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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.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1546-1555, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924353

RESUMO

Sucrose phosphorylase (SPase) gene from Leuconostoc mesenteroides ATCC 12291 was synthesised after codon optimization, and inserted into pET-28a plasmid to generate pET-28a-spase. The recombinant strain Escherichia coli BL21 (DE3)/pET-28a-spase was induced for Spase expression. The recombinant protein Spase was purified and characterized. The specific enzyme activity of SPase was 213.98 U/mg, the purification ratio was 1.47-fold, and the enzyme activity recovery rate was 87.80%. The optimal temperature and the optimal pH of the SPase were identified to be 45 °C and 6.5 respectively, and Km, Vmax and kcat of the SPase for sucrose was 128.8 mmol/L, 2.167 µmol/(mL·min), and 39 237.86 min-1. The recombinant SPase was used for α-arbutin production from hydroquinone and the reaction process was evaluated. The optimal conditions for synthesis of α-arbutin by SPase were 40 g/L hydroquinone, 5:1 molar ratio of sucrose and hydroquinone, and 250 U/mL recombinant SPase at pH 7.0 and 30 °C for 24 h in the dark, and then 500 U/mL glucoamylase was added at 40°C for 2.5 h. Under the optimized process, the yield of α-arbutin reached 98 g/L, and the hydroquinone conversion rate was close to 99%. In summary, the recombinant SPase was cloned and characterized, and its application for α-arbutin production was feasible.


Assuntos
Arbutina , Glucosiltransferases , Microbiologia Industrial , Leuconostoc , Arbutina/biossíntese , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Leuconostoc/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
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
4.
PLoS Genet ; 16(5): e1008807, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32407354

RESUMO

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Histonas/metabolismo , Metilação , Plantas Geneticamente Modificadas/fisiologia , Pólen/genética , Regiões Promotoras Genéticas
5.
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
6.
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
7.
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
8.
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
9.
Proc Natl Acad Sci U S A ; 117(12): 6910-6917, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152121

RESUMO

Auxin is a class of plant hormone that plays a crucial role in the life cycle of plants, particularly in the growth response of plants to ever-changing environments. Since the auxin responses are concentration-dependent and higher auxin concentrations might often be inhibitory, the optimal endogenous auxin level must be closely controlled. However, the underlying mechanism governing auxin homeostasis remains largely unknown. In this study, a UDP-glycosyltransferase (UGT76F1) was identified from Arabidopsis thaliana, which participates in the regulation of auxin homeostasis by glucosylation of indole-3-pyruvic acid (IPyA), a major precursor of the auxin indole-3-acetic acid (IAA) biosynthesis, in the formation of IPyA glucose conjugates (IPyA-Glc). In addition, UGT76F1 was found to mediate hypocotyl growth by modulating active auxin levels in a light- and temperature-dependent manner. Moreover, the transcription of UGT76F1 was demonstrated to be directly and negatively regulated by PIF4, which is a key integrator of both light and temperature signaling pathways. This study sheds a light on the trade-off between IAA biosynthesis and IPyA-Glc formation in controlling auxin levels and reveals a regulatory mechanism for plant growth adaptation to environmental changes through glucosylation of IPyA.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Hipocótilo/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Indóis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Hipocótilo/efeitos dos fármacos , Hipocótilo/metabolismo , Hipocótilo/efeitos da radiação , Indóis/química , Luz , Reguladores de Crescimento de Planta/farmacologia , Plântula , Temperatura
10.
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
11.
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
12.
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
13.
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
14.
Biochemistry ; 59(5): 682-693, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31899625

RESUMO

(1,3)-ß-d-Glucan synthase (GS) catalyzes formation of the linear (1,3)-ß-d-glucan in the fungal cell wall and is a target of clinically approved antifungal antibiotics. The catalytic subunit of GS, FKS protein, does not exhibit significant sequence homology to other glycosyltransferases, and thus, significant ambiguity about its catalytic mechanism remains. One of the major technical barriers in studying GS is the absence of activity assay methods that allow characterization of the lengths and amounts of (1,3)-ß-d-glucan due to its poor solubility in water and organic solvents. Here, we report a successful development of a novel GS activity assay based on size-exclusion chromatography coupled with pulsed amperometric detection and radiation counting (SEC-PAD-RC), which allows for the simultaneous characterization of the amount and length of the polymer product. The assay revealed that the purified yeast GS produces glucan with a length of 6550 ± 760 mer, consistent with the reported degree of polymerization of (1,3)-ß-d-glucan isolated from intact cells. Pre-steady state kinetic analysis revealed a highly efficient but rate-determining chain elongation rate of 51.5 ± 9.8 s-1, which represents the first observation of chain elongation by a nucleotide-sugar-dependent polysaccharide synthase. Coupling the SEC-PAD-RC method with substrate analogue mechanistic probes provided the first unambiguous evidence that GS catalyzes non-reducing end polymerization. On the basis of these observations, we propose a detailed model for the catalytic mechanism of GS. The approaches described here can be used to determine the mechanism of catalysis of other polysaccharide synthases.


Assuntos
Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Saccharomyces cerevisiae/metabolismo , beta-Glucanas/metabolismo , Biocatálise , Cromatografia em Gel , Glucosiltransferases/química , Cinética , Polimerização , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Especificidade da Espécie , beta-Glucanas/química , beta-Glucanas/isolamento & purificação
15.
J Biotechnol ; 309: 107-112, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31926981

RESUMO

Ginsenoside Rh2, a rare protopanaxadiol (PPD)-type triterpene saponin isolated from Panax ginseng, exhibits notable anticancer and immune-system-enhancing activities. Glycosylation catalyzed by uridine diphosphate-dependent glucosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rh2. In this study, UGT73C5 isolated from Arabidopsis thaliana was demonstrated to selectively transfer a glucosyl moiety to the C3 hydroxyl group of PPD to synthesize ginsenoside Rh2. UGT73C5 was coupled with sucrose synthase (SuSy) from A. thaliana to regenerate costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The UGT73C5/SuSy ratio, temperature, pH, cofactor UDP, and PPD concentrations for UGT73C5-SuSy coupled reactions were optimized. Through the stepwise addition of PPD, the maximal ginsenoside Rh2 production was 3.2 mg mL-1, which was the highest yield reported to date. These promising results provided an efficient and cost-effective approach to semisynthesize the highly valuable ginsenoside Rh2.


Assuntos
Arabidopsis/enzimologia , Medicamentos de Ervas Chinesas/metabolismo , Glucosiltransferases/metabolismo , Sapogeninas/metabolismo , Arabidopsis/genética , Técnicas de Cultura Celular por Lotes , Biocatálise , Vias Biossintéticas , Ginsenosídeos/biossíntese , Panax/metabolismo , Sapogeninas/química , Saponinas , Triterpenos , Difosfato de Uridina
16.
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
17.
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
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.
Biochem Biophys Res Commun ; 523(3): 651-657, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31948759

RESUMO

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.


Assuntos
Dextranase/metabolismo , Glucosiltransferases/metabolismo , Leuconostoc mesenteroides/enzimologia , Maltose/metabolismo , Oligossacarídeos/metabolismo , Dextranos/química , Dextranos/metabolismo , Hidrólise , Leuconostoc mesenteroides/química , Leuconostoc mesenteroides/metabolismo , Oligossacarídeos/química , Especificidade por Substrato
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