Sample records for udpg
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Watanabe, Daisuke; Zhou, Yan; Hirata, Aiko; Sugimoto, Yukiko; Takagi, Kenichi; Akao, Takeshi; Ohya, Yoshikazu; Takagi, Hiroshi; Shimoi, Hitoshi
2016-01-01
The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Watanabe, Daisuke; Zhou, Yan; Hirata, Aiko; Sugimoto, Yukiko; Takagi, Kenichi; Akao, Takeshi; Ohya, Yoshikazu; Takagi, Hiroshi
2015-01-01
The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains. PMID:26497456
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Zhang, Tingjing; Liang, Jianqiang; Wang, Panxue; Xu, Ying; Wang, Yutang; Wei, Xinyuan; Fan, Mingtao
2016-10-12
Phloretin-2'-O-glycosyltransferase (P2'GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2'GT of Malus x domestica (MdP2'GT) with a specific activity of 46.82 μkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2'GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2'GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2'GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2'GT displayed the lowest K m and the highest k cat /K m for phloretin and UDPG compared to all previously reported P2'GTs, making MdP2'GT favor phloridzin synthesis the most.
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Zhang, Tingjing; Liang, Jianqiang; Wang, Panxue; Xu, Ying; Wang, Yutang; Wei, Xinyuan; Fan, Mingtao
2016-01-01
Phloretin-2′-O-glycosyltransferase (P2′GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2′GT of Malus x domestica (MdP2′GT) with a specific activity of 46.82 μkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2′GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2′GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2′GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2′GT displayed the lowest Km and the highest kcat/Km for phloretin and UDPG compared to all previously reported P2′GTs, making MdP2′GT favor phloridzin synthesis the most. PMID:27731384
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Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang
2017-12-18
Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.
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NASA Technical Reports Server (NTRS)
Leznicki, A. J.; Bandurski, R. S.
1988-01-01
The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-beta-D-glucose from uridine-5'-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss.
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Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier
2003-01-01
A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase. PMID:12429023
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Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier
2003-03-01
A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.
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Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose 1
Leznicki, Antoni J.; Bandurski, Robert S.
1988-01-01
The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-β-d-glucose from uridine-5′-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss. Images Fig. 4 PMID:11537438
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Dai, Longhai; Li, Jiao; Yao, Peiyuan; Zhu, Yueming; Men, Yan; Zeng, Yan; Yang, Jiangang; Sun, Yuanxia
2017-04-20
Glycosylation is a prominent biological mechanism for structural and functional diversity of natural products. Uridine diphosphate-dependent glycosyltransferases with aglycon promiscuity are generally recognised as effective biocatalysts for glycodiversification of natural products for practical applications. In this study, the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis 168 was explored. Bs-YjiC, with uridine diphosphate glucose (UDPG) as sugar donor, exhibited robust capabilities to glycosylate 19 structurally diverse types of drug-like scaffolds with regio- and stereospecificities and form O-, N- and S-linkage glycosides. Twenty-four glycosides of 17 aglycons were purified from scale-up reactions using Bs-YjiC as a biocatalyst, and their structures were confirmed by nuclear magnetic resonance spectra. Furthermore, a one-pot reaction by coupling Bs-YjiC to sucrose synthase from Arabidopsis thaliana was applied to glycosylate pterostilbene. Without adding the costly UDPG as sugar donor, 9mM (3.8g/L) pterostilbene 4'-O-β-glucoside was obtained by periodic feeding of pterostilbene. These results suggest the aglycon promiscuity of Bs-YjiC and demonstrate its significant application prospect in biosynthesis of valuable natural products. Copyright © 2017 Elsevier B.V. All rights reserved.
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Tang, Qi; Ma, Xiaojun; Mo, Changming; Wilson, Iain W; Song, Cai; Zhao, Huan; Yang, Yanfang; Fu, Wei; Qiu, Deyou
2011-07-05
Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the
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NASA Technical Reports Server (NTRS)
Mar, A.; Dworkin, J.; Oro, J.
1987-01-01
Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.
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Phosphodiester content measured in human liver by in vivo 31P MR spectroscopy at 7 tesla
Clarke, William T.; Valkovič, Ladislav; Levick, Christina; Pavlides, Michael; Barnes, Eleanor; Cobbold, Jeremy F.; Robson, Matthew D.; Rodgers, Christopher T.
2017-01-01
Purpose Phosphorus (31P) metabolites are emerging liver disease biomarkers. Of particular interest are phosphomonoester and phosphodiester (PDE) “peaks” that comprise multiple overlapping resonances in 31P spectra. This study investigates the effect of improved spectral resolution at 7 Tesla (T) on quantifying hepatic metabolites in cirrhosis. Methods Five volunteers were scanned to determine metabolite T1s. Ten volunteers and 11 patients with liver cirrhosis were scanned at 7T. Liver spectra were acquired in 28 min using a 16‐channel 31P array and 3D chemical shift imaging. Concentrations were calculated using γ‐adenosine‐triphosphate (γ‐ATP) = 2.65 mmol/L wet tissue. Results T1 means ± standard deviations: phosphatidylcholine 1.05 ± 0.28 s, nicotinamide‐adenine‐dinucleotide (NAD+) 2.0 ± 1.0 s, uridine‐diphosphoglucose (UDPG) 3.3 ± 1.4 s. Concentrations in healthy volunteers: α‐ATP 2.74 ± 0.11 mmol/L wet tissue, inorganic phosphate 2.23 ± 0.20 mmol/L wet tissue, glycerophosphocholine 2.34 ± 0.46 mmol/L wet tissue, glycerophosphoethanolamine 1.50 ± 0.28 mmol/L wet tissue, phosphocholine 1.06 ± 0.16 mmol/L wet tissue, phosphoethanolamine 0.77 ± 0.14 mmol/L wet tissue, NAD+ 2.37 ± 0.14 mmol/L wet tissue, UDPG 2.00 ± 0.22 mmol/L wet tissue, phosphatidylcholine 1.38 ± 0.31 mmol/L wet tissue. Inorganic phosphate and phosphatidylcholine concentrations were significantly lower in patients; glycerophosphoethanolamine concentrations were significantly higher (P < 0.05). Conclusion We report human in vivo hepatic T1s for phosphatidylcholine, NAD+, and UDPG for the first time at 7T. Our protocol allows high signal‐to‐noise, repeatable measurement of metabolite concentrations in human liver. The splitting of PDE into its constituent peaks at 7T may allow more insight into changes in metabolism. Magn Reson Med 78:2095–2105, 2017. © 2017 The Authors Magnetic Resonance in
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Lei, Min; Wu, Xiangli; Zhang, Jinxia; Wang, Hexiang; Huang, Chenyang
2017-07-01
Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) catalyzes the first step in trehalose synthesis, which involves transfer of glucose from uridine diphosphate glucose (UDPG) to glucose 6-phosphate (G6P) to form trehalose-6-phosphate. To determine the gene and enzymatic characteristics of TPS in Pleurotus ostreatus, we cloned and sequenced the cDNA of PoTPS1, which contains a 1665 bp open reading frame that encodes a 554-amino acid protein with a predicted molecular weight of 62.01 kDa. This gene was expressed in Escherichia coli BL21 and then the recombinant protein was purified and characterized. Results showed that the optimum pH and temperature for the recombinant PoTPS1 were 7.4 and 30 °C, respectively; the K m value against G6P and UDPG were 0.14 and 0.17 mM, respectively, and the V max and K cat values were 91.86 nkat/g and 5.89 s -1 , respectively. Trehalose content was as high as 158.88 mg g -1 dry weight after heat treatment at 40 °C for 15 h, which was consistent with highest TPS1 activity at that time point. This result indicated that PoTPS1 was responsible for trehalose synthesis in P. ostreatus. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Detection of cerebral NAD+ in humans at 7T.
de Graaf, Robin A; De Feyter, Henk M; Brown, Peter B; Nixon, Terence W; Rothman, Douglas L; Behar, Kevin L
2017-09-01
To develop 1 H-based MR detection of nicotinamide adenine dinucleotide (NAD + ) in the human brain at 7T and validate the 1 H results with NAD + detection based on 31 P-MRS. 1 H-MR detection of NAD + was achieved with a one-dimensional double-spin-echo method on a slice parallel to the surface coil transceiver. Perturbation of the water resonance was avoided through the use of frequency-selective excitation. 31 P-MR detection of NAD + was performed with an unlocalized pulse-acquire sequence. Both 1 H- and 31 P-MRS allowed the detection of NAD + signals on every subject in 16 min. Spectral fitting provided an NAD + concentration of 107 ± 28 μM for 1 H-MRS and 367 ± 78 μM and 312 ± 65 μM for 31 P-MRS when uridine diphosphate glucose (UDPG) was excluded and included, respectively, as an overlapping signal. NAD + detection by 1 H-MRS is a simple method that comes at the price of reduced NMR visibility. NAD + detection by 31 P-MRS has near-complete NMR visibility, but it is complicated by spectral overlap with NADH and UDPG. Overall, the 1 H- and 31 P-MR methods both provide exciting opportunities to study NAD + metabolism on human brain in vivo. © 2016 International Society for Magnetic Resonance in Medicine. Magn Reson Med 78:828-835, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.
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Phosphodiester content measured in human liver by in vivo 31 P MR spectroscopy at 7 tesla.
Purvis, Lucian A B; Clarke, William T; Valkovič, Ladislav; Levick, Christina; Pavlides, Michael; Barnes, Eleanor; Cobbold, Jeremy F; Robson, Matthew D; Rodgers, Christopher T
2017-12-01
Phosphorus ( 31 P) metabolites are emerging liver disease biomarkers. Of particular interest are phosphomonoester and phosphodiester (PDE) "peaks" that comprise multiple overlapping resonances in 31 P spectra. This study investigates the effect of improved spectral resolution at 7 Tesla (T) on quantifying hepatic metabolites in cirrhosis. Five volunteers were scanned to determine metabolite T 1 s. Ten volunteers and 11 patients with liver cirrhosis were scanned at 7T. Liver spectra were acquired in 28 min using a 16-channel 31 P array and 3D chemical shift imaging. Concentrations were calculated using γ-adenosine-triphosphate (γ-ATP) = 2.65 mmol/L wet tissue. T 1 means ± standard deviations: phosphatidylcholine 1.05 ± 0.28 s, nicotinamide-adenine-dinucleotide (NAD + ) 2.0 ± 1.0 s, uridine-diphosphoglucose (UDPG) 3.3 ± 1.4 s. Concentrations in healthy volunteers: α-ATP 2.74 ± 0.11 mmol/L wet tissue, inorganic phosphate 2.23 ± 0.20 mmol/L wet tissue, glycerophosphocholine 2.34 ± 0.46 mmol/L wet tissue, glycerophosphoethanolamine 1.50 ± 0.28 mmol/L wet tissue, phosphocholine 1.06 ± 0.16 mmol/L wet tissue, phosphoethanolamine 0.77 ± 0.14 mmol/L wet tissue, NAD + 2.37 ± 0.14 mmol/L wet tissue, UDPG 2.00 ± 0.22 mmol/L wet tissue, phosphatidylcholine 1.38 ± 0.31 mmol/L wet tissue. Inorganic phosphate and phosphatidylcholine concentrations were significantly lower in patients; glycerophosphoethanolamine concentrations were significantly higher (P < 0.05). We report human in vivo hepatic T 1 s for phosphatidylcholine, NAD + , and UDPG for the first time at 7T. Our protocol allows high signal-to-noise, repeatable measurement of metabolite concentrations in human liver. The splitting of PDE into its constituent peaks at 7T may allow more insight into changes in metabolism. Magn Reson Med 78:2095-2105, 2017. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on
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In-silico Investigation of Antitrypanosomal Phytochemicals from Nigerian Medicinal Plants
Setzer, William N.; Ogungbe, Ifedayo V.
2012-01-01
Background Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment. Methods A molecular docking study has been carried out on phytochemical agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacologically to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compounds from 19 species of medicinal plants were investigated using in-silico molecular docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4′ epimerase (TbUDPGE), and ornithine decarboxylase (TbODC). Results This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4′ epimerase (TbUDPGE). Polyphenolic compounds such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins. Conclusions This in-silico molecular docking study has identified potential biomolecular targets of
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Coello, Patricia; Martínez-Barajas, Eleazar
2014-07-01
SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
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Colvin, J R
1980-07-01
The mechanism of formation of cellulose-like microfibrils by a non-soluble, particulate enzyme and uridine diphosphoglucose (UDPG) in a cell-free system from Acetobacter xylinum was studied by transmission electron microscopy and X-ray diffraction. The suspension of particles to which the enzyme is adsorbed is composed of whole, dense ovoids, 50-250 nm long when wet, of fragments of the ovoids, and amorphous substance. There is a typical unit membrane around each ovoid but initially there is no trace of fibrillar material in the suspension. When the suspension of particles is incubated with UDPG, linear wisps of fibrils are produced which associate rapidly to form longer and wider threads, especially in 0.01 M NaCl. There is no visible attachment of the wisps to the particles. After 20 min incubation, threads with the typical morphology of cellulose microfibrils are formed that later tend to become entangled in clumps. The microfibrils are insoluble in hot, aqueous, alkaline solutions and resistant to the action of trypsin, but may be degraded by glusulase. After treatment with 1 M NaOH at 100° C or with cold 18% NaOH they show an X-ray diffraction pattern which resembles that of Cellulose II from mercerized, authentic bacterial cellulose. Incorporation of radioactive glucose into the insoluble residue is enhanced by drying of the cellulose microfibrils before alkaline digestion and especially by the addition of a gross excess of carrier cellulose after incubation. In this system there is no evidence for participation of linear, axial, synthesizing sites on the cell wall of the bacterium or for ordered, organized granules in the assembly of the microfibrils. That is, cellulose-like microfibrils may be formed in a cell-free system without the action of any of the previously suggested cell organelles. In addition, these observations are consistent with a previously described notion of a transient, hydrated, nascent, bacterial cellulose microfibril. The possibility
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Molecular systematics of the subfamily Limenitidinae (Lepidoptera: Nymphalidae)
Dhungel, Bidur
2018-01-01
We studied the systematics of the subfamily Limenitidinae (Lepidoptera: Nymphalidae) using molecular methods to reconstruct a robust phylogenetic hypothesis. The molecular data matrix comprised 205 Limenitidinae species, four outgroups, and 11,327 aligned nucleotide sites using up to 18 genes per species of which seven genes (CycY, Exp1, Nex9, PolII, ProSup, PSb and UDPG6DH) have not previously been used in phylogenetic studies. We recovered the monophyly of the subfamily Limenitidinae and seven higher clades corresponding to four traditional tribes Parthenini, Adoliadini, Neptini, Limenitidini as well as three additional independent lineages. One contains the genera Harma + Cymothoe and likely a third, Bhagadatta, and the other two independent lineages lead to Pseudoneptis and to Pseudacraea. These independent lineages are circumscribed as new tribes. Parthenini was recovered as sister to rest of Limenitidinae, but the relationships of the remaining six lineages were ambiguous. A number of genera were found to be non-monophyletic, with Pantoporia, Euthalia, Athyma, and Parasarpa being polyphyletic, whereas Limenitis, Neptis, Bebearia, Euryphura, and Adelpha were paraphyletic. PMID:29416955
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Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme
NASA Technical Reports Server (NTRS)
Leznicki, A. J.; Bandurski, R. S.
1988-01-01
The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.
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Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose 1
Leznicki, Antoni J.; Bandurski, Robert S.
1988-01-01
The synthesis of indole-3-acetyl-1-O-β-d-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as ρ-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid, and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed. PMID:11537439
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Stereoselectivity in Polyphenol Biosynthesis
NASA Technical Reports Server (NTRS)
Lewis, Norman G.; Davin, Laurence B.
1992-01-01
Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.
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Phosphorus NMR of isolated perfused morris hepatomas
DOE Office of Scientific and Technical Information (OSTI.GOV)
Graham, R.A.; Meyer, R.A.; Brown, T.R.
1986-03-05
The authors are developing techniques for the study of perfused solid tumors by NMR. Tissue-isolated solid hepatomas were grown to 1-2 cm diameter as described previously. The arterial supply was isolated and the tumors perfused (0.5 - 1.0 ml/min) in vitro at 25 C with a 15% suspension of red blood cells in Krebs-Henseliet solution. /sup 31/P-NMR spectra were acquired at 162 MHz in a specially-designed NMR probe using a solenoidal coil. Intracellular pH (monitored from the chemical shift of inorganic phosphate) and ATP levels were stable for up to 6 hrs during perfusion. During 30 min of global ischemia,more » ATP decreased by 75% and pH fell from 7.0 to 6.7. These changes were reversed by 1 hr reperfusion. In addition to ATP and phosphate, the spectra included a large resonance due to phosphomonoesters, as well as peaks consistent with glycerylphosphocholine, glyceryl-phosphoethanolamine, phosphocreatine, NAD, and UDPG. However, the most novel feature of the spectra was the presence of an unidentified peak in the phosphonate region (+ 16.9 ppm). The peak was not present in spectra of muscle, liver, brain, kidney, or fat tissues excised from the same animals. They are presently attempting to identify the compound that gives rise to this peak and to establish its metabolic origin.« less
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Ostrowski, Maciej; Hetmann, Anna; Jakubowska, Anna
2015-09-01
The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-β-D-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA+UDPG↔1-O-IA-glucose+UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC-MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (Km=0.52 mM, Vmax=161 nmol min(-1), kcat/Km=4.36 mM s(-1)) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, D-gluconic acid lactone, an inhibitor of β-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Liu, Nan-Nan; Chi, Zhe; Wang, Qin-Qing; Hong, Jiang; Liu, Guang-Lei; Hu, Zhong; Chi, Zhen-Ming
2017-09-01
After the compositional change of a pullulan production medium, a molecular weight (Mw) of the pullulan produced by Aureobasidium melanogenum P16 was 2.32×10 6 and a pullulan titer was 44.4g/L while a Mw of the pullulan produced by A. melanogenum P16 grown in the initial medium was only 3.47×10 5 and a pullulan titer was 65.3g/L. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase while the decreased pullulan titer was related to the decreased transcriptional levels of the genes encoding 6-P-glucose kinase, glucosyltransferase, α-phosphoglucose mutase, UDPG-pyrophosphorylase and pullulan synthetase. During the 10-L fermentation, when the yeast strain P16 was grown in the initial medium, the pullulan and oligosaccharide titers were 65.5g/L and 7.8g/L, respectively and the Mw of the produced pullulan was 4.42×10 5 while when the yeast strain P16 was grown in the compositionally changed medium, the pullulan and oligosaccharide titers were 46.4g/L and 27.8g/L, respectively and the Mw of the produced pullulan was 2.6×10 6 . Most of the oligosaccharides produced by the yeast strain P16 cultivated in the compositionally changed medium had degree of polymerization of 4 and 5. Therefore, both of the high Mw pullulan and oligosaccharides with high levels were produced by the yeast strain P16. Copyright © 2017 Elsevier B.V. All rights reserved.
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NASA Technical Reports Server (NTRS)
Paliyath, G.; Poovaiah, B. W.
1988-01-01
Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.
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Sengupta, Shinjinee; Lahiri, Sagar; Banerjee, Shakri; Bashistha, Bipasha; Ghosh, Anil K
2011-12-01
Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. 2011 Elsevier B.V. All rights reserved.
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Sato, Vanessa Sayuri; Jorge, João Atílio; Guimarães, Luis Henrique Souza
2016-06-01
The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.
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Mata, Ana Teresa; Jorge, Tiago Filipe; Ferreira, João; do Rosário Bronze, Maria; Branco, Diana; Fevereiro, Pedro; Araújo, Susana; António, Carla
2016-12-16
Trehalose-6-phosphate (T6P) is an important signaling metabolite involved in plant growth control that inhibits the sucrose nonfermenting-1-related protein kinase 1 (SnRK1), a key regulator of energy and carbon metabolism in plants. The quantification of T6P in plant tissues is fundamental to improve our understanding of sugar signaling and the links between plant growth and development in response to stress conditions. However, the almost undetectable levels of T6P together with the complex plant matrix and the presence of T6P isomers such as sucrose-6-phosphate (S6P), makes the detection of this metabolite challenging. This work describes the development and validation of a hydrophilic interaction chromatography (HILIC) method for the on-line coupling with negative ion electrospray (ESI) triple quadrupole tandem mass spectrometry (MS/MS) in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of metabolic intermediates of the biosynthesis of trehalose, including glucose-6-phosphate (G6P), uridine 5-diphospho-glucose (UDPG), T6P (and its isomer S6P). Enhanced signal in the MRM mode and improved chromatographic separation for each compound were obtained using piperidine and methylphosphonic acid as additives in the HILIC mobile phase. The optimized HILIC-ESI-QqQ-MS/MS method increases the range of sensitive analytical methodologies for the quantification of key low-abundant metabolites, and was applied to quantify the fluctuations of S6P, T6P and G6P in Medicago truncatula plants in response to environmental stress. The levels of S6P, T6P, and G6P in M. truncatula plant tissues (roots and leaves) exposed to a water deficit and recovery treatment, ranged from 30 to 150pmolg -1 FW, 16-120pmolg -1 FW, and 330-1690pmolg -1 FW, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.