
Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glicosiltransferases/genética , Proteínas de Plantas/genética , Saponinas/biossíntese , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Retículo Endoplasmático/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Spinacia oleracea/genética
6.
Food Chem ; 318: 126478, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126466

RESUMO

With people's increasing needs for health concern, rutin and emodin in tartary buckwheat have attracted much attention for their antioxidant, anti-diabetic and reducing weight function. However, the biosynthesis of rutin and emodin in tartary buckwheat is still unclear; especially their later glycosylation contributing to make them more stable and soluble is uncovered. Based on tartary buckwheat' genome, the gene structures of 106 UGTs were analyzed; 21 candidate FtUGTs were selected to enzymatic test by comparing their transcript patterns. Among them, FtUGT73BE5 and other 4 FtUGTs were identified to glucosylate flavonol or emodin in vitro; especially rFtUGT73BE5 could catalyze the glucosylation of all tested flavonoids and emodin. Furthermore, the identical in vivo functions of FtUGT73BE5 were demonstrated in tartary buckwheat hairy roots. The transcript profile of FtUGT73BE5 was consistent with the accumulation trend of rutin in plant; this gene may relate to anti-adversity for its transcripts were up-regulated by MeJA, and repressed by ABA.


Assuntos
Emodina/metabolismo , Fagopyrum/genética , Glucosiltransferases/genética , Rutina/biossíntese , Acetatos/farmacologia , Ciclopentanos/farmacologia , Fagopyrum/efeitos dos fármacos , Fagopyrum/metabolismo , Flavonoides/metabolismo , Flavonóis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Estudo de Associação Genômica Ampla , Glucosídeos/metabolismo , Glucosiltransferases/metabolismo , Oxilipinas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Rutina/genética , Rutina/metabolismo
7.
Enzyme Microb Technol ; 135: 109505, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146930

RESUMO

Amylosucrase (ASase) has great industrial potential owing to its multifunctional activities, including transglucosylation, polymerization, and isomerization. In the present study, the properties of Deinococcus geothermalis ASase (DGAS) expressed in Corynebacterium glutamicum (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in Escherichia coli (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity in cDGAS was 10 % higher than that in eDGAS. Analysis of protein secondary structure via circular dichroism spectroscopy revealed that cDGAS had a lower strand/helix ratio than eDGAS. The present results indicate that cDGAS is of greater industrial significance than eDGAS.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Deinococcus/enzimologia , Glucosídeos/biossíntese , Glucosiltransferases/metabolismo , Luteolina/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Deinococcus/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Engenharia Metabólica
8.
PLoS Genet ; 16(2): e1008390, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084124

RESUMO

Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.


Assuntos
DNA de Cinetoplasto/metabolismo , Proteínas de Protozoários/metabolismo , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Trypanosoma brucei brucei/fisiologia , Animais , DNA de Cinetoplasto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Protozoários , Glucosídeos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Leishmania/fisiologia , Mutação , Proteínas de Protozoários/genética , Interferência de RNA , RNA Polimerase II/genética , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo
9.
PLoS One ; 15(2): e0227840, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023283

RESUMO

Flax seed has become consumers' choice for not only polyunsaturated alpha-linolenic fatty acid but also nutraceuticals such as lignans and soluble fiber. There is, however, a major drawback of flax as a source of functional food since the seeds contain significant level of cyanogenic glucosides. The final step of cyanogenic glucoside biosynthesis is mediated by UDP-glucose dependent glucosyltransferase. To date, no flax cyanogenic glucosyl transferase genes have been reported with verified biochemical functionality. Here we present a study on the identification and enzymatic characterization of a first flax cyanogenic glucosyltransferase, LuCGT1. We show that LuCGT1 was highly active towards both aliphatic and aromatic substrates. The LuCGT1 gene is expressed in leaf tissues as well as in developing seeds, and its expression level was drastically reduced in flax mutant lines low in cyanogenic glucosides. Identification of LuCGT1 provides a molecular handle for developing gene specific markers for targeted breeding of low cyanogenic glucosides in flax.


Assuntos
Linho/enzimologia , Linho/genética , Glucosiltransferases/genética , Nitrilos/metabolismo , Regulação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Cinética , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismo
10.
PLoS One ; 15(2): e0228735, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032363

RESUMO

Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.


Assuntos
Glucosiltransferases/genética , Vírus da Influenza A/fisiologia , Células A549 , Sistemas CRISPR-Cas/genética , Edição de Genes , Glucosiltransferases/deficiência , Células HEK293 , Humanos , Macrolídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos
11.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32005733

RESUMO

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, ß-, and γ-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.


Assuntos
Proteínas de Bactérias/genética , Benzopiranos/metabolismo , Glucosiltransferases/genética , Paenibacillus/genética , Polissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Paenibacillus/enzimologia , Paenibacillus/metabolismo , Engenharia de Proteínas , Especificidade por Substrato
12.
Plant Mol Biol ; 102(4-5): 389-401, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31894456

RESUMO

KEY MESSAGE: This study revealed that the Arabidopsis UGT75B1 plays an important role in modulating ABA activity by glycosylation when confronting stress environments. The cellular ABA content and activity can be tightly controlled in several ways, one of which is glycosylation by family 1 UDP-glycosyltransferases (UGTs). Previous analysis has shown UGT75B1 activity towards ABA in vitro. However, the biological role of UGT75B1 remains to be elucidated. Here, we characterized the function of UGT75B1 in abiotic stress responses via ABA glycosylation. GUS assay and qRT-PCR indicated that UGT75B1 is significantly upregulated by adverse conditions, such as osmotic stress, salinity and ABA. Overexpression of UGT75B1 in Arabidopsis leads to higher seed germination rates and seedling greening rates upon exposure to salt and osmotic stresses. In contrast, the big UGT75B1 overexpression plants are more sensitive under salt and osmotic stresses. Additionally, the UGT75B1 overexpression plants showed larger stomatal aperture and more water loss under drought condition, which can be explained by lower ABA levels examined in UGT75B1 OE plants in response to water deficit conditions. Consistently, UGT75B1 ectopic expression leads to downregulation of many ABA-responsive genes under stress conditions, including ABI3, ABI5 newly germinated seedlings and RD29A, KIN1, AIL1 in big plants. In summary, our results revealed that the Arabidopsis UGT75B1 plays an important role in coping with abiotic stresses via glycosylation of ABA.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/fisiologia , Glicosiltransferases/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálise , Secas , Genes de Plantas , Germinação , Glucosiltransferases/genética , Glicosilação , Glicosiltransferases/genética , Pressão Osmótica , Plantas Geneticamente Modificadas/genética , Salinidade , Plântula/genética , Plântula/fisiologia , Cloreto de Sódio , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
13.
Molecules ; 25(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906359

RESUMO

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans.


Assuntos
Deinococcus/enzimologia , Glucosiltransferases/metabolismo , Isoflavonas/química , Extratos Vegetais/química , Soja/química , beta-Glucanas/química , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Vetores Genéticos , Genisteína/química , Glucosiltransferases/genética , Glicosilação , Isoflavonas/biossíntese , Isoflavonas/isolamento & purificação , Isoflavonas/farmacocinética , Espectrometria de Massas , Fitoestrógenos/química , Extratos Vegetais/isolamento & purificação , beta-Glucanas/farmacocinética
14.
Carbohydr Polym ; 231: 115697, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888841

RESUMO

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.


Assuntos
Glucanos/química , Glucosiltransferases/química , Lactobacillus reuteri/enzimologia , Estrutura Molecular , Substituição de Aminoácidos/genética , Dextranos/química , Glucanos/ultraestrutura , Glucosiltransferases/genética , Espectroscopia de Ressonância Magnética , Metilação , Mutação/genética , Engenharia de Proteínas
15.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31968056

RESUMO

Maladaptive responses to stress might play a role in the sensitivity of neurons to stress. To identify novel cellular responses to stress, we performed transcriptional analysis in acutely stressed mouse neurons, followed by functional characterization in Caenorhabditis elegans. In both contexts, we found that the gene GDPGP1/mcp-1 is down-regulated by a variety of stresses. Functionally, the enzyme GDPGP1/mcp-1 protects against stress. Knockdown of GDPGP1 in mouse neurons leads to widespread neuronal cell death. Loss of mcp-1, the single homologue of GDPGP1 in C. elegans, leads to increased degeneration of GABA neurons as well as reduced survival of animals following environmental stress. Overexpression of mcp-1 in neurons enhances survival under hypoxia and protects against neurodegeneration in a tauopathy model. GDPGP1/mcp-1 regulates neuronal glycogen levels, indicating a key role for this metabolite in neuronal stress resistance. Together, our data indicate that down-regulation of GDPGP1/mcp-1 and consequent loss of neuronal glycogen is a maladaptive response that limits neuronal stress resistance and reduces survival.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Glucosiltransferases/genética , Degeneração Neural/genética , Neurônios/metabolismo , Animais , Apoptose/genética , Caenorhabditis elegans/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Glicogênio/genética , Glicogênio/metabolismo , Humanos , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/patologia
16.
Planta ; 251(2): 50, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31950395

RESUMO

MAIN CONCLUSION: Upregulation of the terpenoid pathway and increased ABA content in flax upon Fusarium infection leads to activation of the early plant's response (PR genes, cell wall remodeling, and redox status). Plants have developed a number of defense strategies against the adverse effects of fungi such as Fusarium oxysporum. One such defense is the production of antioxidant secondary metabolites, which fall into two main groups: the phenylpropanoids and the terpenoids. While functions and biosynthesis of phenylpropanoids have been extensively studied, very little is known about the genes controlling the terpenoid synthesis pathway in flax. They can serve as antioxidants, but are also substrates for a plethora of different compounds, including those of regulatory functions, like ABA. ABA's function during pathogen attack remains obscure and often depends on the specific plant-pathogen interactions. In our study we showed that in flax the non-mevalonate pathway is strongly activated in the early hours of pathogen infection and that there is a redirection of metabolites towards ABA synthesis. The elevated synthesis of ABA correlates with flax resistance to F. oxysporum, thus we suggest ABA to be a positive regulator of the plant's early response to the infection.


Assuntos
Ácido Abscísico/metabolismo , Vias Biossintéticas , Linho/metabolismo , Linho/microbiologia , Fusarium/fisiologia , Doenças das Plantas/microbiologia , Plastídeos/metabolismo , Terpenos/metabolismo , Sequência de Bases , DNA Complementar/genética , DNA Fúngico/análise , Linho/genética , Fusarium/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosiltransferases/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Food Chem ; 314: 126212, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31972410

RESUMO

Turanose, a potential novel sweetener in food industry, can be synthesized by Neisseria polysaccharea amylosucrase (NpAS). However, the malt-oligosaccharide byproduct affects the yield. In this study, the NpAS mutant G396S, which was expected to interfer with the extension of glucan by increasing steric hindrance, was obtained. The NpAS and G396S were heterologously expressed in Bacillus subtilis and enzyme properties were analyzed. Results showed that the polymerization activity of G396S was decreased. In addition, the mutant was used in the preparation of turanose. When using 2 M sucrose as substrate, the turanose yield reached 410.4 g·L-1, an increase of 61 g·L-1 compared with that of NpAS. When fructose was added, the optimal fructose concentration for G396S decreased from 0.75 M to 0.5 M. The turanose production reached 523 g·L-1 with the conversion rate of 76.5%. This study contributes the use of turanose in food industry.


Assuntos
Dissacarídeos/metabolismo , Glucosiltransferases/metabolismo , Neisseria/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutose/metabolismo , Glucanos , Glucosiltransferases/genética , Neisseria/genética , Proteínas Recombinantes , Sacarose/metabolismo , Edulcorantes
18.
Carbohydr Res ; 488: 107902, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31911362

RESUMO

Trehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM ß-glucose 1-phosphate (ß-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of ß-phosphoglucomutase to this reaction produced Glc6P from ß-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained.


Assuntos
Glucosiltransferases/metabolismo , Lactococcus lactis/enzimologia , Fosfatos Açúcares/biossíntese , Trealose/análogos & derivados , Leveduras/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Clonagem Molecular , Glucose-6-Fosfatase/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferases/genética , Lactococcus lactis/genética , Fosfatos/metabolismo , Trealose/biossíntese , Leveduras/genética
19.
Protein Expr Purif ; 169: 105571, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31978533

RESUMO

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.


Assuntos
Glucosiltransferases/biossíntese , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Engenharia Genética/métodos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Chaperonas Moleculares/metabolismo , Plasmídeos , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
20.
FASEB J ; 34(1): 1270-1287, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914593

RESUMO

Dysregulation of the adipo-osteogenic differentiation balance of mesenchymal stem cells (MSCs), which are common progenitor cells of adipocytes and osteoblasts, has been associated with many pathophysiologic diseases, such as obesity, osteopenia, and osteoporosis. Growing evidence suggests that lipid metabolism is crucial for maintaining stem cell homeostasis and cell differentiation; however, the detailed underlying mechanisms are largely unknown. Here, we demonstrate that glucosylceramide (GlcCer) and its synthase, glucosylceramide synthase (GCS), are key determinants of MSC differentiation into adipocytes or osteoblasts. GCS expression was increased during adipogenesis and decreased during osteogenesis. Targeting GCS using RNA interference or a chemical inhibitor enhanced osteogenesis and inhibited adipogenesis by controlling the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ). Treatment with GlcCer sufficiently rescued adipogenesis and inhibited osteogenesis in GCS knockdown MSCs. Mechanistically, GlcCer interacted directly with PPARγ through A/B domain and synergistically enhanced rosiglitazone-induced PPARγ activation without changing PPARγ expression, thereby treatment with exogenous GlcCer increased adipogenesis and inhibited osteogenesis. Animal studies demonstrated that inhibiting GCS reduced adipocyte formation in white adipose tissues under normal chow diet and high-fat diet feeding and accelerated bone repair in a calvarial defect model. Taken together, our findings identify a novel lipid metabolic regulator for the control of MSC differentiation and may have important therapeutic implications.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular , Glucosilceramidas/metabolismo , Glucosiltransferases/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , PPAR gama/metabolismo , Animais , Glucosilceramidas/genética , Glucosiltransferases/genética , Humanos , Camundongos , PPAR gama/genética
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