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1.
J Agric Food Chem ; 67(35): 9868-9876, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389242

RESUMO

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.


Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Thermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Alinhamento de Sequência , Thermus/química , Thermus/genética
2.
J Agric Food Chem ; 67(35): 9738-9748, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31411877

RESUMO

The presence of chromium (Cr) in cultivated fields affects carbohydrate metabolism of rice (Oryza sativa L.) and weakens its productivity. Little is known about the molecular mechanism of sucrose metabolism underlying Cr stress response in rice plants. In the present study, the transcriptome map of sucrose metabolism in rice seedlings exposed to both trivalent and hexavalent chromium was investigated using Agilent 4 × 44K rice microarray analysis. Results indicated that Cr exposure (3 days) significantly (p < 0.05) improved sucrose accumulation, and altered the activities of sucrose synthetase, sucrose phosphate phosphatase, and amylosynthease in rice tissues. We identified 119 differentially regulated genes involved in 17 sucrose metabolizing enzymes and found that gene responses in roots were significantly (p < 0.05) stronger than in shoots under both Cr(III) and Cr(VI) treatment. The network maps of gene regulation responsible for sucrose metabolism in rice plants provide a theoretical basis for further cultivating Cr-resistant rice cultivars through molecular genetic improvement.


Assuntos
Cromo/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/metabolismo , Poluentes do Solo/farmacologia , Sacarose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oryza/efeitos dos fármacos , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
3.
Pestic Biochem Physiol ; 159: 98-106, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400791

RESUMO

The cotton aphid, Aphis gossypii Glover, is a destructive global crop pest. Control of A. gossypii has relied heavily on the application of chemical insecticides. The cotton aphid has developed resistance to numerous insecticides, including imidacloprid, which has been widely used to control cotton pests in China since the 1990s. Our objective was to investigate the potential role of UDP-glycosyltransferases (UGTs) in imidacloprid resistance based on transcriptomic and proteomic analyses of field-originated imidacloprid-resistant (IMI_R) and -susceptible (IMI_S) A. gossypii clones. The transcriptomic and proteomic analyses revealed that 12 out of 512 differentially expressed genes and three out of 510 differentially expressed proteins were predicted as UDP-glycosyltransferase (UGT). Based on quantitative real-time PCR analysis, nine UGT genes, UGT343A4, UGT344A15, UGT344A16, UGT344B4, UGT344C7, UGT344C9, UGT344N4, UGT 24541, and UGT7630, were up-regulated in the IMI_R clone compared to the IMI_S clone. Meanwhile, UGT344A16, UGT344B4, UGT344C7, and UGT344N4 were overexpressed at the protein level based on western blot analysis. Furthermore, knockdown of UGT344B4 or UGT344C7 using RNA interference (RNAi) significantly increased sensitivity to imidacloprid in the IMI_R clone. In conclusion, UGTs potentially contributed to imidacloprid resistance in A. gossypii originating from cotton-growing regions of China. These results provide insights into the way we study insecticide resistance in cotton aphids.


Assuntos
Afídeos/efeitos dos fármacos , Glucosiltransferases/metabolismo , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Animais , Afídeos/genética , Afídeos/metabolismo , Glucosiltransferases/genética , Resistência a Inseticidas/genética , Proteômica , Transcriptoma/genética
4.
Gene ; 718: 144073, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31446096

RESUMO

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.


Assuntos
Aspergillus oryzae/metabolismo , Ergosterol/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Triglicerídeos/biossíntese , Aspergillus oryzae/genética , Quitina Sintase/genética , Quitina Sintase/metabolismo , Ergosterol/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Microrganismos Geneticamente Modificados/genética , Triglicerídeos/genética
5.
Phytochemistry ; 166: 112062, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31299395

RESUMO

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.


Assuntos
Biocatálise , Diterpenos/química , Diterpenos/metabolismo , Glucosídeos/química , Glucosiltransferases/metabolismo , Tripterygium/química , Glucosiltransferases/química , Simulação de Acoplamento Molecular , Conformação Proteica
6.
J Agric Food Chem ; 67(33): 9314-9324, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352776

RESUMO

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Trealose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Fermentação , Maltose/metabolismo , Engenharia Metabólica
7.
BMC Plant Biol ; 19(1): 325, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324146

RESUMO

BACKGROUND: The heteroside floridoside is a primary photosynthetic product that is known to contribute to osmotic acclimation in almost all orders of Rhodophyta. However, the encoding genes and enzymes responsible for the synthesis of floridoside and its isomeric form, L- or D-isofloridoside, are poorly studied. RESULTS: Here, four putative trehalose-6-phosphate synthase (TPS) genes, designated as PhTPS1, PhTPS2, PhTPS3, and PhTPS4, were cloned and characterized from the red alga Pyropia haitanensis (Bangiophyceae). The deduced amino acid sequence is similar to the annotated TPS proteins of other organisms, especially the UDP-galactose substrate binding sites of PhTPS1, 2, which are highly conserved. Of these, PhTPS1, 4 are involved in the biosynthesis of floridoside and isofloridoside, with isofloridoside being the main product. PhTPS3 is an isofloridoside phosphate synthase, while PhTPS2 exhibits no activity. When challenged by desiccation, high temperature, and salt stress, PhTPS members were expressed to different degrees, but the responses to thermal stress and desiccation were stronger. CONCLUSIONS: Thus, in P. haitanensis, PhTPSs encode the enzymatical activity of floridoside and isofloridoside phosphate synthase and are crucial for the abiotic stress defense response.


Assuntos
Proteínas de Algas/metabolismo , Glucosiltransferases/metabolismo , Glicerol/análogos & derivados , Rodófitas/fisiologia , Trealose/biossíntese , Proteínas de Algas/genética , Proteínas de Algas/fisiologia , Glucosiltransferases/genética , Glicerol/metabolismo , Filogenia , Rodófitas/enzimologia , Rodófitas/genética , Rodófitas/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
8.
J Agric Food Chem ; 67(32): 8986-8993, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347835

RESUMO

Trehalose plays a crucial role in response to freezing stress in baker's yeast. MAL62, a gene involved in the adenosine diphosphoglucose-dependent trehalose synthesis pathway, can increase trehalose content. However, the difference between MAL62-related trehalose synthesis and traditional uridine diphosphoglucose-dependent trehalose synthesis is not well-understood. MAL62 overexpression showed less effect in enhancing intracellular trehalose compared to TPS1 overexpression. However, MAL62 overexpression elicited trehalose synthesis before fermentation with enhanced maltose metabolism and had a similar effect on cell viability after freezing. Furthermore, MAL62 and TPS1 overexpression in the NTH1 deletion background further strengthened freezing tolerance and improved leavening ability. Our results suggest that the enhancement in freezing tolerance by MAL62 overexpression may involve multiple pathways rather than simply enhancing trehalose synthesis. The results reveal valuable insights into the relationship between maltose metabolism and freezing tolerance and may help to develop better yeast strains for enhancing fermentation characteristics of frozen dough.


Assuntos
Glucosiltransferases/metabolismo , Maltose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , alfa-Glucosidases/metabolismo , Farinha/análise , Farinha/microbiologia , Congelamento , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Trealase/genética , Trealase/metabolismo , Trealose/metabolismo , alfa-Glucosidases/genética
9.
J Agric Food Chem ; 67(32): 8875-8883, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347830

RESUMO

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.


Assuntos
Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Grifola/enzimologia , Micélio/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Grifola/genética , Grifola/crescimento & desenvolvimento , Grifola/metabolismo , Micélio/enzimologia , Micélio/genética , Micélio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
10.
Nat Commun ; 10(1): 2928, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266959

RESUMO

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.


Assuntos
Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Trealose/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia
11.
J Agric Food Chem ; 67(29): 8177-8185, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31290662

RESUMO

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Glucosiltransferases/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Biologia Computacional , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Especificidade por Substrato
12.
Plant Physiol Biochem ; 141: 164-171, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31170640

RESUMO

For a comprehensive understanding of gene expression, enzyme activity and sugar concentrations in response to short-term water deficit in apple (Greensleeves), sugar-modulated gene expression and enzyme activities were analyzed. Water stress resulted in the accumulation of sorbitol, glucose, fructose, galactose and starch, accompanied by a significant reduction in photosynthesis and sucrose concentration. In response to short-term water deficits, the activities of aldose-6-phosphate reductase (A6PR; EC 1.1.1.200), sorbitol dehydrogenase (SDH; EC 1.1.1.14), neutral invertase (NINV; EC 3.2.1.26), sucrose synthase (SUSY; EC 2.4.1.13), and fructokinase (FK; EC 2.7.1.4) were higher, whereas cell wall invertase (CWINV; EC 3.2.1.26) and hexokinase (HK; EC 2.7.1.1) activities were lower. In addition, sucrose phosphate synthase (SPS; EC 2.4.1.14) activity increased during the initial stages of dehydration and then decreased as the drought strengthened. Transcript levels of MdA6PR, MdSDH1/2, MdNINV1/2, MdSUSY3, MdFK1/2/4, MdSOT1/2, MdSUC1-3, MdTMT2/3, MdvGT1, MdpGlcT1-4 were upregulated, whereas transcript levels of MdCWINV1/2, MdHK1/2/3/5, and MdTMT1 were downregulated after 6 days of water stress. These findings suggest that the sorbitol metabolism pathway is induced and high levels of hexose derived from photosynthetic products are transported into vacuoles for adjustment to the water deficit. Our results provide insights into the relationships between sugar levels and sugar-modulated gene and enzyme activity in response to the imposition of short-term water stress.


Assuntos
Metabolismo dos Carboidratos/genética , Secas , Regulação da Expressão Gênica de Plantas , Malus/fisiologia , Folhas de Planta/fisiologia , Parede Celular/metabolismo , Frutas/metabolismo , Glucosiltransferases/metabolismo , Malus/genética , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Sorbitol/metabolismo , Estresse Fisiológico , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo
13.
J Sci Food Agric ; 99(12): 5526-5532, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31102411

RESUMO

BACKGROUND: Trisodium phosphate (TSP), generally recognized as safe (GRAS), could control postharvest diseases and maintain fruit quality. However, changes of fruit quality and sucrose metabolism in harvested jujube after TSP treatment remain largely unknown. In the current study, jujube fruit (cv. sanxing) was used to study the effects of TSP on storage quality and sucrose metabolism during storage at 20 ± 2 °C with 40-50% relative humidity (RH). RESULTS: The results showed that 0.5 g L-1 TSP treatment reduced weight loss and reduced sugar content, suppressed the reduction of fruit firmness, maintained ascorbic acid (AsA) content and inhibited respiratory rate of jujube fruit. In addition, TSP treatment also reduced acid invertase (AI) and neutral invertase (NI) activities in sucrose metabolism in jujube fruit. Sucrose synthase-cleavage (SS-c), sucrose synthase-synthesis (SS-s) and sucrose phosphate synthase (SPS) activities were also suppressed by TSP treatment. CONCLUSION: Treatment with TSP could effectively reduce enzymes activities in sucrose metabolism and maintain storage quality of jujube fruit during storage. © 2019 Society of Chemical Industry.


Assuntos
Conservantes de Alimentos/farmacologia , Frutas/química , Fosfatos/farmacologia , Ziziphus/efeitos dos fármacos , Conservação de Alimentos , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , Glucosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Controle de Qualidade , Sacarose/análise , Ziziphus/química
14.
Curr Top Microbiol Immunol ; 421: 209-227, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123891

RESUMO

The ability of Helicobacter pylori to persist lifelong in the human gastric mucosa is a striking phenomenon. It is even more surprising since infection is typically associated with a vivid inflammatory response. Recent studies revealed the mechanism by which this pathogen inhibits the epithelial responses to IFN-γ and other central inflammatory cytokines in order to abolish an effective antimicrobial defense. The mechanism is based on the modification and depletion of cholesterol by the pathogen's cholesterol-α-glucosyltransferase. It abrogates the assembly of numerous cytokine receptors due to the reduction of lipid rafts. Particularly, the receptors for IFN-γ, IL-22, and IL-6 then fail to assemble properly and to activate JAK/STAT signaling. Consequently, cholesterol depletion prevents the release of antimicrobial peptides, including the highly effective ß-defensin-3. Intriguingly, the inhibition is spatially restricted to heavily infected cells, while the surrounding epithelium continues to respond normally to cytokine stimulation, thus providing a platform of the intense inflammation typically observed in H. pylori infections. It appears that pathogen and host establish a homeostatic balance between tightly colonized and rather inflamed sites. This homeostasis is influenced by the levels of available cholesterol, which potentially exacerbate H. pylori-induced inflammation. The observed blockage of epithelial effector mechanisms by H. pylori constitutes a convincing explanation for the previous failures of T-cell-based vaccination against H. pylori, since infected epithelial cells remain inert upon stimulation by effector cytokines. Moreover, the mechanism provides a rationale for the carcinogenic action of this pathogen in that persistent infection and chronic inflammation represent a pro-carcinogenic environment. Thus, cholesterol-α-glucosyltransferase has been revealed as a central pathogenesis determinant of H. pylori.


Assuntos
Colesterol/deficiência , Infecções por Helicobacter/sangue , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Colesterol/metabolismo , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Glucosiltransferases/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/enzimologia , Humanos
15.
Plant Mol Biol ; 100(3): 215-230, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053988

RESUMO

KEY MESSAGE: Two homologs PsnSuSy1 and PsnSuSy2 from poplar played largely similar but little distinct roles in modulating sink strength, accelerating vegetative growth and modifying secondary growth of plant. Co-overexpression of them together resulted in small but perceptible additive effects. Sucrose synthase (SuSy) acts as a crucial determinant of sink strength by controlling the conversion of sucrose into UDP-glucose, which is not only the sole precursor for cellulose biosynthesis but also an extracellular signaling molecule for plants growth. Therefore, modification of SuSy activity in plants is of utmost importance. We have isolated two SuSy genes from poplar, PsnSuSy1 and PsnSuSy2, which were preferentially expressed in secondary xylem/phloem. To investigate their functions, T2 tobacco transgenic lines of PsnSuSy1 and PsnSuSy2 were generated and then crossed to generate PsnSuSy1/PsnSuSy2 dual overexpression transgenic lines. SuSy activities in all lines were significantly increased though PsnSuSy1/PsnSuSy2 lines only exhibited slightly higher SuSy activities than either PsnSuSy1 or PsnSuSy2 lines. The significantly increased fructose and glucose, engendered by augmented SuSy activities, caused the alternations of many physiological, biochemical measures and phenotypic traits that include accelerated vegetative growth, thickened secondary cell wall, and increased stem breaking force, accompanied with altered expression levels of related pathway genes. The correlation relationships between SuSy activities and many of these traits were statistically significant. However, differences of almost all traits among three types of transgenic lines were insignificant. These findings clearly demonstrated that PsnSuSy1 and PsnSuSy2 had similar but little distinct functions and insubstantial additive effects on modulating sink strength and affecting allocation of carbon elements among secondary cell wall components.


Assuntos
Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucosiltransferases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Parede Celular/ultraestrutura , Celulose/biossíntese , Clorofila/análise , Clonagem Molecular , Perfilação da Expressão Gênica , Glucosiltransferases/metabolismo , Lignina/metabolismo , Floema/metabolismo , Fotossíntese , Polissacarídeos/metabolismo , Populus/genética , Análise de Sequência , Sacarose/metabolismo , Tabaco/citologia , Tabaco/crescimento & desenvolvimento , Xilema/metabolismo
16.
Invest Ophthalmol Vis Sci ; 60(5): 1789-1798, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31022733

RESUMO

Purpose: To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Methods: Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry. Results: Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. Conclusions: We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.


Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Metabolismo dos Lipídeos , Lipidoses/metabolismo , Nervo Óptico/metabolismo , Psicosina/análogos & derivados , Ceramidase Ácida/metabolismo , Idoso , Western Blotting , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Glucosilceramidase/metabolismo , Glucosiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Ceramidase Neutra/metabolismo , Psicosina/metabolismo , beta-Glucosidase/metabolismo
17.
Plant Cell Rep ; 38(8): 869-882, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30963238

RESUMO

KEY MESSAGE: The TPS5 negatively regulates ABA signaling by mediating ROS level and NR activity during seed germination and stomatal closure in Arabidopsis thaliana. Trehalose metabolism is important in plant growth and development and in abiotic stress response. Eleven TPS genes were identified in Arabidopsis, divided into Class I (TPS1-TPS4) and Class II (TPS5-TPS11). Although Class I has been shown to have TPS activity, the function of most members of Class II remains enigmatic. Here, we characterized the biological function of the trehalose-6-phosphate synthase TPS5 in ABA signaling in Arabidopsis. TPS5 expression was induced by ABA and abiotic stress, and expression in epidermal and guard cells was dramatically increased after ABA treatment. Loss-of-function analysis revealed that tps5 mutants (tps5-1 and tps5-cas9) are more sensitive to ABA during seed germination and ABA-mediated stomatal closure. Furthermore, the H2O2 level increased in the tps5-1 and tps5-cas9 mutants, which was consistent with the changes in the expression of RbohD and RbohF, key genes responsible for H2O2 production. Further, TPS5 knockout reduced the amounts of trehalose and other soluble carbohydrates as well as nitrate reductase (NR) activity. In vitro, trehalose and other soluble carbohydrates promoted NR activity, which was blocked by the tricarboxylic acid cycle inhibitor iodoacetic acid. Thus, this study identified that TPS5 functions as a negative regulator of ABA signaling and is involved in altering the trehalose content and NR activity.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Germinação/fisiologia , Glucosiltransferases/fisiologia , Peróxido de Hidrogênio/metabolismo , Estômatos de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
18.
Plant Cell Physiol ; 60(7): 1487-1503, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004494

RESUMO

Plant cells sheath themselves in a complex lattice of polysaccharides, proteins and enzymes forming an integral matrix known as the cell wall. Cellulose microfibrils, the primary component of cell walls, are synthesized at the plasma membrane by CELLULOSE SYNTHASE A (CESA) proteins throughout cellular growth and are responsible for turgor-driven anisotropic expansion. Associations between hormone signaling and cell wall biosynthesis have long been suggested, but recently direct links have been found revealing hormones play key regulatory roles in cellulose biosynthesis. The radially swollen 1 (rsw1) allele of Arabidopsis thaliana CESA1 harbors a single amino acid change that renders the protein unstable at high temperatures. We used the conditional nature of rsw1 to investigate how auxin contributes to isotropic growth. We found that exogenous auxin treatment reduces isotropic swelling in rsw1 roots at the restrictive temperature of 30�C. We also discovered decreases in auxin influx between rsw1 and wild-type roots via confocal imaging of AUX1-YFP, even at the permissive temperature of 19�C. Moreover, rsw1 displayed mis-expression of auxin-responsive and CESA genes. Additionally, we found altered auxin maxima in rsw1 mutant roots at the onset of swelling using DII-VENUS and DR5:vYFP auxin reporters. Overall, we conclude disrupted cell wall biosynthesis perturbs auxin transport leading to altered auxin homeostasis impacting both anisotropic and isotropic growth that affects overall root morphology.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Ácidos Indolacéticos/metabolismo , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Benzamidas/farmacologia , Celulose/biossíntese , Genes de Plantas/genética , Glucosiltransferases/genética , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo
19.
J Agric Food Chem ; 67(19): 5587-5595, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31016980

RESUMO

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production. MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use. In this study, mutant G415P was obtained by directed evolution and S361R/S444E was subsequently generated based on a structure analysis of the region around G415. The t1/2 of G415P and S361R/S444E at 60 °C increased by 3.0- and 3.2-fold, respectively, compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was obtained through a combination of the above mutants, and its t1/2 significantly increased by 19.7-fold. Kinetic and thermodynamic stability results showed that the T50 and Tm values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with those of the wild-type enzyme. When the triple mutant was used in trehalose production, the yield reached 71.6%, higher than the 70.3% achieved with the wild-type. Thus, the mutant has a potential application for industrial trehalose production.


Assuntos
Arthrobacter/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Sequência de Aminoácidos , Arthrobacter/química , Arthrobacter/genética , Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada , Estabilidade Enzimática , Glucosiltransferases/metabolismo , Temperatura Alta , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Especificidade por Substrato
20.
Phytochemistry ; 163: 75-88, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31030081

RESUMO

Cyclocarya paliurus (Batalin) Iljinsk is a medicinal plant belonging to the Juglandaceae family, and its leaves are used for a traditional sweet herbal tea with bioactivity against obesity and hyperglycaemia in China. It contains various bioactive specialised metabolites, such as flavonoids, triterpenes and their glucosides, while no glycosyltransferases (GTs) have been reported in C. paliurus to date. Herein, we identified and cloned the first glucosyltransferase C. paliurus GT1. The expression profiles of C. paliurus GT1 showed very high expression in young leaves, callus and branches, but relatively low expression in old leaves and bark and no expression in root. The recombinant C. paliurus GT1 protein was heterologously expressed in Escherichia coli and exhibited catalytic activity towards multiple flavonoids favouring substrate- and regio-specific biosynthesis. Further enzyme assays indicated a preference for certain hydroxyl group glucosylation by C. paliurus GT1. C. paliurus GT1 actively catalysed the glucosylation of flavones and flavonols, but it was less active towards isoflavones, flavanones or triterpenes. C. paliurus GT1 was also able to catalyse the attachment of sugars to the thiol (S-) or amine (N-) sites on aromatic compounds but not on aliphatic compounds. Molecular docking and site-directed mutagenesis analyses indicated that A43F, V84P, and M201Y dramatically altered the regio-selectivity and activity, and the W283M mutation and deletion of the V309-D320 region enhanced the activity and the formation of disaccharides. Herein, we present the identification and characterization of the first multi-functional glucosyltransferase in C. paliurus and provide a basis for understanding the biosynthesis of flavonoid glucosides. C. paliurus GT1 could be utilized as a synthetic biology tool for the synthesis of O-, N-, or S-glucosylated natural/unnatural products.


Assuntos
Flavonoides/biossíntese , Glucosídeos/biossíntese , Glucosiltransferases/análise , Juglandaceae/química , Flavonoides/química , Glucosídeos/química , Glucosiltransferases/metabolismo , Juglandaceae/metabolismo , Estereoisomerismo , Especificidade por Substrato
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