The Path of Carbon in Photosynthesis XVIII. The Identification of Nucleotide Coenzymes

DOE R&D Accomplishments Database

Buchanan, J. G.; Lynch, V. H.; Benson, A. A.; Calvin, M.; Bradley, D. F.

1953-01-19

The radioactive compounds to be observed when algae or green leaves are allowed to photosynthesize in C{sup 14}O{sub 2} for short periods are almost all phosphorylated derivatives of sugars. Of these, phosphate esters of trioses, sedoheptulose and fructose are the first to incorporate C{sup 14} followed closely by ribulose diphosphate, glucose-6-phosphate and a phosphate of mannose. It has been noted, in earlier papers of this series, that on radiograms of the products of photosynthesis, a dark area appeared in a position occupied by no known sugar phosphate and which gave glucose on acid hydrolysis or on treatment with a phosphatase preparation. This has hitherto been referred to as an 'unknown glucose phosphate'. It was found that this substance was more labile to acid than glucose-l-phosphate, itself a readily hydrolysable phosphate, and furthermore that other labile glucose derivatives were formed as intermediates during the acid hydrolysis. Accumulation of labeled glucose in this area precedes that in sucrose and suggests its synthetic relationship to sucrose phosphate synthesis.

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

PubMed Central

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

The Path of Carbon in Photosynthesis XIX. The Identification of Sucrose Phosphate in Sugar Beet Leaves

DOE R&D Accomplishments Database

Buchanan, J. G.

1952-09-01

The recognition and characterization of a sucrose phosphate as an intermediate in sucrose by synthesis by green plants is described. A tentative structure for this phosphate is proposed and its mode of formation suggested.

Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

PubMed

Li, Zijie; Cai, Li; Qi, Qingsheng; Styslinger, Thomas J; Zhao, Guohui; Wang, Peng George

2011-09-01

We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars d-psicose, d-sorbose, l-tagatose, and l-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to significantly reduce the synthetic cost. Copyright © 2011 Elsevier Ltd. All rights reserved.

Synthesis of cytidine ribonucleotides by stepwise assembly of the heterocycle on a sugar phosphate.

PubMed

Ingar, Abdul-Aziz; Luke, Richard W A; Hayter, Barry R; Sutherland, John D

2003-06-06

Although various syntheses of the nucleic acid bases exist and ribose is a product of the formose reaction, no prebiotically plausible methods for attaching pyrimidine bases to ribose to give nucleosides have been described. Kinetic and thermodynamic factors are thought to mitigate against such condensation reactions in aqueous solution. This inability to produce pyrimidine nucleosides and hence nucleotides is a major stumbling block of the "RNA World" hypothesis and has led to suggestions of alternative nucleic acids as evolutionary precursors to RNA. Here, we show that a process in which the base is assembled in stages on a sugar phosphate can produce cytidine nucleotides. The sequential action of cyanamide and cyanoacetylene on arabinose-3-phosphate produces cytidine-2',3'-cyclophosphate and arabinocytidine-3'-phosphate.

Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

PubMed

Westermayer, Sonja A; Fritz, Georg; Gutiérrez, Joaquín; Megerle, Judith A; Weißl, Mira P S; Schnetz, Karin; Gerland, Ulrich; Rädler, Joachim O

2016-05-01

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions. © 2016 John Wiley & Sons Ltd.

The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

PubMed Central

Shieh, H S; Berman, H M; Dabrow, M; Neidle, S

1980-01-01

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry. PMID:7355129

Enzymatic synthesis of rare sugars with L-rhamnulose-1-phosphate aldolase from Thermotoga maritima MSB8.

PubMed

Li, Zijie; Wu, Xiaoru; Cai, Li; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

2015-09-15

L-Rhamnulose-1-phosphate aldolase from a thermophilic source (Thermotoga maritima MSB8) (RhaDT.mari) was heterologously overexpressed in Escherichia coli and the stereoselectivity of this enzyme with or without Nus tag was investigated. We also applied this enzyme to the synthesis of rare sugars D-psicose, D-sorbose, L-tagatose and L-fructose using our one-pot four-enzyme system. To the best of our knowledge, this is the first use of RhaD from a thermophilic source for rare sugar synthesis and the temperature tolerance of this enzyme paves the path for large scale fermentation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigation of the effects of phosphate fertilizer application on the heavy metal content in agricultural soils with different cultivation patterns.

PubMed

Cheraghi, Mehrdad; Lorestani, Bahareh; Merrikhpour, Hajar

2012-01-01

The use of phosphate fertilizers is essential in agriculture, because they supply farmland with nutrients for growing plants. However, heavy metals might be included as impurities in natural materials and minerals, so heavy metals can also be present in phosphate fertilizers or other chemical fertilizers. The aim of this work was to assess the heavy metal content and contamination status of agricultural soils in the Hamadan province of Iran used for the cultivation of different crops, including cucumber, potatoes, and sugar beet. Surface soil samples were collected and analyzed to determine the total concentration of specific elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn), before the pollution index was calculated for each element. Soils used for the cultivation of the three types of crop were not contaminated with As, Cr, Cu, Pb, or Zn. However, the pollution indices for Cd were 1.1, 4.4, and 3.8 in cucumber, potato, and sugar beet fields, respectively, which indicated moderate, high, and high levels of contamination, respectively. Soils from potato and sugar beet fields were heavily contaminated with Cd, which may have resulted from long-term overuse of phosphate fertilizers.

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

PubMed Central

Heyes, Derren J.; Levy, Colin; Lafite, Pierre; Roberts, Ian S.; Goldrick, Marie; Stachulski, Andrew V.; Rossington, Steven B.; Stanford, Deborah; Rigby, Stephen E. J.; Scrutton, Nigel S.; Leys, David

2009-01-01

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group. PMID:19815542

Genetics Home Reference: DOLK-congenital disorder of glycosylation

MedlinePlus

... called glycosylation, which attaches groups of sugar molecules (oligosaccharides) to proteins. Glycosylation changes proteins in ways that ... to dolichol phosphate in order to build the oligosaccharide chain. Once the chain is formed, dolichol phosphate ...

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations.

PubMed

Zhitnikova, M Y; Shestopalova, A V

2017-11-01

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5'-C5'-C4'-C3') from canonical to alternative conformations and/or C2'-endo → C3'-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.

The role of molecular structure of sugar-phosphate backbone and nucleic acid bases in the formation of single-stranded and double-stranded DNA structures.

PubMed

Poltev, Valeri; Anisimov, Victor M; Danilov, Victor I; Garcia, Dolores; Sanchez, Carolina; Deriabina, Alexandra; Gonzalez, Eduardo; Rivas, Francisco; Polteva, Nina

2014-06-01

Our previous DFT computations of deoxydinucleoside monophosphate complexes with Na(+)-ions (dDMPs) have demonstrated that the main characteristics of Watson-Crick (WC) right-handed duplex families are predefined in the local energy minima of dDMPs. In this work, we study the mechanisms of contribution of chemically monotonous sugar-phosphate backbone and the bases into the double helix irregularity. Geometry optimization of sugar-phosphate backbone produces energy minima matching the WC DNA conformations. Studying the conformational variability of dDMPs in response to sequence permutation, we found that simple replacement of bases in the previously fully optimized dDMPs, e.g. by constructing Pyr-Pur from Pur-Pyr, and Pur-Pyr from Pyr-Pur sequences, while retaining the backbone geometry, automatically produces the mutual base position characteristic of the target sequence. Based on that, we infer that the directionality and the preferable regions of the sugar-phosphate torsions, combined with the difference of purines from pyrimidines in ring shape, determines the sequence dependence of the structure of WC DNA. No such sequence dependence exists in dDMPs corresponding to other DNA conformations (e.g., Z-family and Hoogsteen duplexes). Unlike other duplexes, WC helix is unique by its ability to match the local energy minima of the free single strand to the preferable conformations of the duplex. Copyright © 2013 Wiley Periodicals, Inc.

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate.

PubMed

Taguchi, Yodai; Saburi, Wataru; Imai, Ryozo; Mori, Haruhide

2017-08-01

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0-8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1↔1)-α-d-Manp6P, was synthesized with 51% yield.

The Path of Carbon in Photosynthesis XXI. The Cyclic Regeneration of Carbon Dioxide Acceptor

DOE R&D Accomplishments Database

Bassham, J. A.; Benson, A. A.; Kay, Lorel D.; Harris, Anne Z.; Wilson, A. T.; Calvin, M.

1953-10-01

Photosynthesizing plants have been exposed to C{sup 14}O{sub 2} for short periods of time (0.4 to 15 sec.) and the products of carbon dioxide reduction analyzed by paper chromatography and radio autography. Methods have been developed for the degradation of ribulose and sedoheptulose. These sugars, obtained as their phosphate esters from the above C{sup 14}O{sub 2} exposures and from other experiments, have been degraded and their distribution of radiocarbon determined. The distribution of radiocarbon in these sugars, and other data, indicate that sedoheptulose phosphate and ribulose diphosphates are formed during photosynthesis from triose and hexose phosphates, the latter being synthesized, in turn, by the reduction of 3-phosphoglyceric acid.

Ammonium phosphate as a sole nutritional supplement for the fermentative production of 2,3-butanediol from sugar cane juice.

PubMed

Berbert-Molina, M A; Sato, S; Silveira, M M

2001-01-01

The production of 2,3-butanediol by Klebsiella pneumoniae from sugar cane juice supplemented with different salts was studied. This microorganism is able to degrade sucrose present in sugar cane juice containing ammonium phosphate as the sole nutritional supplement. With a sugar cane juice-based medium containing approximately 180 g sucrose/l and 8.0 g (NH4)2HPO4/l, over 70 g 2,3-butanediol plus acetoin/l were formed. This result is comparable to that achieved with a sugar cane juice-based medium containing several nutrients, although the kinetic profiles of these runs presented significant differences. With the ammonium phosphate-enriched medium, cell growth was initially favoured by both the strong oxygen supply and the higher water activity due to the lower concentration of nutrients. After 14 h, the limitation in some nutrients led to the interruption of cell growth, and decreasing rates for product formation and substrate consumption were observed. During the stationary phase of this run, sucrose was preferentially converted to product, and the substrate was completely depleted after 35 h of the process. With the complete medium, the substrate was totally consumed after 36 h of run. In this case, the higher initial concentration of nutrients reduced the overall process rate but sustained the cell growth for 27 h. Conversion yields of 0.40 g product/g sucrose and productivities close to 2.0 g/l x h were obtained under both conditions.

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetting,M.; Frantom, P.; Blanchard, J.

2008-01-01

The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97 rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structuremore » highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the {beta}-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.« less

Impact of Phosphate, Potassium, Yeast Extract, and Trace Metals on Chitosan and Metabolite Production by Mucor indicus.

PubMed

Safaei, Zahra; Karimi, Keikhosro; Zamani, Akram

2016-08-30

In this study the effects of phosphate, potassium, yeast extract, and trace metals on the growth of Mucor indicus and chitosan, chitin, and metabolite production by the fungus were investigated. Maximum yield of chitosan (0.32 g/g cell wall) was obtained in a phosphate-free medium. Reversely, cell growth and ethanol formation by the fungus were positively affected in the presence of phosphate. In a phosphate-free medium, the highest chitosan content (0.42 g/g cell wall) and cell growth (0.66 g/g sugar) were obtained at 2.5 g/L of KOH. Potassium concentration had no significant effect on ethanol and glycerol yields. The presence of trace metals significantly increased the chitosan yield at an optimal phosphate and potassium concentration (0.50 g/g cell wall). By contrast, production of ethanol by the fungus was negatively affected (0.33 g/g sugars). A remarkable increase in chitin and decrease in chitosan were observed in the absence of yeast extract and concentrations lower than 2 g/L. The maximum chitosan yield of 51% cell wall was obtained at 5 g/L of yeast extract when the medium contained no phosphate, 2.5 g/L KOH, and 1 mL/L trace metal solution.

Laser Raman spectra of mono-, oligo- and polysaccharides in solution

NASA Astrophysics Data System (ADS)

Barrett, T. W.

We examined the Raman spectra of thirteen sugars—seven monosaccharides, two disaccharides, one trisaccharide and three polysaccharides—in the wavelength range 200—1700 cm -1 and (i) varied the phosphate buffered solution from pH 6.0 to 8.5 at constant ionic strength of 0.1 and (ii) varied HCl solutions from pH 0.8 to 5.0. As is to be expected with molecules containing COH groupings, all the molecular spectra are distinct. Of the thirteen sugars examined, only D-fructose 1,6-diphosphate (FDP) demonstrated spectral changes for the pH range 6.0—8.5 in phosphate buffer; but all exhibited band intensity enhancement in HCl at the lower pHs, but not band wavenumber changes. The results indicate that: (i) changes in the pH of the major intracellular buffer, phosphate, toward acidity, are able to hydrolyze the 1-phosphate group of FDP and the relative concentration of fructose 1-phosphate to fructose 6-phosphate is indicated by the intensity ratio of the 982 and 1080 cm -1 bands; (ii) it appears that all phosphate groups of FDP are hydrolyzed at pH 0.8 in HCl; and (iii) although conditions of extreme acidity are able to hydrolyze other sugars examined, there is no major degradation.

Sugar Allocation to Metabolic Pathways is Tightly Regulated and Affects the Virulence of Streptococcus mutans

PubMed Central

Kawada-Matsuo, Miki; Oogai, Yuichi; Komatsuzawa, Hitoshi

2016-01-01

Bacteria take up and metabolize sugar as a carbohydrate source for survival. Most bacteria can utilize many sugars, including glucose, sucrose, and galactose, as well as amino sugars, such as glucosamine and N-acetylglucosamine. After entering the cytoplasm, the sugars are mainly allocated to the glycolysis pathway (energy production) and to various bacterial component biosynthesis pathways, including the cell wall, nucleic acids and amino acids. Sugars are also utilized to produce several virulence factors, such as capsule and lipoteichoic acid. Glutamine-fructose-6-phosphate aminotransferase (GlmS) and glucosamine-6-phosphate deaminase (NagB) have crucial roles in sugar distribution to the glycolysis pathway and to cell wall biosynthesis. In Streptococcus mutans, a cariogenic pathogen, the expression levels of glmS and nagB are coordinately regulated in response to the presence or absence of amino sugars. In addition, the disruption of this regulation affects the virulence of S. mutans. The expression of nagB and glmS is regulated by NagR in S. mutans, but the precise mechanism underlying glmS regulation is not clear. In Staphylococcus aureus and Bacillus subtilis, the mRNA of glmS has ribozyme activity and undergoes self-degradation at the mRNA level. However, there is no ribozyme activity region on glmS mRNA in S. mutans. In this review article, we summarize the sugar distribution, particularly the coordinated regulation of GlmS and NagB expression, and its relationship with the virulence of S. mutans. PMID:28036052

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae.

PubMed

Madhavan, Anjali; Srivastava, Aradhana; Kondo, Akihiko; Bisaria, Virendra S

2012-03-01

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis

PubMed Central

French, Nigel G.; Scoble, Judith A.; Williams, Charlotte C.; Churches, Quentin I.; Frazer, Andrew R.; Taylor, Matthew C.; Coia, Greg; Simpson, Gregory; Turner, Nicholas J.; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed. PMID:29112947

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

PubMed

Hartley, Carol J; French, Nigel G; Scoble, Judith A; Williams, Charlotte C; Churches, Quentin I; Frazer, Andrew R; Taylor, Matthew C; Coia, Greg; Simpson, Gregory; Turner, Nicholas J; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Glucose-Specific Polymer Hydrogels—A Reassessment

PubMed Central

Fazal, Furqan M.; Hansen, David E.

2007-01-01

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (±)-epichlorohydrin in the presence of D-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels were prepared using the following five templates: D-glucose-6-phosphate monobarium salt, D-glucose, L-glucose, barium hydrogen phosphate (BaHPO4), and D-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with D-glucose, L-glucose, D-fructose and D-gluconamide. The extent of analyte sugar binding was determined using 1H-NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol-water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system. PMID:17035016

Glucose-specific poly(allylamine) hydrogels--a reassessment.

PubMed

Fazal, Furqan M; Hansen, David E

2007-01-01

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (+/-)-epichlorohydrin in the presence of d-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels was prepared using the following five templates: d-glucose-6-phosphate monobarium salt, d-glucose, l-glucose, barium hydrogen phosphate (BaHPO(4)), and d-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with d-glucose, l-glucose, d-fructose, and d-gluconamide. The extent of analyte sugar binding was determined using (1)H NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol-water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system.

A Comparative Analysis of the Sugar Phosphate Cyclase Superfamily Involved in Primary and Secondary Metabolism

PubMed Central

Wu, Xiumei; Flatt, Patricia M.; Schlörke, Oliver; Zeeck, Axel; Dairi, Tohru

2011-01-01

Sugar Phosphate Cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, e.g., aromatic amino acids, and clinically relevant secondary metabolites, e.g., aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically-labeled cyclitols revealed that cetoniacytone A, a unique C7N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. Using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homolog gene, cetA, was isolated from the cetoniacytone producer. CetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that may regulate the biosynthesis of a novel set of secondary metabolites. PMID:17195255

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

PubMed

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

PubMed

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

2016-11-01

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

PubMed Central

Heinrichs, Luisa; Schmitz, Jessica; Flügge, Ulf-Ingo; Häusler, Rainer E.

2012-01-01

An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde signaling is discussed. PMID:23233856

High-sugar intake does not exacerbate metabolic abnormalities or cardiac dysfunction in genetic cardiomyopathy.

PubMed

Hecker, Peter A; Galvao, Tatiana F; O'Shea, Karen M; Brown, Bethany H; Henderson, Reney; Riggle, Heather; Gupte, Sachin A; Stanley, William C

2012-05-01

A high-sugar intake increases heart disease risk in humans. In animals, sugar intake accelerates heart failure development by increased reactive oxygen species (ROS). Glucose-6-phosphate dehydrogenase (G6PD) can fuel ROS production by providing reduced nicotinamide adenine dinucleotide phosphate (NADPH) for superoxide generation by NADPH oxidase. Conversely, G6PD also facilitates ROS scavenging using the glutathione pathway. We hypothesized that a high-sugar intake would increase flux through G6PD to increase myocardial NADPH and ROS and accelerate cardiac dysfunction and death. Six-week-old TO-2 hamsters, a non-hypertensive model of genetic cardiomyopathy caused by a δ-sarcoglycan mutation, were fed a long-term diet of high starch or high sugar (57% of energy from sucrose plus fructose). After 24 wk, the δ-sarcoglycan-deficient animals displayed expected decreases in survival and cardiac function associated with cardiomyopathy (ejection fraction: control 68.7 ± 4.5%, TO-2 starch 46.1 ± 3.7%, P < 0.05 for TO-2 starch versus control; TO-2 sugar 58.0 ± 4.2%, NS, versus TO-2 starch or control; median survival: TO-2 starch 278 d, TO-2 sugar 318 d, P = 0.133). Although the high-sugar intake was expected to exacerbate cardiomyopathy, surprisingly, there was no further decrease in ejection fraction or survival with high sugar compared with starch in cardiomyopathic animals. Cardiomyopathic animals had systemic and cardiac metabolic abnormalities (increased serum lipids and glucose and decreased myocardial oxidative enzymes) that were unaffected by diet. The high-sugar intake increased myocardial superoxide, but NADPH and lipid peroxidation were unaffected. A sugar-enriched diet did not exacerbate ventricular function, metabolic abnormalities, or survival in heart failure despite an increase in superoxide production. Copyright © 2012 Elsevier Inc. All rights reserved.

Potential use of sugar binding proteins in reactors for regeneration of CO2 fixation acceptor D-Ribulose-1,5-bisphosphate

PubMed Central

Mahato, Sourav; De, Debojyoti; Dutta, Debajyoti; Kundu, Moloy; Bhattacharya, Sumana; Schiavone, Marc T; Bhattacharya, Sanjoy K

2004-01-01

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations. PMID:15175111

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

PubMed

Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

2017-02-01

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity. Copyright © 2017 American Society for Microbiology.

Starch Biosynthesis in Developing Wheat Grain 1

PubMed Central

Keeling, Peter L.; Wood, John R.; Tyson, R. Huw; Bridges, Ian G.

1988-01-01

We have used 13C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of 13C between carbons atoms 1 and 6 of [1-13C] or [6-13C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the 13C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the 13C sugars were supplied in vivo to the intact plant. The 13C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of 13C in all glucosyl carbons was quantified by 13C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background 13C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues. PMID:16666140

Molecular Data for a Biochemical Model of DNA Radiation Damage: Electron Impact Ionization and Dissociative Ionization of DNA Bases and Sugar-Phosphate Backbone

NASA Technical Reports Server (NTRS)

Dateo, Christopher E.; Fletcher, Graham D.

2004-01-01

As part of the database for building up a biochemical model of DNA radiation damage, electron impact ionization cross sections of sugar-phosphate backbone and DNA bases have been calculated using the improved binary-encounter dipole (iBED) model. It is found that the total ionization cross sections of C3'- and C5'-deoxyribose-phospate, two conformers of the sugar-phosphate backbone, are close to each other. Furthermore, the sum of the ionization cross sections of the separate deoxyribose and phosphate fragments is in close agreement with the C3'- and C5'-deoxyribose-phospate cross sections, differing by less than 10%. Of the four DNA bases, the ionization cross section of guanine is the largest, then in decreasing order, adenine, thymine, and cytosine. The order is in accordance with the known propensity of oxidation of the bases by ionizing radiation. Dissociative ionization (DI), a process that both ionizes and dissociates a molecule, is investigated for cytosine. The DI cross section for the formation of H and (cytosine-Hl)(+), with the cytosine ion losing H at the 1 position, is also reported. The threshold of this process is calculated to be 17.1 eV. Detailed analysis of ionization products such as in DI is important to trace the sequential steps in the biochemical process of DNA damage.

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moretti, Rocco; Chang, Aram; Peltier-Pain, Pauline

2012-03-15

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native d- and l-sugars (both a- and b-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the firstmore » proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.« less

Contamination of commercial cane sugars by some organic acids and some inorganic anions.

PubMed

Wojtczak, Maciej; Antczak, Aneta; Lisik, Krystyna

2013-01-01

The aim of the paper was the identification and the quantitative evaluation of the following inorganic anions: chloride, phosphate, nitrate, nitrite, sulphate and the following organic acids: lactic, acetic, formic, malic and citric in commercial "unrefined" brown cane sugars and in cane raw sugars. The determination was carried out by high performance anion exchange chromatography with conductivity detector HPAEC-CD. The conducted analyses have shown that the content of some inorganic anions and organic acids in cane sugars may be an important criterion of the quality of commercial "unrefined" brown cane sugars. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Path of Carbon in Photosynthesis XVI. Kinetic Relationships of the Intermediates in Steady State Photosynthesis

DOE R&D Accomplishments Database

Benson, A. A.; Kawaguchi, S.; Hayes, P.; Calvin, M.

1952-06-05

A kinetic study of the accumulation of C{sup 14} in the intermediates of steady state photosynthesis in C{sup 14}O{sub 2} provides information regarding the sequence of reactions involved. The work described applied the radio-chromatographic technique for analysis of the labeled early products. The simultaneous carboxylation reaction resulting in malic acid as well as phosphoglycerate is demonstrated in experiments at high light intensity. A comparison of radioactivities in a number of phosphorylated sugars as a function of time reveals concurrent synthesis of fructose and sedoheptulose phosphates followed by that of ribulose phosphates and later by that of glucose phosphates. The possibility that the cleavage of C{sub 4} compounds to C{sub 2} carbon dioxide acceptors may involve C{sub 7} and C{sub 5} sugars and evidence for this mechanism is presented.

New structures and composition of cell wall teichoic acids from Nocardiopsis synnemataformans, Nocardiopsis halotolerans, Nocardiopsis composta and Nocardiopsis metallicus: a chemotaxonomic value.

PubMed

Tul'skaya, Elena M; Shashkov, Alexander S; Streshinskaya, Galina M; Potekhina, Natalia V; Evtushenko, Ludmila I

2014-12-01

The structures of the cell wall teichoic acids (TA) from some species of the genus Nocardiopsis were established by chemical and NMR spectroscopic methods. The cell walls of Nocardiopsis synnemataformans VKM Ac-2518(T) and Nocardiopsis halotolerans VKM Ac-2519(T) both contain two TA with unique structures-poly(polyol phosphate-glycosylpolyol phosphate)-belonging to the type IV TA. In both organisms, the minor TA have identical structures: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-4 of the amino sugar. This structure is found for the first time. The major TA of N. halotolerans has a hitherto unknown structure: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate), the N-acetyl-β-galactosamine being acetalated with pyruvic acid at positions 4 and 6. The major TA of N. synnemataformans is a poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-3 of the amino sugar. The cell walls of Nocardiopsis composta VKM Ac-2520 and N. composta VKM Ac-2521(T) contain only one TA, namely 1,3-poly(glycerol phosphate) partially substituted with N-acetyl-α-glucosamine. The cell wall of Nocardiopsis metallicus VKM Ac-2522(T) contains two TA. The major TA is 1,5-poly(ribitol phosphate), each ribitol unit carrying a pyruvate ketal group at positions 2 and 4. The structure of the minor TA is the same as that of N. composta. The results presented correlate well with the phylogenetic grouping of strains and confirm the species and strain specific features of cell wall TA in members of the genus Nocardiopsis.

Enumeration of sugars and sugar alcohols hydroxyl groups by aqueous-based acetylation and MALDI-TOF mass spectrometry

USDA-ARS?s Scientific Manuscript database

A method is described for enumerating hydroxyl groups on analytes in aqueous media is described, and applied to some common polyalcohols (erythritol, mannitol, and xylitol) and selected carbohydrates. The analytes were derivatized in water with vinyl acetate in presence of sodium phosphate buffer. ...

N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

PubMed Central

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L.

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species. PMID:21655093

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

PubMed

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

Aldolase as a Chirality Intersection of L-Amino Acids and D-Sugars

NASA Astrophysics Data System (ADS)

Munegumi, Toratane

2015-06-01

Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, ( 3S, 4R, 5R), ( 3R, 4R, 5R), ( 3R, 4S, 5R), ( 3S, 4S, 5R), are expected in the nonenzymatic reaction, only the ( 3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

High-Level Production of the Low-Calorie Sugar Sorbitol by Lactobacillus plantarum through Metabolic Engineering▿

PubMed Central

Ladero, Victor; Ramos, Ana; Wiersma, Anne; Goffin, Philippe; Schanck, André; Kleerebezem, Michiel; Hugenholtz, Jeroen; Smid, Eddy J.; Hols, Pascal

2007-01-01

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed. PMID:17261519

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

PubMed

Ruiz-Baca, Estela; Villagómez-Castro, Julio C; Leal-Morales, Carlos A; Sabanero-López, Myrna; Flores-Carreón, Arturo; López-Romero, Everardo

2005-01-01

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates.

PubMed

Blayney, Michelle J; Whitney, Spencer M; Beck, Jennifer L

2011-09-01

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.

Two-dimensional HPLC coupled to ICP-MS and electrospray ionisation (ESI)-MS/MS for investigating the bioavailability in vitro of arsenic species from edible seaweed.

PubMed

Garcia-Sartal, Cristina; Taebunpakul, Sutthinun; Stokes, Emma; Barciela-Alonso, María del Carmen; Bermejo-Barrera, Pilar; Goenaga-Infante, Heidi

2012-04-01

Edible seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four seaweed namely kombu, wakame, nori and sea lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1 h sonication in a water bath followed by HPLC-ICP-MS resulted in 40-61% of the total As to be found in the buffered aqueous extract, of which 86-110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was sea lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in sea lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11-16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93-120% and 41% of the dialyzable As fraction for kombu, wakame, nori and sea lettuce, respectively. Moreover, the relative As species distribution in seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in sea lettuce samples was also confirmed.

Oxidative stress as a mechanism of added sugar-induced cardiovascular disease.

PubMed

Prasad, Kailash; Dhar, Indu

2014-12-01

Added sugars comprising of table sugar, brown sugar, corn syrup, maple syrup, honey, molasses, and other sweeteners in the prepared processed foods and beverages have been implicated in the pathophysiology of cardiovascular diseases. This article deals with the reactive oxygen species (ROS) as a mechanism of sugar-induced cardiovascular diseases. There is an association between the consumption of high levels of serum glucose with cardiovascular diseases. Various sources of sugar-induced generation of ROS, including mitochondria, nicotinamide adenine dinucleotide phosphate-oxidase, advanced glycation end products, insulin, and uric acid have been discussed. The mechanism by which ROS induce the development of atherosclerosis, hypertension, peripheral vascular disease, coronary artery disease, cardiomyopathy, heart failure, and cardiac arrhythmias have been discussed in detail. In conclusion, the data suggest that added sugars induce atherosclerosis, hypertension, peripheral vascular disease, coronary artery disease, cardiomyopathy, heart failure, and cardiac arrhythmias and that these effects of added sugars are mediated through ROS.

Inadequate awareness among chronic kidney disease patients regarding food and drinks containing artificially added phosphate.

PubMed

Shutto, Yoshiko; Shimada, Michiko; Kitajima, Maiko; Yamabe, Hideaki; Saitoh, Yoko; Saitoh, Hisao; Razzaque, Mohammed S

2013-01-01

Hyperphosphatemia is an important determinant of morbidity and mortality in patients with chronic kidney disease (CKD). Patients with CKD are advised to consume a low phosphate diet and are often prescribed phosphate-lowering drug therapy. However, commercially processed food and drinks often contain phosphate compounds, but the phosphate level is not usually provided in the ingredient list, which makes it difficult for CKD patients to choose a correct diet. We conducted a survey of the awareness of food/beverages containing artificially added phosphate among CKD patients undergoing hemodialysis. The subjects were 153 patients (77 males and 76 females; average age 56±11 years) who were randomly selected from the Dialysis Center of Hirosaki City, Japan. The subjects were provided with a list of questions. The survey results showed that 93% of the subjects were aware of the presence of high sugar content in soda, whereas only 25% were aware of the presence of phosphate (phosphoric acid) in such drinks. Despite 78% of the subjects being aware of the detrimental effects of consumption of a high phosphate diet, 43% drank at least 1 to 5 cans of soda per week and about 17% consumed "fast food" once each week. We also assessed the immediate effects of high-phosphate containing carbonated soda consumption by determining urinary calcium, phosphate, protein and sugar contents in overnight fasted healthy volunteers (n = 55; average age 20.7±0.3 years old, 20 males and 35 females). Significantly higher urinary calcium (adjusted using urinary creatinine) excretion was found 2 h after consuming 350 ml of carbonated soda compared to the fasting baseline level (0.15±0.01 vs. 0.09±0.01, p = 0.001). Our survey results suggest that CKD patients undergoing hemodialysis are not adequately aware of the hidden source of phosphate in their diet, and emphasize the need for educational initiatives to raise awareness of this issue among CKD patients.

Inadequate Awareness among Chronic Kidney Disease Patients Regarding Food and Drinks Containing Artificially Added Phosphate

PubMed Central

Shutto, Yoshiko; Shimada, Michiko; Kitajima, Maiko; Yamabe, Hideaki; Saitoh, Yoko; Saitoh, Hisao; Razzaque, Mohammed S.

2013-01-01

Hyperphosphatemia is an important determinant of morbidity and mortality in patients with chronic kidney disease (CKD). Patients with CKD are advised to consume a low phosphate diet and are often prescribed phosphate-lowering drug therapy. However, commercially processed food and drinks often contain phosphate compounds, but the phosphate level is not usually provided in the ingredient list, which makes it difficult for CKD patients to choose a correct diet. We conducted a survey of the awareness of food/beverages containing artificially added phosphate among CKD patients undergoing hemodialysis. The subjects were 153 patients (77 males and 76 females; average age 56±11 years) who were randomly selected from the Dialysis Center of Hirosaki City, Japan. The subjects were provided with a list of questions. The survey results showed that 93% of the subjects were aware of the presence of high sugar content in soda, whereas only 25% were aware of the presence of phosphate (phosphoric acid) in such drinks. Despite 78% of the subjects being aware of the detrimental effects of consumption of a high phosphate diet, 43% drank at least 1 to 5 cans of soda per week and about 17% consumed “fast food” once each week. We also assessed the immediate effects of high-phosphate containing carbonated soda consumption by determining urinary calcium, phosphate, protein and sugar contents in overnight fasted healthy volunteers (n = 55; average age 20.7±0.3 years old, 20 males and 35 females). Significantly higher urinary calcium (adjusted using urinary creatinine) excretion was found 2 h after consuming 350 ml of carbonated soda compared to the fasting baseline level (0.15±0.01 vs. 0.09±0.01, p = 0.001). Our survey results suggest that CKD patients undergoing hemodialysis are not adequately aware of the hidden source of phosphate in their diet, and emphasize the need for educational initiatives to raise awareness of this issue among CKD patients. PMID:24236030

Thermophilic ethanol fermentation from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

PubMed

Rahayu, Farida; Kawai, Yuto; Iwasaki, Yuki; Yoshida, Koichiro; Kita, Akihisa; Tajima, Takahisa; Kato, Junichi; Murakami, Katsuji; Hoshino, Tamotsu; Nakashimada, Yutaka

2017-12-01

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rates of Decomposition of Ribose and other Sugars: Implications for Chemical Evolution

NASA Technical Reports Server (NTRS)

Larralde, Rosa; Robertson, Michael P.; Miller, Stanley L.

1995-01-01

The existence of the RNA world, in which RNA acted as a catalyst as well as an informational macromolecule, assumes a large prebiotic source of ribose or the existence of pre-RNA molecules with backbones different from ribose-phosphate. The generally accepted prebiotic synthesis of ribose, the formose reaction, yields numerous sugars without any selectivity. Even if there were a selective synthesis of ribose, there is still the problem of stability. Sugars are known to be unstable in strong acid or base, but there are few data for neutral solutions. Therefore, we have measured the rate of decomposition of ribose between pH 4 and pH 8 from 40 C to 120 C. The ribose half-lives are very short (73 min at pH 7.0 and 100 C and 44 years at pH 7.0 and 0 C). The other aldopentoses and aldohexoses have half-lives within an order of magnitude of these values, as do 2-deoxyribose, ribose 5-phosphate, and ribose 2,4bisphosphate. These results suggest that the backbone of the first genetic material could not have contained ribose or other sugars because of their instability.

Biogenesis and Metabolic Maintenance of Rubisco.

PubMed

Bracher, Andreas; Whitney, Spencer M; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-04-28

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO 2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO 2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P.

PubMed

Yadav, Umesh Prasad; Ivakov, Alexander; Feil, Regina; Duan, Guang You; Walther, Dirk; Giavalisco, Patrick; Piques, Maria; Carillo, Petronia; Hubberten, Hans-Michael; Stitt, Mark; Lunn, John Edward

2014-03-01

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, has a profound influence on plant metabolism, growth, and development. It has been proposed that Tre6P acts as a signal of sugar availability and is possibly specific for sucrose status. Short-term sugar-feeding experiments were carried out with carbon-starved Arabidopsis thaliana seedlings grown in axenic shaking liquid cultures. Tre6P increased when seedlings were exogenously supplied with sucrose, or with hexoses that can be metabolized to sucrose, such as glucose and fructose. Conditional correlation analysis and inhibitor experiments indicated that the hexose-induced increase in Tre6P was an indirect response dependent on conversion of the hexose sugars to sucrose. Tre6P content was affected by changes in nitrogen status, but this response was also attributable to parallel changes in sucrose. The sucrose-induced rise in Tre6P was unaffected by cordycepin but almost completely blocked by cycloheximide, indicating that de novo protein synthesis is necessary for the response. There was a strong correlation between Tre6P and sucrose even in lines that constitutively express heterologous trehalose-phosphate synthase or trehalose-phosphate phosphatase, although the Tre6P:sucrose ratio was shifted higher or lower, respectively. It is proposed that the Tre6P:sucrose ratio is a critical parameter for the plant and forms part of a homeostatic mechanism to maintain sucrose levels within a range that is appropriate for the cell type and developmental stage of the plant.

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

PubMed

Acket, Sébastien; Degournay, Anthony; Merlier, Franck; Thomasset, Brigitte

2017-06-15

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6]. Copyright © 2017 Elsevier Inc. All rights reserved.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases.

PubMed

Decker, Daniel; Kleczkowski, Leszek A

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP- N -acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P ( K m values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P ( K m of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P ( K m of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P ( K m of 1 mM) and, to some extent, D-Glc-1-P ( K m of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

PubMed Central

Decker, Daniel; Kleczkowski, Leszek A.

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products. PMID:28970843

Short versus long term effects of cyanide on sugar metabolism and transport in dormant walnut kernels.

PubMed

Gerivani, Zahra; Vashaee, Elham; Sadeghipour, Hamid Reza; Aghdasi, Mahnaz; Shobbar, Zahra-Sadat; Azimmohseni, Majid

2016-11-01

Tree seed dormancy release by cold stratification accompanies with the embryo increased gluconeogenesis competence. Cyanide also breaks seed dormancy however, integrated information about its effects on carbon metabolism is lacking. Accordingly, the impacts of HCN on germination, lipid gluconeogenesis and sugar transport capacity of walnut (Juglans regia L.) kernels were investigated during 10-days period prior to radicle protrusion. HCN increased walnut kernel germination and within four days of kernel incubation, hastened the decline of starch, reducing and non-reducing sugars and led to greater activities of alkaline invertase and glucose-6-phosphate dehydrogenase. From four days of kernel incubation onwards, starch and non-reducing sugars accumulated only in the HCN treated axes. Cyanide also increased the activities of phosphoenolpyruvate carboxykinase and glyoxysomal succinate oxidase and led to greater acid invertase activity during the aforementioned period. The expressions of both sucrose transporter (JrSUT1) and H + -ATPase (JrAHA1) genes especially in cotyledons and H + -ATPase activity in kernels were significantly enhanced by exposure to cyanide. Thus in short-term HCN led to prevalence of carbohydrate catabolic events such as oxidative pentose phosphate pathway and possibly glycolysis in dormant walnut kernels. Long-term effects however, are increased gluconeogenesis and enhanced sugar transport capacity of kernels as a prerequisite for germination. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

PubMed

Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

2016-05-01

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

PubMed Central

Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

2016-01-01

Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

PubMed Central

Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M. C.; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Alam, Mohammad Tauqeer; Keller, Markus A.; Breitenbach, Michael; Brindle, Kevin M.; Rabinowitz, Joshua D.; Ralser, Markus

2015-01-01

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism. PMID:25243985

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

PubMed

Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

2016-04-01

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development

PubMed Central

Katz, Ehud; Boo, Kyung Hwan; Kim, Ho Youn; Eigenheer, Richard A.; Phinney, Brett S.; Shulaev, Vladimir; Negre-Zakharov, Florence; Sadka, Avi; Blumwald, Eduardo

2011-01-01

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development. PMID:21841177

Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum.

PubMed

Reynolds, E C; Cai, F; Shen, P; Walker, G D

2003-03-01

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes incorporated into sugar-free chewing gum have been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to compare the ability of CPP-ACP, with that of other forms of calcium, to be retained in supragingival plaque and remineralize enamel subsurface lesions in situ when delivered in a mouthrinse or sugar-free gum in randomized, double-blind trials. In the mouthrinse study, only the CPP-ACP-containing mouthrinse significantly increased plaque calcium and inorganic phosphate levels, and the CPP were immunolocalized to the surfaces of bacterial cells as well as the intercellular matrix. In the chewing gum studies, the gum containing the CPP-ACP, although not containing the most calcium per piece of gum, produced the highest level of enamel remineralization independent of gum-chewing frequency and duration. The CPP could be detected in plaque extracts 3 hrs after subjects chewed the CPP-ACP-containing gum. The results showed that CPP-ACP were superior to other forms of calcium in remineralizing enamel subsurface lesions.

Response surface optimization of the critical medium components for pullulan production by Aureobasidium pullulans FB-1.

PubMed

Singh, Ram Sarup; Singh, Harpreet; Saini, Gaganpreet Kaur

2009-01-01

Culture conditions for pullulan production by Aureobasidium pullulans were optimized using response surface methodology at shake flask level without pH control. In the present investigation, a five-level with five-factor central composite rotatable design of experiments was employed to optimize the levels of five factors significantly affecting the pullulan production, biomass production, and sugar utilization in submerged cultivation. The selected factors included concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride. Using this methodology, the optimal values for concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were 5.31%, 0.11%, 0.07%, 0.05%, and 0.15% (w/v), respectively. This optimized medium has projected a theoretically production of pullulan of 4.44%, biomass yield of 1.03%, and sugar utilization of 97.12%. The multiple correlation coefficient 'R' was 0.9976, 0.9761 and 0.9919 for pullulan production, biomass production, and sugar utilization, respectively. The value of R being very close to one justifies an excellent correlation between the predicted and the experimental data.

Dinuclear copper(II) complexes with {Cu2(mu-hydroxo)bis(mu-carboxylato)}+ cores and their reactions with sugar phosphate esters: A substrate binding model of fructose-1,6-bisphosphatase.

PubMed

Kato, Merii; Tanase, Tomoaki; Mikuriya, Masahiro

2006-04-03

Reactions of CuX2.nH2O with the biscarboxylate ligand XDK (H2XDK = m-xylenediamine bis(Kemp's triacid imide)) in the presence of N-donor auxiliary ligands yielded a series of dicopper(II) complexes, [Cu2(mu-OH)(XDK)(L)2]X (L = N,N,N',N'-tetramethylethylenediamine (tetmen), X = NO3 (1a), Cl (1b); L = N,N,N'-trimethylethylenediamine (tmen), X = NO3 (2a), Cl (2b); L =2,2'-bipyridine (bpy), X = NO3 (3); L = 1,10-phenanthroline (phen), X = NO3 (4); L = 4,4'-dimethyl-2,2'-bipyridine (Me2bpy), X = NO3 (5); L = 4-methyl-1,10-phenanthroline (Mephen), X = NO3 (6)). Complexes 1-6 were characterized by X-ray crystallography (Cu...Cu = 3.1624(6)-3.2910(4) A), and the electrochemical and magnetic properties were also examined. Complexes 3 and 4 readily reacted with diphenyl phosphoric acid (HDPP) or bis(4-nitrophenyl) phosphoric acid (HBNPP) to give [Cu2(mu-phosphate)(XDK)(L)2]NO3 (L = bpy, phosphate = DPP (11); L = phen, phosphate = DPP (12), BNPP (13)), where the phsophate diester bridges the two copper ions in a mu-1,3-O,O' bidentate fashion (Cu...Cu = 4.268(3)-4.315(1) A). Complexes 4 and 6 with phen and Mephen have proven to be good precursors to accommodate a series of sugar monophosphate esters (Sugar-P) onto the biscarboxylate-bridged dicopper centers, yielding [Cu2(mu-Sugar-P)(XDK)(L)2] (Sugar-P = alpha-D-Glc-1-P (23a and b), D-Glc-6-P (24a and b), D-Man-6-P (25a), D-Fru-6-P (26a and b); L = phen (a), Mephen (b)) and [Cu2(mu-Gly-n-P)(XDK)(Mephen)2] (Gly-n-P = glycerol n-phosphate; n = 2 (21), 3 (22)), where Glc, Man, and Fru are glucose, mannose, and fructose, respectively. The structure of [Cu2(mu-MNPP)(XDK)(phen)2(CH3OH)] (20) was characterized as a reference compound (H2MNPP = 4-nitrophenyl phosphoric acid). Complexes 4 and 6 also reacted with d-fructose 1,6-bisphosphate (D-Fru-1,6-P2) to afford the tetranuclear copper(II) complexes formulated as [Cu4(mu-D-Fru-1,6-P2)(XDK)2(L)4] (L = phen (27a), Mephen (27b)). The detailed structure of 27a was determined by X-ray crystallography to involve two different tetranuclear complexes with alpha- and beta-anomers of D-Fru-1,6-P2, [Cu4(mu-alpha-D-Fru-1,6-P2)(XDK)2(phen)4] and [Cu4(mu-beta-D-Fru-1,6-P2)(XDK)2(phen)4], in which the D-Fru-1,6-P2 tetravalent anion bridges the two [Cu2(XDK)(phen)2]2+ units through the C1 and C6 phosphate groups in a mu-1,3-O,O' bidentate fashion (Cu...Cu = 4.042(2)-4.100(2) A). Notably, the structure with alpha-D-Fru-1,6-P2 demonstrated the presence of a strong hydrogen bond between the C2 hydroxyl group and the C1 phosphate oxygen atom, which may support the previously proposed catalytic mechanism in the active site of fructose-1,6-bisphosphatase.

Dietary phosphorus burden increases cariogenesis independent of vitamin D uptake.

PubMed

Goodson, J Max; Shi, Ping; Mumena, Chrispinus H; Haq, Afrozul; Razzaque, Mohammed S

2017-03-01

An association between excessive sugar consumption and dental decay, particularly in children, has been well-established. In this study we have analyzed whether consumption of phosphorus, an important ingredient of sugary drinks, has any association with the evolvement of dental decay. Food consumption, gingival redness and dental decay were evaluated in a total of 8,317 children with the mean age of 9.99+0.68 years with a strong gender bias (p<0.0005); boys being more affected than girls. Our results showed a significant association (p=0.044) between dental decay and calorie-adjusted sugar intake. Presence of gingival inflammation also correlated (p=0.008) with the formation of dental decay. In addition, decayed teeth were positively associated with increased salivary levels of adiponectin (p=0.0002) and matrix degrading MMP-9 (p=0.015), while negatively associated with salivary levels of the vascular endothelial growth factor (VEGF; p=0.008). More importantly, we found a statistically significant correlation (p=0.0008) between calorie-adjusted dietary phosphorus intake and occurrence of dental decay. Our analyzed results also showed a significantly high percentage of dental decay in children who consumed a diet, low in sugar but high in phosphorus (6.58%, n=661), compared to those who consumed a low sugar and low phosphorus containing diet (5.02%, n=413). Finally, we did not find any significant association between vitamin D uptake and the genesis of dental decay. From these results, we concluded that both high sugar and high phosphate consumption can influence evolvement of dental decay, and that, a healthier diet could be achieved by reducing consumption of dental cariogenic dietary factors, sugar and phosphate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism

PubMed Central

Muñoz-Bertomeu, Jesús; Bermúdez, María Angeles; Segura, Juan; Ros, Roc

2011-01-01

Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions. PMID:21068209

Methods for the determination of intracellular levels of ribose phosphates.

PubMed

Camici, Marcella; Tozzi, Maria Grazia; Ipata, Piero Luigi

2006-10-31

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes.

PubMed

Howlett, Robert; Anttonen, Katri; Read, Nicholas; Smith, Margaret C M

2018-04-01

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1 - mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1 - strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Metabolomics of Clostridial Biofuel Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rabinowitz, Joshua D; Aristilde, Ludmilla; Amador-Noguez, Daniel

2015-09-08

Members of the genus Clostridium collectively have the ideal set of the metabolic capabilities for fermentative biofuel production: cellulose degradation, hydrogen production, and solvent excretion. No single organism, however, can effectively convert cellulose into biofuels. Here we developed, using metabolomics and isotope tracers, basic science knowledge of Clostridial metabolism of utility for future efforts to engineer such an organism. In glucose fermentation carried out by the biofuel producer Clostridium acetobutylicum, we observed a remarkably ordered series of metabolite concentration changes as the fermentation progressed from acidogenesis to solventogenesis. In general, high-energy compounds decreased while low-energy species increased during solventogenesis. Thesemore » changes in metabolite concentrations were accompanied by large changes in intracellular metabolic fluxes, with pyruvate directed towards acetyl-CoA and solvents instead of oxaloacetate and amino acids. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources from biomass production into solvent production. In contrast to C. acetobutylicum, which is an avid fermenter, C. cellulolyticum metabolizes glucose only slowly. We find that glycolytic intermediate concentrations are radically different from fast fermenting organisms. Associated thermodynamic and isotope tracer analysis revealed that the full glycolytic pathway in C. cellulolyticum is reversible. This arises from changes in cofactor utilization for phosphofructokinase and an alternative pathway from phosphoenolpyruvate to pyruvate. The net effect is to increase the high-energy phosphate bond yield of glycolysis by 150% (from 2 to 5) at the expense of lower net flux. Thus, C. cellulolyticum prioritizes glycolytic energy efficiency over speed. Degradation of cellulose results in other sugars in addition to glucose. Simultaneous feeding of stable isotope-labeled glucose and unlabeled pentose sugars (xylose or arabinose) to C. acetobutylicum revealed that, as expected, glucose was preferred, with the pentose sugar selectively assimilated into the pentose phosphate pathway (PPP). Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among these pentose sugars, with arabinose utilized preferentially over xylose. Pentose catabolism occurred via the phosphoketolase pathway (PKP), an alternative route of pentose catabolism that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate. Taken collectively, these findings reveal two hierarchies in Clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP. Thus, in addition to massively expanding the available data on Clostridial metabolism, we identified three key regulatory points suitable for targeting in future bioengineering efforts: phosphofructokinase for enhancing fermentation, the pyruvate-oxaloacetate node for controlling solventogenesis, and the phosphoketolase reaction for driving pentose catabolism.« less

Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

NASA Technical Reports Server (NTRS)

Degani, CH.; Kawatsuji, M.; Halmann, M.

1975-01-01

It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

The Calvin Cycle Inevitably Produces Sugar-Derived Reactive Carbonyl Methylglyoxal During Photosynthesis: A Potential Cause of Plant Diabetes

PubMed Central

Takagi, Daisuke; Inoue, Hironori; Odawara, Mizue; Shimakawa, Ginga; Miyake, Chikahiro

2014-01-01

Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O2 evolution (R = 0.88; P < 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves. PMID:24406631

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

PubMed Central

Fraenkel, D. G.; Banerjee, Santimoy

1972-01-01

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf). PMID:4560065

Alkaline Hydrothermal Vents as Electrochemical Reactors Driving an Auto-Trophic Origin of Life

NASA Astrophysics Data System (ADS)

Camprubi, E.; Lane, N.

2017-07-01

We report the reduction of CO2 to formaldehyde under simulated alkaline hydrothermal conditions. Formaldehyde is transformed into relevant sugars via the formose reaction. Acetyl phosphate can also be synthesised and phosphorylates organic molecules.

Lethality of a Heat- and Phosphate-Catalyzed Glucose By-Product to Escherichia coli O157:H7 and Partial Protection Conferred by the rpoS Regulon

PubMed Central

Byrd, Jeffrey J.; Cheville, Ann M.; Bose, Jeffrey L.; Kaspar, Charles W.

1999-01-01

A by-product of glucose produced during sterilization (121°C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25°C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25°C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4°C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media. PMID:10347019

Two Site-Directed Mutations are Required for the Conversion of a Sugar Dehydratase into an Aminotransferase¶

PubMed Central

Cook, Paul D.; Kubiak, Rachel L.; Toomey, Daniel P.; Holden, Hazel M.

2009-01-01

l-colitose and d-perosamine are unusual sugars found in the O-antigens of some Gram-negative bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella enterica, among others. The biosynthetic pathways for these two sugars begin with the formation of GDP-mannose from d-mannose-1-phosphate and GTP followed by the subsequent dehydration and oxidation of GDP-mannose to yield GDP-4-keto-6-deoxymannose. Following the production of GDP-4-keto-6-deoxymannose, the two pathways diverge. In the case of GDP-perosamine biosynthesis, the next step involves an amination reaction at the C-4′ position of the sugar, whereas in GDP-colitose production, the 3′-hydroxyl group is removed. The enzymes catalyzing these reactions are GDP-perosamine synthase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), respectively. Both of these enzymes are pyridoxal-5′-phosphate (PLP)-dependent and their three-dimensional structures place them into the well-characterized aspartate aminotransferase superfamily. A comparison of the active site architecture of ColD from Escherichia coli (Strain 5a, type O55:H7) to that of GDP-perosamine synthase from Caulobacter crescentus CB15, suggested that only two mutations would be required to convert ColD into an aminotransferase. Here we present a combined structural and functional analysis of the ColD S187N/H188K mutant protein that, indeed, has been converted from a dehydratase into an aminotransferase. PMID:19402712

Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

PubMed Central

Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

2012-01-01

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

Phosphate Solubilization and Gene Expression of Phosphate-Solubilizing Bacterium Burkholderia multivorans WS-FJ9 under Different Levels of Soluble Phosphate.

PubMed

Zeng, Qingwei; Wu, Xiaoqin; Wang, Jiangchuan; Ding, Xiaolei

2017-04-28

Phosphate-solubilizing bacteria (PSB) have the ability to dissolve insoluble phosphate and enhance soil fertility. However, the growth and mineral phosphate solubilization of PSB could be affected by exogenous soluble phosphate and the mechanism has not been fully understood. In the present study, the growth and mineral phosphate-solubilizing characteristics of PSB strain Burkholderia multivorans WS-FJ9 were investigated at six levels of exogenous soluble phosphate (0, 0.5, 1, 5, 10, and 20 mM). The WS-FJ9 strain showed better growth at high levels of soluble phosphate. The phosphate-solubilizing activity of WS-FJ9 was reduced as the soluble phosphate concentration increased, as well as the production of pyruvic acid. Transcriptome profiling of WS-FJ9 at three levels of exogenous soluble phosphate (0, 5, and 20 mM) identified 446 differentially expressed genes, among which 44 genes were continuously up-regulated when soluble phosphate concentration was increased and 81 genes were continuously down-regulated. Some genes related to cell growth were continuously up-regulated, which would account for the better growth of WS-FJ9 at high levels of soluble phosphate. Genes involved in glucose metabolism, including glycerate kinase, 2-oxoglutarate dehydrogenase, and sugar ABC-type transporter, were continuously down-regulated, which indicates that metabolic channeling of glucose towards the phosphorylative pathway was negatively regulated by soluble phosphate. These findings represent an important first step in understanding the molecular mechanisms of soluble phosphate effects on the growth and mineral phosphate solubilization of PSB.

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

PubMed

Vincent, Florence; Yates, David; Garman, Elspeth; Davies, Gideon J; Brannigan, James A

2004-01-23

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

Entropy and charge in molecular evolution--the case of phosphate

NASA Technical Reports Server (NTRS)

Arrhenius, G.; Sales, B.; Mojzsis, S.; Lee, T.; Bada, J. L. (Principal Investigator)

1997-01-01

Biopoesis, the creation of life, implies molecular evolution from simple components, randomly distributed and in a dilute state, to form highly organized, concentrated systems capable of metabolism, replication and mutation. This chain of events must involve environmental processes that can locally lower entropy in several steps; by specific selection from an indiscriminate mixture, by concentration from dilute solution, and in the case of the mineral-induced processes, by particular effectiveness in ordering and selective reaction, directed toward formation of functional biomolecules. Numerous circumstances provide support for the notion that negatively charged molecules were functionally required and geochemically available for biopoesis. Sulfite ion may have been important in bisulfite complex formation with simple aldehydes, facilitating the initial concentration by sorption of aldehydes in positively charged surface active minerals. Borate ion may have played a similar, albeit less investigated role in forming charged sugar complexes. Among anionic species, oligophosphate ions and charged phosphate esters are likely to have been of even more wide ranging importance, reflected in the continued need for phosphate in a proposed RNA world, and extending its central role to evolved biochemistry. Phosphorylation is shown to result in selective concentration by surface sorption of compounds, otherwise too dilute to support condensation reactions. It provides protection against rapid hydrolysis of sugars and, by selective concentration, induces the oligomerization of aldehydes. As a manifestation of life arisen, phosphate already appears in an organic context in the oldest preserved sedimentary record.

Carbohydrate and energy-yielding metabolism in non-conventional yeasts.

PubMed

Flores, C L; Rodríguez, C; Petit, T; Gancedo, C

2000-10-01

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.

The activity of Rubisco's molecular chaperone, Rubisco activase, in leaf extracts

USDA-ARS?s Scientific Manuscript database

Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the “molecular chiropractic” activity of Rubisco activase (activase). To make the study of activas...

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

1998-03-10

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant Zymomonas

DOEpatents

Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

1998-01-27

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant zymomonas

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Physiological role of glucose-6-phosphate dehydrogenase in cold acclimation of strawberry (Fragaria × ananassa)

NASA Astrophysics Data System (ADS)

Zhang, Yong; Yu, Dingqun; Luo, Ya; Wang, Xiaorong; Chen, Qing; Sun, Bo; Wang, Yan; Liu, Zejing; Tang, Haoru

2018-04-01

In recent years, there has been an increasing interest in study of new resistance mechanism in fruit trees. All these regard the climate change and subsequent fruit production. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway (OPPP), and the expression of this enzyme is related to different biotic and abiotic stresses. Under accumulation of low temperature stress, the significant increase in G6PDH activity was found to be closely correlated to the levels of antioxidant enzymes, malondialdehyde (MDA) contents, sugar contents as well as changes of superoxide (O2•-). It is suggested that the enhancement of cold resistance of strawberry, which induced by cold acclimation, related to the significant increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate reactive oxygen species (ROS); on the other hand, it may be involved in the activation of antioxidant enzymes, and accelerates many other important NADPH-dependent enzymatic reactions. Then further result in the elevation of membrane stability and cold resistance of strawberry. Interestingly, even though the plants were placed again under a temperature of 25°C for 1 d, the higher cold resistance, enzyme activities and soluble sugar content acquired.

Reaction of cytidine nucleotides with cyanoacetylene: support for the intermediacy of nucleoside-2',3'-cyclic phosphates in the prebiotic synthesis of RNA.

PubMed

Crowe, Michael A; Sutherland, John D

2006-06-01

A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5'-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2',3'-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2',3'-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2'- and 3'-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5'-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2'p and C3'p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA.

Ionization Cross Sections and Dissociation Channels of the DNA Sugar-Phosphate Backbone by Electron Collisions

NASA Technical Reports Server (NTRS)

Dateo, Christopher; Huo, Winifred M.; Fletcher, Graham D.

2004-01-01

It has been suggested that the genotoxic effects of ionizing radiation in living cells are not caused by the highly energetic incident radiation, but rather are induced by less energetic secondary species generated, the most abundant of which are free electrons.' The secondary electrons will further react to cause DNA damage via indirect and direct mechanisms. Detailed knowledge of these mechanisms is ultimately important for the development of global models of cellular radiation damage. We are studying one possible mechanism for the formation cf DNA strand breaks involving dissociative ionization of the DNA sugar-phosphate backbone induced by secondary electron co!lisions. We will present ionization cross sections at electron collision energies between threshold and 10 KeV using the improved binary encounter dipole (iBED) formulation' Preliminary results of the possible dissociative ionization pathways will be presented. It is speculated that radical fragments produced from the dissociative ionization can further react, providing a possible mechanism for double strand breaks and base damage.

Changes in sugar content and related enzyme activities in table grape (Vitis vinifera L.) in response to foliar selenium fertilizer.

PubMed

Zhu, Shuaimeng; Liang, Yinli; An, Xiaojuan; Kong, Fanchao; Gao, Dekai; Yin, Hongfei

2017-09-01

Spraying selenium (Se) fertilizer is an effective method for Se-enriched fruit production. Sugar content in fruit is the major factor determining berry quality. However, changes in sugar metabolism in response to Se fertilizer are unclear. Hence, this study was conducted to identify the effects of Se fertilizer on sugar metabolism and related enzyme activities of grape berries. Additionally, production of leaves with and without Se fertilizer was also investigated. Acid invertase (AI) activity, total soluble sugar and Se content in berries, and photosynthetic rate in leaves produced under Se fertilizer treatments were higher than that of control. Glucose and fructose were the primary sugars in berries, with a trace of sucrose. In both berries and leaves, neutral invertase activity was lower than AI, there was no significant difference in neutral invertase, sucrose synthase and sucrose phosphate synthase between Se fertilizer-treated and control. In berries, AI showed a significant positive correlation with glucose and fructose; also Se content was significantly correlated with sugar content. AI played an important role in the process of sugar accumulation in berries; high AI activity in berries and photosynthetic rate in leaves could explain the mechanism by which Se fertilizer affected sugar accumulation in berries. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

NASA Astrophysics Data System (ADS)

Coggins, Adam J.; Powner, Matthew W.

2017-04-01

Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions.

Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit.

PubMed

Dai, Zhan Wu; Léon, Céline; Feil, Regina; Lunn, John E; Delrot, Serge; Gomès, Eric

2013-03-01

Changes in carbohydrate metabolism during grape berry development play a central role in shaping the final composition of the fruit. The present work aimed to identify metabolic switches during grape development and to provide insights into the timing of developmental regulation of carbohydrate metabolism. Metabolites from central carbon metabolism were measured using high-pressure anion-exchange chromatography coupled to tandem mass spectrometry and enzymatic assays during the development of grape berries from either field-grown vines or fruiting cuttings grown in the greenhouse. Principal component analysis readily discriminated the various stages of berry development, with similar trajectories for field-grown and greenhouse samples. This showed that each stage of fruit development had a characteristic metabolic profile and provided compelling evidence that the fruit-bearing cuttings are a useful model system to investigate regulation of central carbon metabolism in grape berry. The metabolites measured showed tight coordination within their respective pathways, clustering into sugars and sugar-phosphate metabolism, glycolysis, and the tricarboxylic acid cycle. In addition, there was a pronounced shift in metabolism around veraison, characterized by rapidly increasing sugar levels and decreasing organic acids. In contrast, glycolytic intermediates and sugar phosphates declined before veraison but remained fairly stable post-veraison. In summary, these detailed and comprehensive metabolite analyses revealed the timing of important switches in primary carbohydrate metabolism, which could be related to transcriptional and developmental changes within the berry to achieve an integrated understanding of grape berry development. The results are discussed in a meta-analysis comparing metabolic changes in climacteric versus non-climacteric fleshy fruits.

Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit

PubMed Central

Gomès, Eric

2013-01-01

Changes in carbohydrate metabolism during grape berry development play a central role in shaping the final composition of the fruit. The present work aimed to identify metabolic switches during grape development and to provide insights into the timing of developmental regulation of carbohydrate metabolism. Metabolites from central carbon metabolism were measured using high-pressure anion-exchange chromatography coupled to tandem mass spectrometry and enzymatic assays during the development of grape berries from either field-grown vines or fruiting cuttings grown in the greenhouse. Principal component analysis readily discriminated the various stages of berry development, with similar trajectories for field-grown and greenhouse samples. This showed that each stage of fruit development had a characteristic metabolic profile and provided compelling evidence that the fruit-bearing cuttings are a useful model system to investigate regulation of central carbon metabolism in grape berry. The metabolites measured showed tight coordination within their respective pathways, clustering into sugars and sugar-phosphate metabolism, glycolysis, and the tricarboxylic acid cycle. In addition, there was a pronounced shift in metabolism around veraison, characterized by rapidly increasing sugar levels and decreasing organic acids. In contrast, glycolytic intermediates and sugar phosphates declined before veraison but remained fairly stable post-veraison. In summary, these detailed and comprehensive metabolite analyses revealed the timing of important switches in primary carbohydrate metabolism, which could be related to transcriptional and developmental changes within the berry to achieve an integrated understanding of grape berry development. The results are discussed in a meta-analysis comparing metabolic changes in climacteric versus non-climacteric fleshy fruits. PMID:23364938

Purification of Rubisco Activase from Leaves or after Expression in Escherichia coli.

USDA-ARS?s Scientific Manuscript database

Rubisco activase is a molecular chaperone that modulates the activation state of Rubisco by catalyzing the ATP-dependent removal of tightly-bound inhibitory sugar-phosphates from Rubisco’s catalytic sites. This chapter reports methods developed for the purification of native and recombinant Rubisco...

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

PubMed Central

Bolton, P. G.; Dean, A. C. R.

1972-01-01

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined. PMID:4342213

Pentose phosphates in nucleoside interconversion and catabolism.

PubMed

Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L

2006-03-01

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars.

PubMed

Popowska, Magdalena; Osińska, Magdalena; Rzeczkowska, Magdalena

2012-04-01

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

Effects of microbial phytase on apparent and standardized total tract digestibility of calcium in calcium supplements fed to growing pigs.

PubMed

González-Vega, J C; Walk, C L; Stein, H H

2015-05-01

An experiment was conducted to test the hypothesis that differences in the apparent total tract digestibility (ATTD) and standardized total tract digestibility (STTD) of Ca exist among Ca supplements and that inclusion of microbial phytase increases the ATTD and STTD of Ca. One hundred and four growing barrows (average initial BW of 17.73 ± 2.53 kg) were allotted to a randomized complete block design with 13 dietary treatments and 8 pigs per treatment. A basal diet containing corn, cornstarch, potato protein isolate, soybean oil, calcium carbonate, monosodium phosphate, vitamins, and minerals was formulated. Five additional diets were formulated by adding monocalcium phosphate (MCP), dicalcium phosphate (DCP), calcium carbonate, Lithothamnium calcareum Ca, or a high-Ca sugar beet co-product to the basal diet at the expense of cornstarch. Six additional diets that were similar to the previous 6 diets with the exception that they also contained 500 units per kilogram of microbial phytase were also formulated. A Ca-free diet was used to determine basal endogenous losses of Ca. Feces were collected using the marker-to-marker approach. Results indicated that regardless of inclusion of microbial phytase, MCP had the greatest (P < 0.05) ATTD and STTD of Ca. The ATTD and STTD of Ca in DCP were greater (P < 0.05) than in calcium carbonate, L. calcareumC a, or in the sugar beet co-product, but no differences were observed among the ATTD and STTD of Ca in calcium carbonate, L. calcareum Ca, or sugar beet co-product. Inclusion of microbial phytase increased (P < 0.05) the ATTD and STTD of Ca in the diets, but this was not the case in the Ca supplements. Regardless of inclusion of microbial phytase, the ATTD of P was greater ( P< 0.05) in pigs fed basal, MCP, or DCP diets than in pigs fed calcium carbonate, L. calcareum Ca, or the sugar beet co-product, but pigs fed calcium carbonate diets had greater ( P< 0.05) ATTD of P than pigs fed L. calcareumCa or the sugar beet co-product. Regardless of Ca source, inclusion of microbial phytase increased (P < 0.001) the ATTD of P. In conclusion, MCP has the greatest ATTD and STTD of Ca among the calcium supplements used in this experiment, followed by DCP. Basal, MCP, and DCP diets had greater ATTD of P than the other diets, and inclusion of microbial phytase increased the ATTD and STTD of Ca and the ATTD of P in the diets.

Changes in the sugar content of sweet sorghum stems under natural conditions during winter in saline soil of the Yellow River Delta

NASA Astrophysics Data System (ADS)

Li, Ying; Yuan, Fang; Wang, Baoshan

2018-02-01

In order to investigate the maximum storage period during their natural growth state, the sweet sorghum (Sorghum bicolor L. Moench) stems of four cultivars were analyzed to determine changes in contents of water, total sugars, main soluble sugars and the enzyme activity. From early November 2016 to late January 2017, the decrease in the total sugar content and the contents of sucrose, glucose and fructose slowed down, and the enzyme activities (sucrose synthase and sucrose phosphate synthase) involving sucrose metabolism in the stem remained stable. However, these indicators decreased significantly after the end of January 2017. Low temperatures and a dry environment were conducive to the storage of the sweet sorghum stems. During the winter (from early November 2016 to late January 2017) in northern China, the sweet sorghum plants can be stored naturally in the field via regulating sowing dates, which saves a lot of storage space and production costs for bioethanol company.

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

PubMed Central

Bouma, C L; Reizer, J; Reizer, A; Robrish, S A; Thompson, J

1997-01-01

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum. PMID:9209025

Quantitative trait loci, epigenetics, sugars, and microRNAs: quaternaries in phosphate acquisition and use

USDA-ARS?s Scientific Manuscript database

Phosphorus (P) is a critical element for plant growth and is frequently the limiting nutrient in many soils. Continued production and application of P fertilizer relies on a nonrenewable resource which will peak in about 2050. This will result in significantly increased cost, particularly for develo...

On-The-Move Nutrient Delivery System (NDS): User Acceptability of Rotary Flow Control Version

DTIC Science & Technology

2009-04-01

Following: Salt, Sodium Citrate, Monopotassium Phosphate, Sodium Benzoate , Potassium Sorbate, FD & C Yellow #5. Faslln,,, Flow Mallifold 126.00 g (4.44 oz...Fat Cholesterol Sodium 440mg Potassium Total Carbohydrate Dietary Fiber Sugars Protein Vitamin A Vitamin C Calcium lron Phosphorus 260 o Og Og Omg 18

Hawkmoths use nectar sugar to reduce oxidative damage from flight.

PubMed

Levin, E; Lopez-Martinez, G; Fane, B; Davidowitz, G

2017-02-17

Nectar-feeding animals have among the highest recorded metabolic rates. High aerobic performance is linked to oxidative damage in muscles. Antioxidants in nectar are scarce to nonexistent. We propose that nectarivores use nectar sugar to mitigate the oxidative damage caused by the muscular demands of flight. We found that sugar-fed moths had lower oxidative damage to their flight muscle membranes than unfed moths. Using respirometry coupled with δ 13 C analyses, we showed that moths generate antioxidant potential by shunting nectar glucose to the pentose phosphate pathway (PPP), resulting in a reduction in oxidative damage to the flight muscles. We suggest that nectar feeding, the use of PPP, and intense exercise are causally linked and have allowed the evolution of powerful fliers that feed on nectar. Copyright © 2017, American Association for the Advancement of Science.

Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9.

PubMed

Lin, I Winnie; Sosso, Davide; Chen, Li-Qing; Gase, Klaus; Kim, Sang-Gyu; Kessler, Danny; Klinkenberg, Peter M; Gorder, Molly K; Hou, Bi-Huei; Qu, Xiao-Qing; Carter, Clay J; Baldwin, Ian T; Frommer, Wolf B

2014-04-24

Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

The Effect of Lipopolysaccharide Core Oligosaccharide Size on the Electrostatic Binding of Antimicrobial Proteins to Models of the Gram Negative Bacterial Outer Membrane

PubMed Central

2016-01-01

Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate-rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different-sized core oligosaccharide regions we demonstrate the role of uncharged sugars in blocking short-range electrostatic interactions between the cationic antibiotics and the vulnerable anionic phosphate groups. PMID:27003358

Sucrose dependent mineral phosphate solubilization in Enterobacter asburiae PSI3 by heterologous overexpression of periplasmic invertases.

PubMed

Kumar, Chanchal; Wagh, Jitendra; Archana, G; Naresh Kumar, G

2016-12-01

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.

Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

PubMed

Hithell, Gordon; Shaw, Daniel J; Donaldson, Paul M; Greetham, Gregory M; Towrie, Michael; Burley, Glenn A; Parker, Anthony W; Hunt, Neil T

2016-05-05

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

Content and Vacuole/Extravacuole Distribution of Neutral Sugars, Free Amino Acids, and Anthocyanin in Protoplasts 1

PubMed Central

Wagner, George J.

1979-01-01

Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. γ-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents. Images PMID:16660921

Single Zymomonas mobilis strain for xylose and arabinose fermentation

DOEpatents

Zhang, M.; Chou, Y.C.; Picataggio, S.K.; Finkelstein, M.

1998-12-01

This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol. 6 figs.

Single zymomonas mobilis strain for xylose and arabinose fermentation

DOEpatents

Zhang, Min; Chou, Yat-Chen; Picataggio, Stephen K.; Finkelstein, Mark

1998-01-01

This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol.

The Path of Carbon in Photosynthesis IV. The Identity and Sequence of the Intermediates in Sucrose Synthesis

DOE R&D Accomplishments Database

Calvin, M.; Benson, A.

1948-12-14

The synthesis of sucrose from C{sup 14}0{sub 2} by green algae has been investigated and the intermediates separated by the method of paper chromatography. It is shown that sucrose is the first free sugar appearing during photosynthesis. It is apparently formed by condensation of the glucose-I-phosphate and a fructose phosphate. A series of radioautographs of paper chromatograms of extracts from plants which have photosynthesized for different periods of time has been prepared. The results indicate that 2-phosphoglyceric acid is the first product synthesized from C0{sub 2} during photosynthesis.

Dietary fat mediates hyperglycemia and the glucogenic response to increased protein consumption in an insect, Manduca sexta L.

PubMed

Thompson, S N

2004-08-04

Many insects display non-homeostatic regulation over blood sugar level. The concentration of trehalose varies dramatically depending on physiological and nutritional state. In the absence of dietary carbohydrate, blood trehalose in larvae of the lepidopteran insect Manduca sexta is maintained by gluconeogenesis and is dependent on dietary protein consumption. In the present study, the effect of dietary fat on the glucogenic response of insects to increased dietary protein was examined by NMR analysis of (2-13C)pyruvate metabolism. Last instar larvae were maintained on a carbohydrate-free chemically defined artificial diet having variable levels of casein with and without corn oil. Gluconeogenic flux, the ratio of the rate of gluconeogenesis to the rate of glycolysis, was estimated from the 13C distribution in trehalose arising by gluconeogenesis and the 13C enrichment of alanine due to pyruvate cycling. Insects grew well on carbohydrate-free diets and growth increased with increasing dietary protein level. At all dietary protein levels, larvae grew better on diets with fat. Without dietary fat, larvae were glucogenic but displayed low blood trehalose concentrations, <30 mM, regardless of protein consumption. When fat was included in the diet, however, gluconeogenic flux and blood trehalose level increased sharply in response to increased dietary protein level, with trehalose concentrations >50 mM at higher levels of protein consumption. When offered a choice of a high carbohydrate and a high protein diet, larvae maintained on diets with fat displayed a food preference related to blood sugar level. Those with low blood sugar fed on carbohydrate, while those with high blood sugar preferred protein. Trehalose synthesized from (2-13C)pyruvate exhibited asymmetry in the 13C distribution in individual glucose molecules, indicating a disequilibrium at the triose phosphate isomerase-catalyzed step of the gluconeogenic pathway. In trehalose from larvae on diets with fat, the asymmetric 13C distribution was higher than in trehalose from insects on diets lacking fat. This may partially result from isotopic disequilibrium when unenriched glycerol is metabolized to dihydroxyacetone phosphate following fat hydrolysis. The asymmetry in 13C distribution, however, also occurred in insects on diets without fat and decreased with increased gluconeogenic flux suggesting that true disequilibrium between the triose phosphates is the principal reason for the asymmetry.

Convergent Starvation Signals and Hormone Crosstalk in Regulating Nutrient Mobilization upon Germination in Cereals[C][W

PubMed Central

Hong, Ya-Fang; Ho, Tuan-Hua David; Wu, Chin-Feng; Ho, Shin-Lon; Yeh, Rong-Hwei; Lu, Chung-An; Chen, Peng-Wen; Yu, Lin-Chih; Chao, Annlin; Yu, Su-May

2012-01-01

Germination is a unique developmental transition from metabolically quiescent seed to actively growing seedling that requires an ensemble of hydrolases for coordinated nutrient mobilization to support heterotrophic growth until autotrophic photosynthesis is established. This study reveals two crucial transcription factors, MYBS1 and MYBGA, present in rice (Oryza sativa) and barley (Hordeum vulgare), that function to integrate diverse nutrient starvation and gibberellin (GA) signaling pathways during germination of cereal grains. Sugar represses but sugar starvation induces MYBS1 synthesis and its nuclear translocation. GA antagonizes sugar repression by enhancing conuclear transport of the GA-inducible MYBGA with MYBS1 and the formation of a stable bipartite MYB-DNA complex to activate the α-amylase gene. We further discovered that not only sugar but also nitrogen and phosphate starvation signals converge and interconnect with GA to promote the conuclear import of MYBS1 and MYBGA, resulting in the expression of a large set of GA-inducible but functionally distinct hydrolases, transporters, and regulators associated with mobilization of the full complement of nutrients to support active seedling growth in cereals. PMID:22773748

Pyrolytic sugars from cellulosic biomass.

PubMed

Kuzhiyil, Najeeb; Dalluge, Dustin; Bai, Xianglan; Kim, Kwang Ho; Brown, Robert C

2012-11-01

Depolymerization of cellulose offers the prospect of inexpensive sugars from biomass. Breaking the glycosidic bonds of cellulose to liberate glucose has usually been pursued by acid or enzymatic hydrolysis although a purely thermal depolymerization route to sugars is also possible. Fast pyrolysis of pure cellulose yields primarily the anhydrosugar levoglucosan (LG) whereas the presence of naturally occurring alkali and alkaline earth metals (AAEMs) in biomass strongly catalyzes ring-breaking reactions that favor formation of light oxygenates. Here, we show a method of significantly increasing the yield of sugars from biomass by purely thermal means through infusion of certain mineral acids (phosphoric and sulfuric acid) into the biomass to convert the AAEMs into thermally stable salts (particularly potassium sulfates and phosphates). These salts not only passivate AAEMs that normally catalyze fragmentation of pyranose rings, but also buffer the system at pH levels that favor glycosidic bond breakage. It appears that AAEM passivation contributes to 80 % of the enhancement in LG yield while the buffering effect of the acid salts contributes to the balance of the enhancement. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High Level Ethanol from Sugar Cane Molasses by a New Thermotolerant Saccharomyces cerevisiae Strain in Industrial Scale.

PubMed

Fadel, M; Keera, Abeer A; Mouafi, Foukia E; Kahil, Tarek

2013-01-01

A new local strain of S. cerevisiae F-514, for ethanol production during hot summer season, using Egyptian sugar cane molasses was applied in Egyptian distillery factory. The inouluum was propagated through 300 L, 3 m(3), and 12 m(3) fermenters charged with diluted sugar cane molasses containing 4%-5% sugars. The yeast was applied in fermentation vessels 65 m(3) working volume to study the varying concentrations of urea, DAP, orthophosphoric acid (OPA), and its combinations as well as magnesium sulfate and inoculum size. The fermenter was allowed to stay for a period of 20 hours to give time for maximum conversion of sugars into ethanol. S. cerevisiae F-514 at molasses sugar level of 18% (w/v), inoculum size of 20% (v/v) cell concentration of 3.0 × 10(8)/mL, and combinations of urea, diammonium phosphate (DAP), orthophosphoric acid (OPA), and magnesium sulfate at amounts of 20, 10, 5, and 10 kg/65 m(3) working volume fermenters, respectively, supported maximum ethanol production (9.8%, v/v), fermentation efficiency (FE) 88.1%, and remaining sugars (RS) 1.22%. The fermentation resulted 13.4 g dry yeast/L contained 34.6% crude protein and 8.2% ash. By selecting higher ethanol yielding yeast strain and optimizing, the fermentation parameters both yield and economics of the fermentation process can be improved.

Improved treatment of nucleosides and nucleotides in the OPLS-AA force field

NASA Astrophysics Data System (ADS)

Robertson, Michael J.; Tirado-Rives, Julian; Jorgensen, William L.

2017-09-01

DFT calculations have been used to develop improved descriptions of the torsional energetics for nucleosides and nucleotides in the OPLS-AA force field. Scans of nucleotide dihedral angles (γ, χ, and β) and methyl phosphates provided the bases for the new torsional parameters. In addition, the angle-bending parameters of phosphodiesters and ribose were updated, and adjustments were made to existing carbohydrate torsions to better capture the sugar puckering landscape of ribose. MD simulations of nucleosides with the new parameters demonstrate a significant improvement in the ribose sugar puckering and χ angle distributions. Additionally, energy-minimization of protein-nucleotide crystal structures with the new parameters produced accurate poses.

Expression Patterns of Genes Involved in Sugar Metabolism and Accumulation during Apple Fruit Development

PubMed Central

Cheng, Lailiang

2012-01-01

Both sorbitol and sucrose are imported into apple fruit from leaves. The metabolism of sorbitol and sucrose fuels fruit growth and development, and accumulation of sugars in fruit is central to the edible quality of apple. However, our understanding of the mechanisms controlling sugar metabolism and accumulation in apple remains quite limited. We identified members of various gene families encoding key enzymes or transporters involved in sugar metabolism and accumulation in apple fruit using homology searches and comparison of their expression patterns in different tissues, and analyzed the relationship of their transcripts with enzyme activities and sugar accumulation during fruit development. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase, cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase are high, and the resulting high enzyme activities are responsible for the rapid utilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugar accumulation. As the fruit continues to grow due to cell expansion, the transcript levels and activities of these enzymes are down-regulated, with concomitant accumulation of fructose and elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1 and MdTMT2; the excess carbon is converted into starch. At the late stage of fruit development, sucrose accumulation is enhanced, consistent with the elevated expression of sucrose-phosphate synthase (SPS), MdSPS5 and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentally regulated. This represents a comprehensive analysis of the genes involved in sugar metabolism and accumulation in apple, which will serve as a platform for further studies on the functions of these genes and subsequent manipulation of sugar metabolism and fruit quality traits related to carbohydrates. PMID:22412983

Exogenous sucrose supply changes sugar metabolism and reduces photosynthesis of sugarcane through the down-regulation of Rubisco abundance and activity.

PubMed

Lobo, Ana Karla Moreira; de Oliveira Martins, Marcio; Lima Neto, Milton Costa; Machado, Eduardo Caruso; Ribeiro, Rafael Vasconcelos; Silveira, Joaquim Albenisio Gomes

2015-05-01

Photosynthetic modulation by sugars has been known for many years, but the biochemical and molecular comprehension of this process is lacking. We studied how the exogenous sucrose supplied to leaves could affect sugar metabolism in leaf, sheath and stalk and inhibit photosynthesis in four-month old sugarcane plants. Exogenous sucrose 50mM sprayed on attached leaves strongly impaired the net CO2 assimilation (PN) and decreased the instantaneous carboxylation efficiency (PN/Ci), suggesting that the impairment in photosynthesis was caused by biochemical restrictions. The photosystem II activity was also affected by excess sucrose as indicated by the reduction in the apparent electron transport rate, effective quantum yield and increase in non-photochemical quenching. In leaf segments, sucrose accumulation was related to increases in the activities of soluble acid and neutral invertases, sucrose synthase and sucrose phosphate synthase, whereas the contents of fructose increased and glucose slightly decreased. Changes in the activities of sucrose hydrolyzing and synthesizing enzymes in leaf, sheath and stalk and sugar profile in intact plants were not enough to identify which sugar(s) or enzyme(s) were directly involved in photosynthesis modulation. However, exogenous sucrose was able to trigger down-regulation in the Rubisco abundance, activation state and enzymatic activity. Despite the fact that PN/Ci had been notably decreased by sucrose, in vitro activity and abundance of PEPCase did not change, suggesting an in vivo modulation of this enzyme. The data reveal that sucrose and/or other derivative sugars in leaves inhibited sugarcane photosynthesis by down-regulation of Rubisco synthesis and activity. Our data also suggest that sugar modulation was not exerted by a feedback mechanism induced by the accumulation of sugars in immature sugarcane stalk. Copyright © 2015. Published by Elsevier GmbH.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

PubMed

Dadashipour, Mohammad; Iwamoto, Mariko; Hossain, Mohammad Murad; Akutsu, Jun-Ichi; Zhang, Zilian; Kawarabayasi, Yutaka

2018-05-15

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways. IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially. Copyright © 2018 American Society for Microbiology.

Theoretical and experimental quantification of doubly and singly differential cross sections for electron-induced ionization of isolated tetrahydrofuran molecules

DOE PAGES

Champion, Christophe; Quinto, Michele A.; Bug, Marion U.; ...

2014-07-29

Electron-induced ionization of the commonly used surrogate of the DNA sugar-phosphate backbone, namely, the tetrahydrofuran molecule, is here theoretically described within the 1 st Born approximation by means of quantum-mechanical approach. Comparisons between theory and recent experiments are reported in terms of doubly and singly differential cross sections.

Lack of Awareness among Future Medical Professionals about the Risk of Consuming Hidden Phosphate-Containing Processed Food and Drinks

PubMed Central

Shutto, Yoshiko; Shimada, Michiko; Kitajima, Maiko; Yamabe, Hideaki; Razzaque, Mohammed S.

2011-01-01

Phosphate toxicity is an important determinant of mortality in patients with chronic kidney disease (CKD), particularly those undergoing hemodialysis treatments. CKD patients are advised to take a low phosphate-containing diet, and are additionally prescribed with phosphate-lowering drugs. Since these patients usually seek guidance from their physicians and nurses for their dietary options, we conducted a survey to determine the levels of awareness regarding the high phosphate content in commercially processed food and drinks among medical and nursing students at the Hirosaki University School of Medicine in Japan. For this survey, 190 medical and nursing students (average age 21.7±3 years) were randomly selected, and provided with a list of questions aimed at evaluating their awareness of food and drinks containing artificially added phosphate ingredients. While 98.9% of these students were aware of the presence of sugar in commercially available soda drinks, only 6.9% were aware of the presence of phosphate (phosphoric acid). Similarly, only 11.6% of these students were aware of the presence of phosphate in commercially processed food, such as hamburgers and pizza. Moreover, around two thirds of the surveyed students (67.7%) were unaware of the harmful effects of unrestricted consumption of phosphate-containing food and drinks. About 28% of the surveyed students consume such “fast food” once a week, while 40% drink at least 1∼5 cans of soda drinks/week. After realizing the potential long-term risks of consuming excessive phosphate-containing food and drinks, 40.5% of the survey participants considered reducing their phosphate intake by minimizing the consumption of commercially processed “fast food” items and soda drinks. Moreover, another 48.4% of students showed interest in obtaining more information on the negative health effects of consuming excessive amounts of phosphate. This survey emphasizes the need for educational initiative to raise awareness of the health risks posed by excessive consumption of phosphate additives. PMID:22220204

Lack of awareness among future medical professionals about the risk of consuming hidden phosphate-containing processed food and drinks.

PubMed

Shutto, Yoshiko; Shimada, Michiko; Kitajima, Maiko; Yamabe, Hideaki; Razzaque, Mohammed S

2011-01-01

Phosphate toxicity is an important determinant of mortality in patients with chronic kidney disease (CKD), particularly those undergoing hemodialysis treatments. CKD patients are advised to take a low phosphate-containing diet, and are additionally prescribed with phosphate-lowering drugs. Since these patients usually seek guidance from their physicians and nurses for their dietary options, we conducted a survey to determine the levels of awareness regarding the high phosphate content in commercially processed food and drinks among medical and nursing students at the Hirosaki University School of Medicine in Japan. For this survey, 190 medical and nursing students (average age 21.7±3 years) were randomly selected, and provided with a list of questions aimed at evaluating their awareness of food and drinks containing artificially added phosphate ingredients. While 98.9% of these students were aware of the presence of sugar in commercially available soda drinks, only 6.9% were aware of the presence of phosphate (phosphoric acid). Similarly, only 11.6% of these students were aware of the presence of phosphate in commercially processed food, such as hamburgers and pizza. Moreover, around two thirds of the surveyed students (67.7%) were unaware of the harmful effects of unrestricted consumption of phosphate-containing food and drinks. About 28% of the surveyed students consume such "fast food" once a week, while 40% drink at least 1∼5 cans of soda drinks/week. After realizing the potential long-term risks of consuming excessive phosphate-containing food and drinks, 40.5% of the survey participants considered reducing their phosphate intake by minimizing the consumption of commercially processed "fast food" items and soda drinks. Moreover, another 48.4% of students showed interest in obtaining more information on the negative health effects of consuming excessive amounts of phosphate. This survey emphasizes the need for educational initiative to raise awareness of the health risks posed by excessive consumption of phosphate additives.

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Concentration of simple aldehydes by sulfite-containing double-layer hydroxide minerals: implications for biopoesis

NASA Technical Reports Server (NTRS)

Pitsch, S.; Krishnamurthy, R.; Arrhenius, G.; Bada, J. L. (Principal Investigator)

2000-01-01

Environmental conditions play an important role in conceptual studies of prebiotically relevant chemical reactions that could have led to functional biomolecules. The necessary source compounds are likely to have been present in dilute solution, raising the question of how to achieve selective concentration and to reach activation. With the assumption of an initial 'RNA World', the questions of production, concentration, and interaction of aldehydes and aldehyde phosphates, potential precursors of sugar phosphates, come into the foreground. As a possible concentration process for simple, uncharged aldehydes, we investigated their adduct formation with sulfite ion bound in the interlayer of positively charged expanding-sheet-structure double-layer hydroxide minerals. Minerals of this type, initially with chloride as interlayer counter anion, have previously been shown to induce concentration and subsequent aldolization of aldehyde phosphates to form tetrose, pentose, and hexose phosphates. The reversible uptake of the simple aldehydes formaldehyde, glycolaldehyde, and glyceraldehyde by adduct formation with the immobilized sulfite ions is characterized by equilibrium constants of K=1.5, 9, and 11, respectively. This translates into an observable uptake at concentrations exceeding 50 mM.

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

PubMed Central

Lunn, John E.; Feil, Regina; Hendriks, Janneke H. M.; Gibon, Yves; Morcuende, Rosa; Osuna, Daniel; Scheible, Wolf-Rüdiger; Carillo, Petronia; Hajirezaei, Mohammad-Reza; Stitt, Mark

2006-01-01

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol·g−1FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol·g−1FW at the end of the night, which rose to 108 pmol·g−1FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 μM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118–11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis. PMID:16551270

Unraveling the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress by ¹H NMR-based metabolomics.

PubMed

Ye, Yangfang; Wang, Xin; Zhang, Limin; Lu, Zhenmei; Yan, Xiaojun

2012-07-01

Nicotine can cause oxidative damage to organisms; however, some bacteria, for example Pseudomonas sp. HF-1, are resistant to such oxidative stress. In the present study, we analyzed the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress using ¹H NMR spectroscopy coupled with multivariate data analysis. We found that the dominant metabolites in Pseudomonas sp. HF-1 were eight aliphatic organic acids, six amino acids, three sugars and 11 nucleotides. After 18 h of cultivation, 1 g/L nicotine caused significant elevation of sugar (glucose, trehalose and maltose), succinate and nucleic acid metabolites (cytidine, 5'-CMP, guanine 2',3'-cyclic phosphate and adenosine 2',3'-cyclic phosphate), but decrease of glutamate, putrescine, pyrimidine, 2-propanol, diethyl ether and acetamide levels. Similar metabolomic changes were induced by 2 g/L nicotine, except that no significant change in trehalose, 5'-UMP levels and diethyl ether were found. However, 3 g/L nicotine led to a significant elevation in the two sugars (trehalose and maltose) levels and decrease in the levels of glutamate, putrescine, pyrimidine and 2-propanol. Our findings indicated that nicotine resulted in the enhanced nucleotide biosynthesis, decreased glucose catabolism, elevated succinate accumulation, severe disturbance in osmoregulation and complex antioxidant strategy. And a further increase of nicotine level was a critical threshold value that triggered the change of metabolic flow in Pseudomonas sp. HF-1. These findings revealed the comprehensive insights into the metabolic response of nicotine-degrading bacteria to nicotine-induced oxidative toxicity.

Influence of transport energization on the growth yield of Escherichia coli.

PubMed

Muir, M; Williams, L; Ferenci, T

1985-09-01

The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaerobic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-protein-dependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products.

Influence of transport energization on the growth yield of Escherichia coli.

PubMed Central

Muir, M; Williams, L; Ferenci, T

1985-01-01

The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaerobic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-protein-dependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products. PMID:3928598

The Path of Carbon in Photosynthesis XV. Ribulose and Sedoheptulose

DOE R&D Accomplishments Database

Benson, A. A.; Bassham, J. A.; Calvin, M.; Hall, A. G.; Hirsch, H.; Kawaguchi, S.; Lynch, V.; Tolbert, N. E.

1952-01-01

The intermediates of carbon dioxide reduction by plants include phosphorylated derivatives of hydroxy acids and sugars. Their identification became possible when the use of labeled carbon dioxide permitted discrimination between the earliest products and the many other components of photosynthetic tissues. A number of compounds were identified by virtue of the chemical and physical properties of the radioactive compounds in tracer amounts and by direct comparison of these properties with those of suspected known metabolic intermediates. It became apparent that several labeled compounds found in short exposures to radioactive carbon dioxide were not substances previously identified as metabolic intermediates. Two phosphate esters in particular were observed in the products of the first few seconds of steady-state photosynthesis by all the photosynthetic microorganisms and higher plants examined in this laboratory. These esters have been isolated by paper chromatography in tracer quantities and enzymatically hydrolyzed to give two sugars, ribulose and sedoheptulose. This paper contains a description of the chemical identification of these sugars and some observations and suggestions regarding the function of their esters. The general importance of these compounds in photosynthesis was summarized before their identification. The products of photosynthesis with C{sup 14}O{sub 2} by each plant included phosphate esters of the same two then unknown compounds in addition to those of the expected glucose, fructose, dihydroxyacetone and glyceric acid. As the time of steady-state photosynthesis in C{sup 14}O{sub 2} decreased, the fractions of total fixed radiocarbon in the esters of the two unidentified compounds increased.

The rare sugar d-allose acts as a triggering molecule of rice defence via ROS generation

PubMed Central

Akimitsu, Kazuya

2013-01-01

Only d-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to d-allose. d-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-d-allose, a structural derivative of d-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding d-allose kinase to increase d-allose 6-phosphate synthesis were more sensitive to d-allose, but E. coli AlsI encoding d-allose 6-phosphate isomerase expression to decrease d-allose 6-phosphate reduced sensitivity. A d-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, d-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of d-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of d-allose to d-allose 6-phosphate, and treatment with d-allose might prove to be useful for reducing disease development in rice. PMID:24014866

The rare sugar D-allose acts as a triggering molecule of rice defence via ROS generation.

PubMed

Kano, Akihito; Fukumoto, Takeshi; Ohtani, Kouhei; Yoshihara, Akihide; Ohara, Toshiaki; Tajima, Shigeyuki; Izumori, Ken; Tanaka, Keiji; Ohkouchi, Takeo; Ishida, Yutaka; Nishizawa, Yoko; Ichimura, Kazuya; Tada, Yasuomi; Gomi, Kenji; Akimitsu, Kazuya

2013-11-01

Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice.

The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness

PubMed Central

Henry, Clémence; Bledsoe, Samuel W.; Siekman, Allison; Kollman, Alec; Waters, Brian M.; Feil, Regina; Stitt, Mark; Lagrimini, L. Mark

2014-01-01

Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed. PMID:25271261

Thalassospira sp. Isolated from the Oligotrophic Eastern Mediterranean Sea Exhibits Chemotaxis toward Inorganic Phosphate during Starvation▿†

PubMed Central

Hütz, Annemarie; Schubert, Karin; Overmann, Jörg

2011-01-01

The eastern Mediterranean Sea represents an ultraoligotrophic environment where soluble phosphate limits the growth of bacterioplankton. Correspondingly, genes coding for high-affinity phosphate uptake systems and for organophosphonate utilization are highly prevalent in the plankton metagenome. Chemotaxis toward inorganic phosphate constitutes an alternative strategy to cope with phosphate limitation, but so far has only been demonstrated for two bacterial pathogens and an archaeon, and not in any free-living planktonic bacterium. In the present study, bacteria affiliated with the genus Thalassospira were found to constitute a regular, low-abundance member of the bacterioplankton that can be detected throughout the water column of the eastern Mediterranean Sea. A representative (strain EM) was isolated in pure culture and exhibited a strong positive chemotaxis toward inorganic phosphate that was induced exclusively in phosphate-starved cultures. Phosphate-depleted cells were 2-fold larger than in exponentially growing cultures, and 43% of the cells retained their motility even during prolonged starvation over 10 days. In addition, Thalassospira sp. strain EM was chemotactically attracted by complex substrates (yeast extract and peptone), amino acids, and 2-aminoethylphosphonate but not by sugar monomers. Similarly to the isolate from the eastern Mediterranean, chemotaxis toward phosphate was observed in starved cultures of the other two available isolates of the genus, T. lucentensis DSM 14000T and T. profundimaris WP0211T. Although Thalassospira sp. represents only up to 1.2% of the total bacterioplankton community in the water column of the eastern Mediterranean Sea, its chemotactic behavior potentially leads to an acceleration of nutrient cycling and may also explain the persistence of marine copiotrophs in this extremely nutrient-limited environment. PMID:21602377

Thalassospira sp. isolated from the oligotrophic eastern Mediterranean Sea exhibits chemotaxis toward inorganic phosphate during starvation.

PubMed

Hütz, Annemarie; Schubert, Karin; Overmann, Jörg

2011-07-01

The eastern Mediterranean Sea represents an ultraoligotrophic environment where soluble phosphate limits the growth of bacterioplankton. Correspondingly, genes coding for high-affinity phosphate uptake systems and for organophosphonate utilization are highly prevalent in the plankton metagenome. Chemotaxis toward inorganic phosphate constitutes an alternative strategy to cope with phosphate limitation, but so far has only been demonstrated for two bacterial pathogens and an archaeon, and not in any free-living planktonic bacterium. In the present study, bacteria affiliated with the genus Thalassospira were found to constitute a regular, low-abundance member of the bacterioplankton that can be detected throughout the water column of the eastern Mediterranean Sea. A representative (strain EM) was isolated in pure culture and exhibited a strong positive chemotaxis toward inorganic phosphate that was induced exclusively in phosphate-starved cultures. Phosphate-depleted cells were 2-fold larger than in exponentially growing cultures, and 43% of the cells retained their motility even during prolonged starvation over 10 days. In addition, Thalassospira sp. strain EM was chemotactically attracted by complex substrates (yeast extract and peptone), amino acids, and 2-aminoethylphosphonate but not by sugar monomers. Similarly to the isolate from the eastern Mediterranean, chemotaxis toward phosphate was observed in starved cultures of the other two available isolates of the genus, T. lucentensis DSM 14000T and T. profundimaris WP0211T. Although Thalassospira sp. represents only up to 1.2% of the total bacterioplankton community in the water column of the eastern Mediterranean Sea, its chemotactic behavior potentially leads to an acceleration of nutrient cycling and may also explain the persistence of marine copiotrophs in this extremely nutrient-limited environment.

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon.

PubMed

Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling; Liu, Wenge

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar '203Z' and its near-isogenic line (NIL) 'SW' (in the '203Z' background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening.

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon

PubMed Central

Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar ‘203Z’ and its near-isogenic line (NIL) ‘SW’ (in the ‘203Z’ background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening. PMID:29324867

Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization.

PubMed

Lugtenberg, B J; Kravchenko, L V; Simons, M

1999-10-01

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.

Energy and Fructose From Beverages Sweetened With Sugar or High-Fructose Corn Syrup Pose a Health Risk for Some People12

PubMed Central

Bray, George A.

2013-01-01

Sugar intake in the United States has increased by >40 fold since the American Revolution. The health concerns that have been raised about the amounts of sugar that are in the current diet, primarily as beverages, are the subject of this review. Just less than 50% of the added sugars (sugar and high-fructose corn syrup) are found in soft drinks and fruit drinks. The intake of soft drinks has increased 5-fold between 1950 and 2000. Most meta-analyses have shown that the risk of obesity, diabetes, cardiovascular disease, and metabolic syndrome are related to consumption of beverages sweetened with sugar or high-fructose corn syrup. Calorically sweetened beverage intake has also been related to the risk of nonalcoholic fatty liver disease, and, in men, gout. Calorically sweetened beverages contribute to obesity through their caloric load, and the intake of beverages does not produce a corresponding reduction in the intake of other food, suggesting that beverage calories are “add-on” calories. The increase in plasma triglyceride concentrations by sugar-sweetened beverages can be attributed to fructose rather than glucose in sugar. Several randomized trials of sugar-containing soft drinks versus low-calorie or calorie-free beverages show that either sugar, 50% of which is fructose, or fructose alone increases triglycerides, body weight, visceral adipose tissue, muscle fat, and liver fat. Fructose is metabolized primarily in the liver. When it is taken up by the liver, ATP decreases rapidly as the phosphate is transferred to fructose in a form that makes it easy to convert to lipid precursors. Fructose intake enhances lipogenesis and the production of uric acid. By worsening blood lipids, contributing to obesity, diabetes, fatty liver, and gout, fructose in the amounts currently consumed is hazardous to the health of some people. PMID:23493538

Energy and fructose from beverages sweetened with sugar or high-fructose corn syrup pose a health risk for some people.

PubMed

Bray, George A

2013-03-01

Sugar intake in the United States has increased by >40 fold since the American Revolution. The health concerns that have been raised about the amounts of sugar that are in the current diet, primarily as beverages, are the subject of this review. Just less than 50% of the added sugars (sugar and high-fructose corn syrup) are found in soft drinks and fruit drinks. The intake of soft drinks has increased 5-fold between 1950 and 2000. Most meta-analyses have shown that the risk of obesity, diabetes, cardiovascular disease, and metabolic syndrome are related to consumption of beverages sweetened with sugar or high-fructose corn syrup. Calorically sweetened beverage intake has also been related to the risk of nonalcoholic fatty liver disease, and, in men, gout. Calorically sweetened beverages contribute to obesity through their caloric load, and the intake of beverages does not produce a corresponding reduction in the intake of other food, suggesting that beverage calories are "add-on" calories. The increase in plasma triglyceride concentrations by sugar-sweetened beverages can be attributed to fructose rather than glucose in sugar. Several randomized trials of sugar-containing soft drinks versus low-calorie or calorie-free beverages show that either sugar, 50% of which is fructose, or fructose alone increases triglycerides, body weight, visceral adipose tissue, muscle fat, and liver fat. Fructose is metabolized primarily in the liver. When it is taken up by the liver, ATP decreases rapidly as the phosphate is transferred to fructose in a form that makes it easy to convert to lipid precursors. Fructose intake enhances lipogenesis and the production of uric acid. By worsening blood lipids, contributing to obesity, diabetes, fatty liver, and gout, fructose in the amounts currently consumed is hazardous to the health of some people.

On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

PubMed

Linck, Annabell; Vu, Xuan-Khang; Essl, Christine; Hiesl, Charlotte; Boles, Eckhard; Oreb, Mislav

2014-05-01

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara

Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations inmore » primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.« less

Metabolic profiling reveals altered sugar and secondary metabolism in response to UGPase overexpression in Populus

DOE PAGES

Payyavula, Raja S.; Tschaplinski, Timothy J.; Jawdy, Sara; ...

2014-10-07

Background: UDP-glucose pyrophopharylase (UGPase) is a sugar metabolizing enzyme (E.C. 2.7.7.9) that catalyzes a reversible reaction of UDP-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate glucose. UDP-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. The functional role of UGPase in woody plants such as Populus is poorly understood. Results: We characterized the functional role of UGPase in Populus deltoides by overexpressing a native gene. Overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. Metabolomic analyses showed that manipulation of UGPase results in perturbations inmore » primary as well as secondary metabolism resulting in reduced sugar and starch levels and increased phenolics such as caffeoyl- and feruloyl conjugates. While cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. Conclusions: These results demonstrate that UGPase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism outside of cell wall biosynthesis of Populus.« less

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells*

PubMed Central

Tan, Bo; Dong, Sucai; Shepard, Robert L.; Kays, Lisa; Roth, Kenneth D.; Geeganage, Sandaruwan; Kuo, Ming-Shang; Zhao, Genshi

2015-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD+ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD+ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed. PMID:25944913

Hydrothermal synthesis of cathode materials

NASA Astrophysics Data System (ADS)

Chen, Jiajun; Wang, Shijun; Whittingham, M. Stanley

A number of cathodes are being considered for the next generation of lithium ion batteries to replace the expensive LiCoO 2 presently used. Besides the layered oxides, such as LiNi yMn yCo 1-2 yO 2, a leading candidate is lithium iron phosphate with the olivine structure. Although this material is inherently low cost, a manufacturing process that produces electrochemically active LiFePO 4 at a low cost is also required. Hydrothermal reactions are one such possibility. A number of pure phosphates have been prepared using this technique, including LiFePO 4, LiMnPO 4 and LiCoPO 4; this method has also successfully produced mixed metal phosphates, such as LiFe 0.33Mn 0.33Co 0.33PO 4. Ascorbic acid was found to be better than hydrazine or sugar at preventing the formation of ferric ions in aqueous media. When conductive carbons are added to the reaction medium excellent electrochemical behavior is observed.

The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during the rehydration phase.

PubMed Central

Bernacchia, G; Schwall, G; Lottspeich, F; Salamini, F; Bartels, D

1995-01-01

Transketolases, key enzymes of the reductive and oxidative pentose phosphate pathways, are responsible for the synthesis of sugar phosphate intermediates. Here we report the first molecular analysis of transketolase genes from plants. Three distinct classes of transketolase-encoding cDNA clones were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum. One class represented by the transcript tkt3 is constitutively expressed in leaves and roots under all physiological conditions tested. By biochemical analysis and protein sequencing of purified transketolase, it was shown that tkt3 is expressed in three enzymatically active isoforms. An intriguing discovery was that accumulation of the two other transketolase transcripts, tkt7 and tkt10, is preferentially associated with the rehydration process of the desiccated plant; whereas tkt10 is only expressed in leaves, tkt7 was detected in leaves and roots. This observation suggests a possible role for these transketolases in the conversion of sugars, which are a major phenomenon in the rehydration process. Despite an abundant level of tkt7 and tkt10 transcripts in rehydrating leaves, proteins could not be isolated. This is due in part to a translational control mechanism acting on the loading of mRNAs to polysomes. Images PMID:7859749

Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala)

PubMed Central

Zhang, Wen; Lunn, John E.; Feil, Regina; Wang, Yufei; Zhao, Jingjing; Tao, Hongxia; Zhao, Zhengyang

2017-01-01

ABSTRACT Trehalose-6-phosphate (Tre6P) is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple (Malus domestica, Borkh. cv. Gala). Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple. PMID:28069587

Ionization energies of aqueous nucleic acids: photoelectron spectroscopy of pyrimidine nucleosides and ab initio calculations.

PubMed

Slavícek, Petr; Winter, Bernd; Faubel, Manfred; Bradforth, Stephen E; Jungwirth, Pavel

2009-05-13

Vertical ionization energies of the nucleosides cytidine and deoxythymidine in water, the lowest ones amounting in both cases to 8.3 eV, are obtained from photoelectron spectroscopy measurements in aqueous microjets. Ab initio calculations employing a nonequilibrium polarizable continuum model quantitatively reproduce the experimental spectra and provide molecular interpretation of the individual peaks of the photoelectron spectrum, showing also that lowest ionization originates from the base. Comparison of calculated vertical ionization potentials of pyrimidine bases, nucleosides, and nucleotides in water and in the gas phase underlines the dramatic effect of bulk hydration on the electronic structure. In the gas phase, the presence of sugar and, in particular, of phosphate has a strong effect on the energetics of ionization of the base. Upon bulk hydration, the ionization potential of the base in contrast becomes rather insensitive to the presence of the sugar and phosphate, which indicates a remarkable screening ability of the aqueous solvent. Accurate aqueous-phase vertical ionization potentials provide a significant improvement to the corrected gas-phase values used in the literature and represent important information in assessing the threshold energies for photooxidation and oxidation free energies of solvent-exposed DNA components. Likewise, such energetic data should allow improved assessment of delocalization and charge-hopping mechanisms in DNA ionized by radiation.

Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala).

PubMed

Zhang, Wen; Lunn, John E; Feil, Regina; Wang, Yufei; Zhao, Jingjing; Tao, Hongxia; Guo, Yanping; Zhao, Zhengyang

2017-02-15

Trehalose-6-phosphate (Tre6P) is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple ( Malus domestica , Borkh. cv. Gala). Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple. © 2017. Published by The Company of Biologists Ltd.

Cross-talk between Phosphate Starvation and Other Environmental Stress Signaling Pathways in Plants

PubMed Central

Baek, Dongwon; Chun, Hyun Jin; Yun, Dae-Jin; Kim, Min Chul

2017-01-01

The maintenance of inorganic phosphate (Pi) homeostasis is essential for plant growth and yield. Plants have evolved strategies to cope with Pi starvation at the transcriptional, post-transcriptional, and post-translational levels, which maximizes its availability. Many transcription factors, miRNAs, and transporters participate in the Pi starvation signaling pathway where their activities are modulated by sugar and phytohormone signaling. Environmental stresses significantly affect the uptake and utilization of nutrients by plants, but their effects on the Pi starvation response remain unclear. Recently, we reported that Pi starvation signaling is affected by abiotic stresses such as salt, abscisic acid, and drought. In this review, we identified transcription factors, such as MYB, WRKY, and zinc finger transcription factors with functions in Pi starvation and other environmental stress signaling. In silico analysis of the promoter regions of Pi starvation-responsive genes, including phosphate transporters, microRNAs, and phosphate starvation–induced genes, suggest that their expression may be regulated by other environmental stresses, such as hormones, drought, cold, heat, and pathogens as well as by Pi starvation. Thus, we suggest the possibility of cross-talk between Pi starvation signaling and other environmental stress signaling pathways. PMID:29047263

Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates, phosphate, sugars and hormones play in lateral root formation?

PubMed Central

Fusconi, Anna

2014-01-01

Background Arbuscular mycorrhizae (AMs) form a widespread root–fungus symbiosis that improves plant phosphate (Pi) acquisition and modifies the physiology and development of host plants. Increased branching is recognized as a general feature of AM roots, and has been interpreted as a means of increasing suitable sites for colonization. Fungal exudates, which are involved in the dialogue between AM fungi and their host during the pre-colonization phase, play a well-documented role in lateral root (LR) formation. In addition, the increased Pi content of AM plants, in relation to Pi-starved controls, as well as changes in the delivery of carbohydrates to the roots and modulation of phytohormone concentration, transport and sensitivity, are probably involved in increasing root system branching. Scope This review discusses the possible causes of increased branching in AM plants. The differential root responses to Pi, sugars and hormones of potential AM host species are also highlighted and discussed in comparison with those of the non-host Arabidopsis thaliana. Conclusions Fungal exudates are probably the main compounds regulating AM root morphogenesis during the first colonization steps, while a complex network of interactions governs root development in established AMs. Colonization and high Pi act synergistically to increase root branching, and sugar transport towards the arbusculated cells may contribute to LR formation. In addition, AM colonization and high Pi generally increase auxin and cytokinin and decrease ethylene and strigolactone levels. With the exception of cytokinins, which seem to regulate mainly the root:shoot biomass ratio, these hormones play a leading role in governing root morphogenesis, with strigolactones and ethylene blocking LR formation in the non-colonized, Pi-starved plants, and auxin inducing them in colonized plants, or in plants grown under high Pi conditions. PMID:24227446

Mathematical modeling of the central carbohydrate metabolism in Arabidopsis reveals a substantial regulatory influence of vacuolar invertase on whole plant carbon metabolism.

PubMed

Nägele, Thomas; Henkel, Sebastian; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G

2010-05-01

A mathematical model representing metabolite interconversions in the central carbohydrate metabolism of Arabidopsis (Arabidopsis thaliana) was developed to simulate the diurnal dynamics of primary carbon metabolism in a photosynthetically active plant leaf. The model groups enzymatic steps of central carbohydrate metabolism into blocks of interconverting reactions that link easily measurable quantities like CO(2) exchange and quasi-steady-state levels of soluble sugars and starch. When metabolite levels that fluctuate over diurnal cycles are used as a basic condition for simulation, turnover rates for the interconverting reactions can be calculated that approximate measured metabolite dynamics and yield kinetic parameters of interconverting reactions. We used experimental data for Arabidopsis wild-type plants, accession Columbia, and a mutant defective in vacuolar invertase, AtbetaFruct4, as input data. Reducing invertase activity to mutant levels in the wild-type model led to a correct prediction of increased sucrose levels. However, additional changes were needed to correctly simulate levels of hexoses and sugar phosphates, indicating that invertase knockout causes subsequent changes in other enzymatic parameters. Reduction of invertase activity caused a decline in photosynthesis and export of reduced carbon to associated metabolic pathways and sink organs (e.g. roots), which is in agreement with the reported contribution of vacuolar invertase to sink strength. According to model parameters, there is a role for invertase in leaves, where futile cycling of sucrose appears to have a buffering effect on the pools of sucrose, hexoses, and sugar phosphates. Our data demonstrate that modeling complex metabolic pathways is a useful tool to study the significance of single enzyme activities in complex, nonintuitive networks.

Producing glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion

DOE PAGES

Bacik, John -Paul; Klesmith, Justin R.; Whitehead, Timothy A.; ...

2015-09-09

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium andmore » solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Furthermore, greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.« less

Distinctly Different Glass Transition Behaviors of Trehalose Mixed with Na2HPO 4 or NaH 2PO 4: Evidence for its Molecular Origin.

PubMed

Weng, Lindong; Elliott, Gloria D

2015-07-01

The present study is aimed at understanding how the interactions between sugar molecules and phosphate ions affect the glass transition temperature of their mixtures, and the implications for pharmaceutical formulations. The glass transition temperature (Tg) and the α-relaxation temperature (Tα) of dehydrated trehalose/sodium phosphate mixtures (monobasic or dibasic) were determined by differential scanning calorimetry and dynamic mechanical analysis, respectively. Molecular dynamics simulations were also conducted to investigate the microscopic interactions between sugar molecules and phosphate ions. The hydrogen-bonding characteristics and the self-aggregation features of these mixtures were quantified and compared. Thermal analysis measurements demonstrated that the addition of NaH2PO4 decreased both the glass transition temperature and the α-relaxation temperature of the dehydrated trehalose/NaH2PO4 mixture compared to trehalose alone while both Tg and Tα were increased by adding Na2HPO4 to pure trehalose. The hydrogen-bonding interactions between trehalose and HPO4(2-) were found to be stronger than both the trehalose-trehalose hydrogen bonds and those formed between trehalose and H2PO4(-). The HPO4(2-) ions also aggregated into smaller clusters than H2PO4(-) ions. The trehalose/Na2HPO4 mixture yielded a higher T g than pure trehalose because marginally self-aggregated HPO4(2-) ions established a strengthened hydrogen-bonding network with trehalose molecules. In contrast H2PO4(-) ions served only as plasticizers, resulting in a lower Tg of the mixtures than trehalose alone, creating large-sized ionic pockets, weakening interactions, and disrupting the original hydrogen-bonding network amongst trehalose molecules.

The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness.

PubMed

Henry, Clémence; Bledsoe, Samuel W; Siekman, Allison; Kollman, Alec; Waters, Brian M; Feil, Regina; Stitt, Mark; Lagrimini, L Mark

2014-11-01

Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Distinctly Different Glass Transition Behaviors of Trehalose Mixed with Na2HPO4 or NaH2PO4: Evidence for its Molecular Origin

PubMed Central

Weng, Lindong; Elliott, Gloria D.

2015-01-01

Purpose The present study is aimed at understanding how the interactions between sugar molecules and phosphate ions affect the glass transition temperature of their mixtures, and the implications for pharmaceutical formulations. Methods The glass transition temperature (Tg) and the α-relaxation temperature (Tα) of dehydrated trehalose/sodium phosphate mixtures (monobasic or dibasic) were determined by differential scanning calorimetry and dynamic mechanical analysis, respectively. Molecular dynamics simulations were also conducted to investigate the microscopic interactions between sugar molecules and phosphate ions. The hydrogen-bonding characteristics and the self-aggregation features of these mixtures were quantified and compared. Results Thermal analysis measurements demonstrated that the addition of NaH2PO4 decreased both the glass transition temperature and the α-relaxation temperature of the dehydrated trehalose/NaH2PO4 mixture compared to trehalose alone while both Tg and Tα were increased by adding Na2HPO4 to pure trehalose. The hydrogen-bonding interactions between trehalose and HPO42− were found to be stronger than both the trehalose-trehalose hydrogen bonds and those formed between trehalose and H2PO4−. The HPO42− ions also aggregated into smaller clusters than H2PO4− ions. Conclusions The trehalose/Na2HPO4 mixture yielded a higher Tg than pure trehalose because marginally self-aggregated HPO42− ions established a strengthened hydrogen-bonding network with trehalose molecules. In contrast H2PO4− ions served only as plasticizers, resulting in a lower Tg of the mixtures than trehalose alone, creating large-sized ionic pockets, weakening interactions, and disrupting the original hydrogen-bonding network amongst trehalose molecules. PMID:25537342

ADP-Glucose Pyrophosphorylase Is Activated by Posttranslational Redox-Modification in Response to Light and to Sugars in Leaves of Arabidopsis and Other Plant Species1[w

PubMed Central

Hendriks, Janneke H.M.; Kolbe, Anna; Gibon, Yves; Stitt, Mark; Geigenberger, Peter

2003-01-01

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It was recently shown that potato (Solanum tuberosum) tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular Cys bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farré, P. Geigenberger [2002] Plant Cell 14: 2191–2213). We show here that AGPase is also subject to posttranslational regulation in leaves of pea (Pisum sativum), potato, and Arabidopsis. Conversion is accompanied by an increase in activity, which involves changes in the kinetic properties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer when preilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves via the petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced in the starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPB monomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novel posttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels in the leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recycling of phosphate during photosynthetic carbon fixation and sucrose synthesis. PMID:12972664

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

PubMed

da Silva, F R; Vettore, A L; Kemper, E L; Leite, A; Arruda, P

2001-09-25

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Effect of Repeated Acid Challenges on the Color Stability of Resin-Infiltrated Enamel White Spot Lesions

DTIC Science & Technology

2016-06-01

plaque or exogenous acid sources (e.g. sugar and soda) outweigh the protective factors of saliva (calcium, phosphate, antibacterial factors, and...ecological catastrophes? Microbiology, 149(Pt 2), 279-294. Martignon S, Ekstrand KR, Ellwood R. (2006). Efficacy of sealing proximal early active ...oral hydrogen-ion concentrations associated with dental caries activity . Journal of Dental Research, 23(4), 257-266. Stephan RM. (1947). pH and

Reversal of infectious mononucleosis-associated suppressor T cell activity by D-mannose

PubMed Central

1983-01-01

Epstein-Barr virus-induced infectious mononucleosis (IM) is associated with the activation of suppressor T lymphocytes that profoundly inhibit immunoglobulin (Ig) production in vitro. We have examined the nature of signals operating in the interaction between IM suppressor T cells and their targets, and explored the possibility that a lectin-like receptor molecule and its specific sugar might provide specificity to this interaction. When D-mannose or some of its derivatives, including alpha- methyl-D-mannoside, mannose-6-phosphate, and mannan, were added to suppressed cultures containing IM T lymphocytes and pokeweed mitogen (PWM)-stimulated normal mononuclear cells, a significant enhancement of Ig production was observed. These sugars had little or no effect on Ig production by the PWM-stimulated responder cells alone and thus the enhanced Ig production could be attributed to the reversal of suppression in the co-cultures by these sugars. This was further confirmed by the observation that the sugars were effective only if present during the first 24 h of culture, a time when IM suppressor T cells exert their principal effect. The effect of sugars on Ig production by suppressed cultures was similar to that achieved by decreasing by about fourfold the number of IM T cells in culture. The effect of the sugars is unlikely to represent a form of nonspecific toxicity, since inhibited cultures become responders in the presence of the sugar. Furthermore, toxicity restricted to the suppressor T cells is unlikely, since preincubation of the T cells with the sugars did not reduce their subsequent ability to suppress in secondary indicator cultures. In addition, there was no correlation between the effect of the sugars on T cell proliferation and their effect on T cell-mediated suppression. The reversal of suppression by sugars was dose dependent and demonstrated stereo-specificity in that L-mannose was without effect while D-mannose reversed suppression. These data indicate that D- mannose and some of its derivatives consistently reverse suppression of Ig production by IM T cells and strongly suggest a role for saccharides as critical components in the cellular receptors involved in certain physiologic immune cell interactions. PMID:6225821

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salminen, S.O.; Streeter, J.G.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with /sup 14/C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O/sub 2/ (2% in the gas phase). Uptake and conversion of /sup 14/C to CO/sub 2/ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO/sub 2/, and fructose was respired at a rate of at least double that of glucose. Sucrose and glucose were converted to CO/sub 2/ at a very low but measurable rate (<0.1more » nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO/sub 2/ at a rate 30 times greater than the conversion of carbon Number 6 to CO/sub 2/, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.« less

Citramalic acid and salicylic acid in sugar beet root exudates solubilize soil phosphorus

PubMed Central

2011-01-01

Background In soils with a low phosphorus (P) supply, sugar beet is known to intake more P than other species such as maize, wheat, or groundnut. We hypothesized that organic compounds exuded by sugar beet roots solubilize soil P and that this exudation is stimulated by P starvation. Results Root exudates were collected from plants grown in hydroponics under low- and high-P availability. Exudate components were separated by HPLC, ionized by electrospray, and detected by mass spectrometry in the range of mass-to-charge ratio (m/z) from 100 to 1000. Eight mass spectrometric signals were enhanced at least 5-fold by low P availability at all harvest times. Among these signals, negative ions with an m/z of 137 and 147 were shown to originate from salicylic acid and citramalic acid. The ability of both compounds to mobilize soil P was demonstrated by incubation of pure substances with Oxisol soil fertilized with calcium phosphate. Conclusions Root exudates of sugar beet contain salicylic acid and citramalic acid, the latter of which has rarely been detected in plants so far. Both metabolites solubilize soil P and their exudation by roots is stimulated by P deficiency. These results provide the first assignment of a biological function to citramalic acid of plant origin. PMID:21871058

Hyperphosphatemia after sodium phosphate laxatives in low risk patients: Prospective study

PubMed Central

Casais, Marcela Noemi; Rosa-Diez, Guillermo; Pérez, Susana; Mansilla, Elina Noemi; Bravo, Susana; Bonofiglio, Francisco Carlos

2009-01-01

AIM: To establish the frequency of hyperphosphatemia following the administration of sodium phosphate laxatives in low-risk patients. METHODS: One hundred consecutive ASA I-II individuals aged 35-74 years, who were undergoing colonic cleansing with oral sodium phosphate (OSP) before colonoscopy were recruited for this prospective study. Exclusion criteria: congestive heart failure, chronic kidney disease, diabetes, liver cirrhosis, intestinal obstruction, decreased bowel motility, increased bowel permeability, and hyperparathyroidism. The day before colonoscopy, all the participants entered a 24-h period of diet that consisted of 4 L of clear fluids with sugar or honey and 90 mL (60 g) of OSP in two 45-mL doses, 5 h apart. Serum phosphate was measured before and after the administration of the laxative. RESULTS: The main demographic data (mean ± SD) were: age, 58.9 ± 8.4 years; height, 163.8 ± 8.6 cm; weight, 71 ± 13 kg; body mass index, 26 ± 4; women, 66%. Serum phosphate increased from 3.74 ± 0.56 to 5.58 ± 1.1 mg/dL, which surpassed the normal value (2.5-4.5 mg/dL) in 87% of the patients. The highest serum phosphate was 9.6 mg/dL. Urea and creatinine remained within normal limits. Post-treatment OSP serum phosphate concentration correlated inversely with glomerular filtration rate (P < 0.007, R2 = 0.0755), total body water (P < 0.001, R2 = 0.156) and weight (P < 0.013, R2 = 0.0635). CONCLUSION: In low-risk, well-hydrated patients, the standard dose of OSP-laxative-induced hyperphosphatemia is related to body weight. PMID:20014460

L-Arabinose isomerase and its use for biotechnological production of rare sugars.

PubMed

Xu, Zheng; Li, Sha; Feng, Xiaohai; Liang, Jinfeng; Xu, Hong

2014-11-01

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.

Interaction of Tl +3 with mononucleotides: metal ion binding and sugar conformation

NASA Astrophysics Data System (ADS)

Nafisi, Sh.; Mohajerani, N.; Hadjiakhoondi, A.; Monajemi, M.; Garib, F.

2001-05-01

The interaction of Tl 3+ with sodium salts of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), cytidine-5'-monophosphate (5'-CMP), thymidine 5'-monophosphate (5'-dTMP) in ratios 1 and 2 have been studied in neutral pH. The solid complexes were isolated and characterized by Fourier transform infrared (FTIR) and 1H NMR spectroscopy. In the Tl 2(AMP) 3, Tl 3+ binds directly to N-7 and indirectly to the N-1 position of the pyrimidine ring and phosphate group with sugar moiety in C2'-endoanti. The crystalline salt of Tl 2(GMP) 3 show direct Tl-N-7 and Tl-PO 3(inner-sphere) binding. The conformation of ribose moiety in Tl 2(GMP) 3 is C3'-endoanti. In the Tl 2(CMP) 3, Tl 3+ bind directly to N-3 and PO32- (inner-sphere). The conformation of ribose moiety in Tl 2(CMP) 3 is C2'-endoanti. In the Tl 2(dTMP) 3, Tl 3+ bind indirectly to carbonyl group. The sugar moiety in Tl 2(dTMP) 3 is C3'-endoanti.

The Mycobacterium tuberculosis Complex has a Pathway for the Biosynthesis of 4-Formamido-4,6-Dideoxy-d-Glucose.

PubMed

Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M

2018-05-15

Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

The intrinsic basicity of the phosphate backbone exceeds that of uracil and thymine residues: protonation of the phosphate moiety is preferred over the nucleobase for pdThd and pUrd.

PubMed

Wu, R R; Hamlow, L A; He, C C; Nei, Y-W; Berden, G; Oomens, J; Rodgers, M T

2017-11-22

The gas-phase conformations of the protonated forms of thymidine-5'-monophosphate and uridine-5'-monophosphate, [pdThd+H] + and [pUrd+H] + , are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and electronic structure calculations. The IRMPD action spectra of [pdThd+H] + and [pUrd+H] + are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Low-energy conformations of [pdThd+H] + and [pUrd+H] + and their relative stabilities are computed at the MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers indicate that the dominant conformers of [pdThd+H] + and [pUrd+H] + populated in the experiments are protonated at the phosphate oxo oxygen atom, with a syn nucleobase orientation that is stabilized by strong P[double bond, length as m-dash]OH + O2 and P-OHO4' hydrogen-bonding interactions, and C2'-endo sugar puckering. Minor abundance of conformers protonated at the O2 carbonyl of the nucleobase residue may also contribute for [pdThd+H] + , but do not appear to be important for [pUrd+H] + . Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of thymidine and uridine, [dThd+H] + and [Urd+H] + , and the deprotonated forms of pdThd and pUrd, [pdThd-H] - and [pUrd-H] - , provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Most interestingly, the thymine and uracil nucleobases remain in their canonical forms for [pdThd+H] + and [pUrd+H] + , unlike [dThd+H] + and [Urd+H] + , where protonation occurs on the nucleobases and induces tautomerization of the thymine and uracil residues.

Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases.

PubMed

Liu, Chunliang; Dunaway-Mariano, Debra; Mariano, Patrick S

2017-03-10

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (K i ) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The K i of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

PubMed

Benning, C; Huang, Z H; Gage, D A

1995-02-20

Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

Tetranuclear copper(II) complexes bridged by alpha-D-glucose-1-phosphate and incorporation of sugar acids through the Cu4 core structural changes.

PubMed

Kato, Merii; Sah, Ajay Kumar; Tanase, Tomoaki; Mikuriya, Masahiro

2006-08-21

Tetranuclear copper(II) complexes containing alpha-D-glucose-1-phosphate (alpha-D-Glc-1P), [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(bpy)4(H2O)2]X3 [X = NO3 (1a), Cl (1b), Br (1c)], and [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(phen)4(H2O)2](NO3)3 (2) were prepared by reacting the copper(II) salt with Na2[alpha-D-Glc-1P] in the presence of diimine ancillary ligands, and the structure of 2 was characterized by X-ray crystallography to comprise four {Cu(phen)}2+ fragments connected by the two sugar phosphate dianions in 1,3-O,O' and 1,1-O mu4-bridging fashion as well as a mu-hydroxo anion. The crystal structure of 2 involves two chemically independent complex cations in which the C2 enantiomeric structure for the trapezoidal tetracopper(II) framework is switched according to the orientation of the alpha-D-glucopyranosyl moieties. Temperature-dependent magnetic susceptibility data of 1a indicated that antiferromagnetic spin coupling is operative between the two metal ions joined by the hydroxo bridge (J = -52 cm(-1)) while antiferromagnetic interaction through the Cu-O-Cu sugar phosphate bridges is weak (J = -13 cm(-1)). Complex 1a readily reacted with carboxylic acids to afford the tetranuclear copper(II) complexes, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-CA)2(bpy)4](NO3)2 [CA = CH3COO (3), o-C6H4(COO)(COOH) (4)]. Reactions with m-phenylenediacetic acid [m-C6H4(CH2COOH)2] also gave the discrete tetracopper(II) cationic complex [Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)(CH2COOH))2(bpy)4](NO3)2 (5a) as well as the cluster polymer formulated as {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)2)(bpy)4](NO3)2}n (5b). The tetracopper structure of 1a is converted into a symmetrical rectangular core in complexes 3, 4, and 5b, where the hydroxo bridge is dissociated and, instead, two carboxylate anions bridge another pair of Cu(II) ions in a 1,1-O monodentate fashion. The similar reactions were applied to incorporate sugar acids onto the tetranuclear copper(II) centers. Reactions of 1a with delta-D-gluconolactone, D-glucuronic acid, or D-glucaric acid in dimethylformamide resulted in the formation of discrete tetracopper complexes with sugar acids, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-SA)2(bpy)4](NO3)2 [SA = D-gluconate (6), D-glucuronate (7), D-glucarateH (8a)]. The structures of 6 and 7 were determined by X-ray crystallography to be almost identical with that of 3 with additional chelating coordination of the C-2 hydroxyl group of D-gluconate moieties (6) or the C-5 cyclic O atom of D-glucuronate units (7). Those with D-glucaric acid and D-lactobionic acid afforded chiral one-dimensional polymers, {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-glucarate)(bpy)4](NO3)2}n (8b) and {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-lactobionate)(bpy)4(H2O)2](NO3)3}n (9), respectively, in which the D-Glc-1P-bridged tetracopper(II) units are connected by sugar acid moieties through the C-1 and C-6 carboxylate O atoms in 8b and the C-1 carboxylate and C-6 alkoxy O atoms of the gluconate chain in 9. When complex 7 containing d-glucuronate moieties was heated in water, the mononuclear copper(II) complex with 2-dihydroxy malonate, [Cu(mu-O2CC(OH)2CO2)(bpy)] (10), and the dicopper(II) complex with oxalate, [Cu2(mu-C2O4)(bpy)2(H2O)2](NO3)2 (11), were obtained as a result of oxidative degradation of the carbohydrates through C-C bond cleavage reactions.

Sugar fermentation in probiotic bacteria--an in vitro study.

PubMed

Hedberg, M; Hasslöf, P; Sjöström, I; Twetman, S; Stecksén-Blicks, C

2008-12-01

Food supplemented with probiotic bacteria is a rapidly growing sector of the market. The aim of the present study was to evaluate and compare the acid production of selected probiotic strains available in commercial products. Six Lactobacillus strains (Lactobacillus plantarum 299v and 931; Lactobacillus rhamnosus GG and LB21; Lactobacillus paracasei subsp. paracasei F19, and Lactobacillus reuteri PTA 5289) were cultivated at 37 degrees C in an anaerobic atmosphere on Man, Rogosa, Shape (MRS) agar for 48 h or MRS broth for 16 h. After centrifugation, the cells were washed and resuspended in sterile phosphate-buffered saline and immediately subjected to a fermentation assay with 12 different carbohydrates (nine sugars and three sugar alcohols) in microtiter plates with a pH indicator. The plates were examined for color changes after 24, 48, and 72 h of incubation under aerobic and anaerobic conditions. Three scores were used: negative (pH > 6.8); weak (pH 5.2-6.8), and positive (pH < 5.2). The strains were characterized with the API 50 CH system to confirm their identity. L. plantarum fermented all the sugars except for melibiose, raffinose, and xylitol. Both L. rhamnosus strains were generally less active although L. rhamnosus GG was slightly more active than strain LB21 in the 5% CO(2) setting. The latter strain exhibited negative reactions for sucrose, maltose, arabinose, and sorbitol under anaerobic conditions. The assays with L. paracasei and L. reuteri had negative or weak reactions for all tested sugars under both aerobic and anaerobic conditions. The metabolic capacity to form acid from dietary sugars differed significantly between the various probiotic strains.

Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production.

PubMed

Reshamwala, Shamlan M S; Pagar, Sandip K; Velhal, Vishal S; Maranholakar, Vijay M; Talangkar, Vishal G; Lali, Arvind M

2014-12-01

Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting D-glucose to D-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing D-glucose and equimolar sodium phosphite resulted in d-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

PubMed Central

Fettke, Joerg; Leifels, Lydia; Brust, Henrike; Herbst, Karoline; Steup, Martin

2012-01-01

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-14C]glucose 1-phosphate, [U-14C]sucrose, [U-14C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-14C]sucrose plus unlabelled equimolar glucose 1-phosphate. 14C-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 14C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-14C]glucose 1-phosphate or adenosine-[U-14C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro 14C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells. PMID:22378944

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

PubMed

Fettke, Joerg; Leifels, Lydia; Brust, Henrike; Herbst, Karoline; Steup, Martin

2012-05-01

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹⁴C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹⁴-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹⁴C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹⁴C]glucose 1-phosphate or adenosine-[U-¹⁴C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹⁴C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

Structure of the inositol-1-phosphate cytidylyltransferase from Thermotoga maritima.

PubMed

Kurnasov, Oleg V; Luk, Hung-Jie Daniel; Roberts, Mary F; Stec, Boguslaw

2013-09-01

The unique steps in the synthesis of an unusual osmolyte in hyperthermophiles, di-myo-inositol-1,1'-phosphate (DIP), involve the production of CDP-inositol and its condensation with an inositol-1-phosphate molecule to form phosphorylated DIP. While many organisms fuse both activities into a single enzyme, the two are separate in Thermotoga maritima. The crystal structure of the T. maritima inositol-1-phosphate cytidylyltransferase, which as a soluble protein may transiently associate with its membrane-embedded partner phospho-DIP synthase (P-DIPS), has now been obtained. The structure shows a conserved motif of sugar nucleotide transferases (COG1213) with a structurally reinforced C-terminal Cys bonded to the core of the protein. A bound arsenosugar identifies the location of the active site for inositol 1-phosphate. Based on homologous structures from several species and the identification of the crucial conserved aspartate residue, a catalytic mechanism for this enzyme is proposed as well as a mode for its association with P-DIPS. This structure imposes constraints on the mode of association, communication and temperature activation of two separate enzymes in T. maritima. For the first time, a working model for the membrane-bound P-DIPS unit has been constructed. This sheds light on the functioning of the phosphatidylserine and phosphatidylinositol synthases involved in many physiological processes that are homologous to P-DIPS. This work provides fresh insights into the synthesis of the unusual thermoprotective compound DIP in hyperthermophiles.

Induction of Hexose-Phosphate Translocator Activity in Spinach Chloroplasts.

PubMed Central

Quick, W. P.; Scheibe, R.; Neuhaus, H. E.

1995-01-01

Many environmental and experimental conditions lead to accumulation of carbohydrates in photosynthetic tissues. This situation is typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly to leaves. In this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for several days. These chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability to perform starch synthesis from exogenous glucose-6-phosphate (Glc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Glucose-1-phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Glc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a novel Glc-6-P-translocator capacity is present in the envelope membrane, exhibiting an apparent Km of 0.54 mM and a Vmax of 2.9 [mu]mol Glc-6-P mg-1 chlorophyll h-1. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. The generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates is not supported by these results. A physiological role for a Glc-6-P translocator in green plastids is presented with reference to the source/sink function of the leaf. PMID:12228584

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed Central

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells. PMID:9003425

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.

The Path of Carbon in Photosynthesis XII. Some Temperature Effects

DOE R&D Accomplishments Database

Ouellet, C.

1951-06-25

The photosynthetic assimilation of radioactive carbon dioxide for two-minute periods by Scenedesmus has bee studied at temperatures ranging from 25? to 44? C. All labeled intermediates cease to be formed at about 45? C. With rising temperature, the radioactivity reaching the sugar phosphate reservoirs decreases regularly while there is a sharp maximum in sucrose at 37? C. and a less pronounced one in malic and aspartic acids about 40? C. A tentative interpretation of these effects is offered.

Transcriptome and metabolome analyses of sugar and organic acid metabolism in Ponkan (Citrus reticulata) fruit during fruit maturation.

PubMed

Lin, Qiong; Wang, Chengyang; Dong, Wencheng; Jiang, Qing; Wang, Dengliang; Li, Shaojia; Chen, Ming; Liu, Chunrong; Sun, Chongde; Chen, Kunsong

2015-01-01

Ponkan (Citrus reticulata Blanco cv. Ponkan) is an important mandarin citrus in China. However, the low ratio of sugars to organic acids makes it less acceptable for consumers. In this work, three stages (S120, early development stage; S195, commercial harvest stage; S205, delayed harvest stage) of Ponkan fruit were selected for study. Among 28 primary metabolites analyzed in fruit, sugars increased while organic acids in general decreased. RNA-Seq analysis was carried out and 19,504 genes were matched to the Citrus clementina genome, with 85 up-regulated and 59 down-regulated genes identified during fruit maturation. A sucrose phosphate synthase (SPS) gene was included in the up-regulated group, and this was supported by the transcript ratio distribution. Expression of two asparagine transferases (AST), and a specific ATP-citrate lyase (ACL) and glutamate decarboxylase (GAD) members increased during fruit maturation. It is suggested that SPS, AST, ACL and GAD coordinately contribute to sugar accumulation and organic acid degradation during Ponkan fruit maturation. Both the glycolysis pathway and TCA cycle were accelerated during later maturation, indicating the flux change from sucrose metabolism to organic acid metabolism was enhanced, with citrate degradation occurring mainly through the gamma-aminobutyric acid (GABA) and acetyl-CoA pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

PubMed

Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

2017-07-01

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering of Corynebacterium glutamicum for growth and succinate production from levoglucosan, a pyrolytic sugar substrate.

PubMed

Kim, Eun-Mi; Um, Youngsoon; Bott, Michael; Woo, Han Min

2015-10-01

Thermochemical processing provides continuous production of bio-oils from lignocellulosic biomass. Levoglucosan, a pyrolytic sugar substrate C6H10O5 in a bio-oil, has been used for ethanol production using engineered Escherichia coli. Here we provide the first example for succinate production from levoglucosan with Corynebacterium glutamicum, a well-known industrial amino acid producer. Heterologous expression of a gene encoding a sugar kinase from Lipomyces starkeyi, Gibberella zeae or Pseudomonas aeruginosa was employed for levoglucosan conversion in C. glutamicum because the wild type was unable to utilize levoglucosan as sole carbon source. As result, expression of a levoglucosan kinase (LGK) of L. starkeyi only enabled growth with levoglucosan as sole carbon source in CgXII minimal medium by catalyzing conversion of levoglucosan to glucose-6-phosphate. Subsequently, the lgk gene was expressed in an aerobic succinate producer of C. glutamicum, strain BL-1. The recombinant strain showed a higher succinate yield (0.25 g g(-1)) from 2% (w/v) levoglucosan than the reference strain BL-1 from 2% (w/v) glucose (0.19 g g(-1)), confirming that levoglucosan is an attractive carbon substrate for C. glutamicum producer strains. In summary, we demonstrated that a pyrolytic sugar could be a potential carbon source for microbial cell factories. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of oral D-tagatose, a stereoisomer of D-fructose, on liver metabolism in man as examined by 31P-magnetic resonance spectroscopy.

PubMed

Buemann, B; Gesmar, H; Astrup, A; Quistorff, B

2000-10-01

D-tagatose, which is a stereoisomer of D-fructose, is phosphorylated to D-tagatose-1-phosphate by fructokinase in the liver. Because of a slow degradation rate of D-tagatose-1-phosphate, this substance may accumulate, and ingested D-tagatose may therefore cause a longer lasting reduction in inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in the liver compared with D-fructose. Similar to what is seen in patients with hereditary fructose intolerance, this may increase purine nucleotide degradation and thereby increase uric acid production. The effect of 30 g D-tagatose or D-fructose administered orally on ketohexose-1-phosphates, ATP, and Pi levels in the liver was studied by 31P-magnetic resonance spectroscopy (PMRS) in 5 young male volunteers. Blood and urine were collected to detect a possible increased uric acid production. A peak at 5.2 ppm assigned as D-tagatose-1-phosphate equivalent to about 1 mmol/L was found in the spectrum within 30 minutes after D-tagatose was administered in all subjects. Concomitantly, ATP was reduced by about 12% (P < .05). Both effects had vanished after 150 minutes. Serum uric acid concentration was increased by 17% 50 minutes after D-tagatose (P < .05) and did not reach baseline level when the experiment was terminated 230 minutes after the load. Although renal fractional extraction of uric acid decreased by approximately 12%, this could not explain the acute hyperuricemic effect of D-tagatose. No changes in 31PMRS spectra or serum uric acid concentration were found after D-fructose. These results suggest that a moderate intake of D-tagatose may affect liver metabolism by phosphate trapping despite the fact that the sugar may only be incompletely absorbed in the gut.

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

PubMed Central

Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

2013-01-01

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine.

PubMed

Biswas, Pradip K; Behrman, Edward J; Gopalan, Venkat

2017-04-01

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

PubMed Central

Führing, Jana Indra; Cramer, Johannes Thomas; Schneider, Julia; Baruch, Petra; Gerardy-Schahn, Rita; Fedorov, Roman

2015-01-01

In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens. PMID:25860585

Production of biomass/energy crops on phosphatic clay soils in central Florida

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stricker, J.A.; Prine, G.M.; Woodard, K.R.

1993-12-31

Phosphatic clay is a byproduct of phosphate mining. Presently more than 40,470 ha have been created, most in central Florida, and about 810 ha are being added each year. Phosphatic clays have high fertility and high water holding capacity, reducing fertilization costs and producing high yields without irrigation. Based on 10 years of research, scientists have selected tall annual-regenerating perennial C-4 grasses as having the greatest potential for biomass production in Florida. The purpose of this work was to determine the feasibility of growing these tall perennial grasses for biomass on phosphatic clay. Elephantgrass, sugarcane and energycane, and erianthus weremore » planted in duplicate replications on phosphatic clay soil in late August, 1986. yield was measured by one harvest in December or January each year for four years. Nitrogen fertilization included 112 kg ha{sup {minus}1} the first year followed by 134 kg ha{sup {minus}1} for the next three years. Nitrogen is the only supplemental nutrient needed to grow all tall grass crops on phosphatic clay. The average annual oven dry matter yield over the 4-yr period was 36.3 Mg ha{sup {minus}1} for PI 300086 elephantgrass, 45.2 for N51 elephantgrass, 42.5 for L79-1002 energycane, 49.0 for US72-1153 energycane, 49.7 for US78-1009 sugarcane, 52.2 for US56-9 sugarcane, 56.2 for CP72-1210 sugarcane, and 48.8 for 1K-7647 erianthus. More recent work has utilized domestic sewage sludge as a nitrogen source for the tall grasses. Preliminary sugar yields of selected sugarcane accessions & sweet sorghum were 4.7 Mg ha{sup {minus}1} for CP72-1210, 12.5 for US67-2022, 3.4 for US78-1009 and 1.3 Mg ha{sup {minus}1} for sweet sorghum. The high yields of the tall grasses grown on phosphatic clay with low inputs indicate a great potential for these crops as a source of renewable energy. A sustainable cropping system may be maintained by utilizing municipal sewage sludge as a nitrogen source with tall grasses on phosphatic clay.« less

Chemical intervention in plant sugar signalling increases yield and resilience

NASA Astrophysics Data System (ADS)

Griffiths, Cara A.; Sagar, Ram; Geng, Yiqun; Primavesi, Lucia F.; Patel, Mitul K.; Passarelli, Melissa K.; Gilmore, Ian S.; Steven, Rory T.; Bunch, Josephine; Paul, Matthew J.; Davis, Benjamin G.

2016-12-01

The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a ‘signalling-precursor’ concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function.

Optimization of a novel sequential alkalic and metal salt pretreatment for enhanced delignification and enzymatic saccharification of corn cobs.

PubMed

Sewsynker-Sukai, Yeshona; Gueguim Kana, E B

2017-11-01

This study presents a sequential sodium phosphate dodecahydrate (Na 3 PO 4 ·12H 2 O) and zinc chloride (ZnCl 2 ) pretreatment to enhance delignification and enzymatic saccharification of corn cobs. The effects of process parameters of Na 3 PO 4 ·12H 2 O concentration (5-15%), ZnCl 2 concentration (1-5%) and solid to liquid ratio (5-15%) on reducing sugar yield from corn cobs were investigated. The sequential pretreatment model was developed and optimized with a high coefficient of determination value (0.94). Maximum reducing sugar yield of 1.10±0.01g/g was obtained with 14.02% Na 3 PO 4 ·12H 2 O, 3.65% ZnCl 2 and 5% solid to liquid ratio. Scanning electron microscopy (SEM) and Fourier Transform Infrared analysis (FTIR) showed major lignocellulosic structural changes after the optimized sequential pretreatment with 63.61% delignification. In addition, a 10-fold increase in the sugar yield was observed compared to previous reports on the same substrate. This sequential pretreatment strategy was efficient for enhancing enzymatic saccharification of corn cobs. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, C.W.B.; Das Gupta, S.K.; Mattai, J.

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition at temperatures similar to the transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. {sup 2}H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and {sup 13}C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicting very little motion atmore » the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. The authors postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluididty of the hydrophobic region in the L{sub {lambda}} phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.« less

The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz, Z.; Xavier, K; Miller, S

2009-01-01

Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (??)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology withmore » aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.« less

Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

NASA Astrophysics Data System (ADS)

Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

2017-06-01

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

The level of sugars and synthesis of trehalose in Ascaris suum tissues.

PubMed

Dmitryjuk, M; Łopieńska-Biernat, E; Farjan, M

2009-09-01

The activities of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) were observed in muscles, individual parts of the reproductive system and haemolymph of Ascaris suum. The highest activity of TPS was detected in the upper uterus, while the lowest activity of TPS was detected in the ovary and oviduct of the nematode. Relatively high activity was detected in muscles, haemolymph and two remaining parts of the uterus. The TPP activity was the highest in lower length of the uterus, following muscles, ovary, central and upper uterus. The lowest activity of TPP was detected in the haemolymph and oviduct of A. suum. Besides TPS and TPP, trehalose was also detected in the studied tissues except the cuticle and the intestine. Glucose was present in all organs, but the highest concentration was found in the cuticle and intestine.

Molecular mechanical studies of proflavine and acridine orange intercalation.

PubMed Central

Dearing, A; Weiner, P; Kollman, P A

1981-01-01

Previous workers have reported that proflavine and acridine orange form various structurally different complexes with the dinucleoside phosphates rCpG and dCpG, with uniform C3'-endo and mixed C3'-endo (3'-5') C2'-endo sugar puckers being observed. We present theoretical calculations, based on the method of molecular mechanics, which support the experimental observations. The results suggest that the mixed C3'-edo (3'-5') C2'-endo pucker conformation isi intrinsically more stable than the uniform C3'-endo conformation, but that the additional stabilisation gained from specific, hydrogen bonding, interactions between nucleic acid and solvent, or intramolecularly within the nucleic acid, can lead to the adoption of the latter conformation, or of variants between the two. The role played by hydrogen bonding between amino-groups and nucleic acid phosphate appears more subtle than previously supposed. PMID:7232221

Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol.

PubMed

van der Woude, Aniek D; Perez Gallego, Ruth; Vreugdenhil, Angie; Puthan Veetil, Vinod; Chroumpi, Tania; Hellingwerf, Klaas J

2016-04-08

Erythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803. By functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates. After several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium.

Effects of salinity on physicochemical properties of Alaska pollock surimi after repeated freeze-thaw cycles.

PubMed

Kang, E J; Hunt, A L; Park, J W

2008-06-01

The effects of residual salt in surimi on physicochemical properties as affected by various freeze and thaw (FT) cycles were examined. Fresh Alaska pollock surimi was mixed with 4.0% sugar and 5.0% sorbitol, along with 8 combinations of salt (0.4%, 0.6%, 0.8%, and 1.0% NaCl) and sodium polyphosphate (0.25% and 0.5%), vacuum-packed, and stored at -18 degrees C until used. FT cycles (0, 6, and 9) were used to mimic long-term frozen storage. At the time of gel preparation, each treatment was appropriately adjusted to maintain 2% salt and 78% moisture. The pH decreased as residual salt increased during frozen storage. Salt extractable protein (SEP) decreased (P < 0.05) as FT cycles extended from 0 to 9. Regardless of residual salt and phosphate concentration during frozen storage, whiteness value (L*- 3b*) decreased (P < 0.05) as FT cycles extended, except for samples with 0.4% salt/0.5% phosphate and 0.6% salt/0.25% phosphate. Water retention ability (WRA) and texture significantly (P < 0.05) decreased at higher salt content (0.8% and 1.0%) after 9 FT cycles, indicating higher residual salt concentration can shorten the shelf life of frozen surimi. Our study revealed lower residual salt concentration and higher phosphate concentration are likely to extend the shelf life of frozen surimi.

A Binary-Encounter-Bethe Approach to Simulate DNA Damage by the Direct Effect

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cucinotta, Francis A.

2013-01-01

The DNA damage is of crucial importance in the understanding of the effects of ionizing radiation. The main mechanisms of DNA damage are by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research in this area, many questions on the formation of DNA damage remains. To refine existing DNA damage models, an approach based on the Binary-Encounter-Bethe (BEB) model was developed[1]. This model calculates differential cross sections for ionization of the molecular orbitals of the DNA bases, sugars and phosphates using the electron binding energy, the mean kinetic energy and the occupancy number of the orbital. This cross section has an analytic form which is quite convenient to use and allows the sampling of the energy loss occurring during an ionization event. To simulate the radiation track structure, the code RITRACKS developed at the NASA Johnson Space Center is used[2]. This code calculates all the energy deposition events and the formation of the radiolytic species by the ion and the secondary electrons as well. We have also developed a technique to use the integrated BEB cross section for the bases, sugar and phosphates in the radiation transport code RITRACKS. These techniques should allow the simulation of DNA damage by ionizing radiation, and understanding of the formation of double-strand breaks caused by clustered damage in different conditions.

Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress.

PubMed

Debnath, Mousumi; Ashwath, Nanjappa; Hill, Camilla Beate; Callahan, Damien L; Dias, Daniel Anthony; Jayasinghe, Nirupama Samanmalie; Midmore, David James; Roessner, Ute

2018-05-10

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na + and compatible organic solutes, and more K + in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

PubMed

Deroover, Sofie; Ghillebert, Ruben; Broeckx, Tom; Winderickx, Joris; Rolland, Filip

2016-06-01

Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Peach leaf curl disease shifts sugar metabolism in severely infected leaves from source to sink.

PubMed

Moscatello, Stefano; Proietti, Simona; Buonaurio, Roberto; Famiani, Franco; Raggi, Vittorio; Walker, Robert P; Battistelli, Alberto

2017-03-01

Peach leaf curl is a disease that affects the leaves of peach trees, and in severe cases all of the leaf can be similarly affected. This study investigated some effects of this disease on the metabolism of peach leaves in which all parts of the leaf were infected. These diseased leaves contained very little chlorophyll and performed little or no photosynthesis. Compared to uninfected leaves, diseased leaves possessed higher contents of fructose and especially glucose, but lowered contents of sucrose, sorbitol and especially starch. The activities of soluble acid invertase, neutral invertase, sorbitol dehydrogenase and sucrose synthase were all higher in diseased leaves, whereas, those of aldose-6-phosphate reductase and sucrose phosphate synthase were lower. The activities of hexokinase and fructokinase were little changed. In addition, immunblots showed that the contents of Rubisco and ADP-glucose phosphorylase were reduced in diseased leaves, whereas, the content of phosphoenolpyruvate carboxylase was increased. The results show that certain aspects of the metabolism of diseased leaves are similar to immature sink leaves. That is photosynthetic function is reduced, the leaf imports rather than exports sugars, and the contents of non-structural carbohydrates and enzymes involved in their metabolism are similar to sink leaves. Further, the effects of peach leaf curl on the metabolism of peach leaves are comparable to the effects of some other diseases on the metabolism of photosynthetic organs of other plant species. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Development and application of microbial selective plugging processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenneman, G.E.; Gevertz, D.; Davey, M.E.

1995-12-31

Phillips Petroleum Company recently completed a microbial selective plugging (MSP) pilot at the North Burbank Unit (NBU), Shidler, Oklahoma. Nutrients were selected for the pilot that could stimulate indigenous microflora in the reservoir brine to grow and produce exopolymer. It was found that soluble corn starch polymers (e.g., maltodextrins) stimulated the indigenous bacteria to produce exopolymer, whereas simple sugars (e.g., glucose and sucrose), as well as complex media (e.g., molasses and Nutrient Broth), did not. Injection of maltodextrin into rock cores in the presence of indigenous NBU bacteria resulted in stable permeability reductions (> 90%) across the entire length, whilemore » injection of glucose resulted only in face plugging. In addition, it was found that organic phosphate esters (OPE) served as a preferable source of phosphorus for the indigenous bacteria, since orthophosphates and condensed phosphates precipitated in NBU brine at reservoir temperature (45{degrees}C). Injection of maltodextrin and ethyl acid phosphate into a producing well stimulated an increase in maltodextrin utilizing bacteria (MUB) in the back-flowed, produced fluid. Additional screens of indigenous and nonindigenous bacteria yielded several nonindigenous isolates that could synthesize polymer when growing in brine containing 6% NaCl at 45{degrees}C.« less

Remineralisation by chewing sugar-free gums in a randomised, controlled in situ trial including dietary intake and gauze to promote plaque formation.

PubMed

Cochrane, N J; Shen, P; Byrne, S J; Walker, G D; Adams, G G; Yuan, Y; Reynolds, C; Hoffmann, B; Dashper, S G; Reynolds, E C

2012-01-01

Remineralisation has been shown to be an effective mechanism of preventing the progression of enamel caries. The aim of this double-blind, randomised, cross-over in situ study was to compare enamel remineralisation by chewing sugar-free gum with or without casein phosphopeptide amorphous calcium phosphate (CPP-ACP) where the enamel lesions were exposed to dietary intake and some were covered with gauze to promote plaque formation. Participants wore removable palatal appliances containing 3 recessed enamel half-slabs with subsurface lesions covered with gauze and 3 without gauze. Mineral content was measured by transverse microradiography, and plaque composition was analysed by real-time polymerase chain reaction. For both the gauze-free and gauze-covered lesions, the greatest amount of remineralisation was produced by the CPP-ACP sugar-free gum, followed by the gum without CPP-ACP and then the no-gum control. Recessing the enamel in the appliance allowed plaque accumulation without the need for gauze. There was a trend of less remineralisation and greater variation in mineral content for the gauze-covered lesions. The cell numbers of total bacteria and streptococci were slightly higher in the plaque from the gauze-covered enamel for 2 of the 3 treatment legs; however, there was no significant difference in Streptococcus mutans cell numbers. In conclusion, chewing sugar-free gum containing CPP-ACP promoted greater levels of remineralisation than a sugar-free gum without CPP-ACP or a no-gum control using an in situ remineralisation model including dietary intake irrespective of whether gauze was used to promote plaque formation or not. Copyright © 2012 S. Karger AG, Basel.

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars

PubMed Central

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-01-01

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. PMID:28298443

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry1[W][OA

PubMed Central

Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan; LaClair, Russell W.; Shachar-Hill, Yair

2010-01-01

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-13CFru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO3]−/or [H2PO4]− ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure 13C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments. PMID:20442274

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

PubMed

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-05-05

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

PubMed Central

Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

2015-01-01

The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric.

PubMed

Hubbard, N L; Pharr, D M; Huber, S C

1990-09-01

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO(2) respired during ripening was positively correlated with sugar accumulation (R(2) = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO(2) was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.

Role of Sucrose Phosphate Synthase in Sucrose Biosynthesis in Ripening Bananas and Its Relationship to the Respiratory Climacteric 1

PubMed Central

Hubbard, Natalie L.; Pharr, D. Mason; Huber, Steven C.

1990-01-01

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO2 respired during ripening was positively correlated with sugar accumulation (R2 = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO2 was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP. PMID:16667688

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

PubMed

Korte, Jan; Alber, Marina; Trujillo, Carolina M; Syson, Karl; Koliwer-Brandl, Hendrik; Deenen, René; Köhrer, Karl; DeJesus, Michael A; Hartman, Travis; Jacobs, William R; Bornemann, Stephen; Ioerger, Thomas R; Ehrt, Sabine; Kalscheuer, Rainer

2016-12-01

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice

PubMed Central

Koliwer-Brandl, Hendrik; Hartman, Travis; Jacobs, William R.; Ioerger, Thomas R.; Ehrt, Sabine

2016-01-01

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors. PMID:27936238

Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

PubMed

Saier, M H; Keeler, D K; Feucht, B U

1982-03-10

Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

PubMed Central

Mostafa, Yasser S.; Alamri, Saad A.

2012-01-01

Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH2PO4 was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid. PMID:23961184

Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms.

PubMed

Cook, G M; Kearns, D B; Russell, J B; Reizer, J; Saier, M H

1995-09-01

Streptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme IIs for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl beta-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlc rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

A Snapshot of the Plant Glycated Proteome

PubMed Central

Bilova, Tatiana; Lukasheva, Elena; Brauch, Dominic; Greifenhagen, Uta; Paudel, Gagan; Tarakhovskaya, Elena; Frolova, Nadezhda; Mittasch, Juliane; Balcke, Gerd Ulrich; Tissier, Alain; Osmolovskaya, Natalia; Vogt, Thomas; Wessjohann, Ludger A.; Birkemeyer, Claudia; Milkowski, Carsten; Frolov, Andrej

2016-01-01

Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs. PMID:26786108

From chaperonins to Rubisco assembly and metabolic repair.

PubMed

Hayer-Hartl, Manajit

2017-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO 2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP-dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries. © 2017 The Protein Society.

Gas Chromatography/Mass Spectrometry-Based Metabolite Profiling of Nutrients and Antinutrients in Eight Lens and Lupinus Seeds (Fabaceae).

PubMed

Farag, Mohamed A; Khattab, Amira R; Ehrlich, Anja; Kropf, Matthias; Heiss, Andreas G; Wessjohann, Ludger A

2018-04-25

Lens culinaris and several Lupinus species are two legumes regarded as potential protein resources aside from their richness in phytochemicals. Consequently, characterization of their metabolite composition seems warranted to be considered as a sustainable commercial functional food. This study presents a discriminatory holistic approach for metabolite profiling in accessions of four lentil cultivars and four Lupinus species via gas chromatography/mass spectrometry. A total of 107 metabolites were identified, encompassing organic and amino acids, sugars, and sterols, along with antinutrients, viz., alkaloids and sugar phosphates. Among the examined specimens, four nutritionally valuable accessions ought to be prioritized for future breeding to include Lupinus hispanicus, enriched in organic ( ca. 11.7%) and amino acids ( ca. 5%), and Lupinus angustifolius, rich in sucrose ( ca. 40%), along with two dark-colored lentil cultivars 'verte du Puy' and 'Black Beluga' enriched in peptides. Antinutrient chemicals were observed in Lupinus polyphyllus, owing to its high alkaloid content. Several species-specific markers were also revealed using multivariate data analyses.

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering.

PubMed

Nic Lochlainn, Laura; Caffrey, Patrick

2009-01-01

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.

Sn-Beta zeolites with borate salts catalyse the epimerization of carbohydrates via an intramolecular carbon shift

PubMed Central

Gunther, William R.; Wang, Yuran; Ji, Yuewei; Michaelis, Vladimir K.; Hunt, Sean T.; Griffin, Robert G.; Román-Leshkov, Yuriy

2012-01-01

Carbohydrate epimerization is an essential technology for the widespread production of rare sugars. In contrast to other enzymes, most epimerases are only active on sugars substituted with phosphate or nucleotide groups, thus drastically restricting their use. Here we show that Sn-Beta zeolite in the presence of sodium tetraborate catalyses the selective epimerization of aldoses in aqueous media. Specifically, a 5 wt% aldose (for example, glucose, xylose or arabinose) solution with a 4:1 aldose:sodium tetraborate molar ratio reacted with catalytic amounts of Sn-Beta yields near-equilibrium epimerization product distributions. The reaction proceeds by way of a 1,2 carbon shift wherein the bond between C-2 and C-3 is cleaved and a new bond between C-1 and C-3 is formed, with C-1 moving to the C-2 position with an inverted configuration. This work provides a general method of performing carbohydrate epimerizations that surmounts the main disadvantages of current enzymatic and inorganic processes. PMID:23047667

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027Wmore » revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Shanklin, John; Khusnutdinova, Anna

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Electron-Impact Ionization and Dissociative Ionization of Biomolecules

NASA Technical Reports Server (NTRS)

Huo, Winifred M.; Chaban, Galina M.; Dateo, Christopher E.

2006-01-01

It is well recognized that secondary electrons play an important role in radiation damage to humans. Particularly important is the damage of DNA by electrons, potentially leading to mutagenesis. Molecular-level study of electron interaction with DNA provides information on the damage pathways and dominant mechanisms. Our study of electron-impact ionization of DNA fragments uses the improved binary-encounter dipole model and covers DNA bases, sugar phosphate backbone, and nucleotides. An additivity principle is observed. For example, the sum of the ionization cross sections of the separate deoxyribose and phosphate fragments is in close agreement with the C3(sup prime)- and C5 (sup prime)-deoxyribose-phospate cross sections, differing by less than 5%. Investigation of tandem double lesion initiated by electron-impact dissociative ionization of guanine, followed by proton reaction with the cytosine in the Watson-Crick pair, is currently being studied to see if tandem double lesion can be initiated by electron impact. Up to now only OH-induced tandem double lesion has been studied.

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE PAGES

Huang, Lili; Shanklin, John; Khusnutdinova, Anna; ...

2016-06-20

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

The regulation of nucleotide metabolism of immune cells: papaverine induced nucleotide breakdown.

PubMed

Sheppard, H; Sass, S; Tsien, W H

1980-06-01

During a period of prelabeling of mouse thymus cells with any nucleoside at 4 degrees C, nucleoside phosphates accumulated, but no nucleic acid synthesis occurred. Elevating the temperature to 37 degrees C then led to incorporation into the respective nucleic acid reaching a maximum in 5--15 min. Papaverine inhibited this incorporation (IC50:50 muM) and caused an efflux of label into the medium as a nonphosphorylated product. The responses of the different nucleotide phosphate pools showed more dependency on the base then the sugar moeity. The effect of papaverine could not be altered or mimicked by deprivation of oxygen, glucose, or calcium. Mouse spleen cells responded like thymocytes to papaverine, but rat GH3 pituitary cell DNA syntesis was only transiently inhibited with no concomitant efflux of 3H into the medium. As expected, thymus cellular adenosine triphosphate (ATP), determined by the luciferin-luciferase reaction, decreased in the presence of papaverine; suprisingly, extracellular ATP fell as well. The results suggest that decreases in cellular ATP of mouse thymus cells leads to reductions of all nucleoside phosphates and the efflux of the resultant nucleosides. Papaverine may effect a decrease in the ATP levels by activating a phosphohydrolase rather than, or in addition to, the previously suggested inhibition of mitochondrial electron transport.

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, M.; Beevers, H.

1979-07-01

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase,more » sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO/sub 2/ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.« less

Characterization of Sugar Contents and Sucrose Metabolizing Enzymes in Developing Leaves of Hevea brasiliensis

PubMed Central

Zhu, Jinheng; Qi, Jiyan; Fang, Yongjun; Xiao, Xiaohu; Li, Jiuhui; Lan, Jixian; Tang, Chaorong

2018-01-01

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants. PMID:29449852

Short communication: Development of a rapid laboratory method to polymerize lactose to nondigestible carbohydrates.

PubMed

Kuechel, A F; Schoenfuss, T C

2018-04-01

Nondigestible carbohydrates with a degree of polymerization between 3 and 10 (oligosaccharides) are commonly used as dietary fiber ingredients in the food industry, once they have been confirmed to have positive effects on human health by regulatory authorities. These carbohydrates are produced through chemical or enzymatic synthesis. Polylactose, a polymerization product of lactose and glucose, has been produced by reactive extrusion using a twin-screw extruder, with citric acid as the catalyst. Trials using powdered cheese whey permeate as the lactose source for this reaction were unsuccessful. The development of a laboratory method was necessary to investigate the effect of ingredients present in permeate powder that could be inhibiting polymerization. A Mars 6 Microwave Digestion System (CEM Corp., Matthews, NC) was used to heat and polymerize the sugars. The temperatures had to be lowered from extrusion conditions to produce a caramel-like product and not decompose the sugars. Small amounts of water had to be added to the reaction vessels to allow consistent heating of sugars between vessels. Elevated levels of water (22.86 and 28.57%, vol/wt) and calcium phosphate (0.928 and 1.856%, wt/wt) reduced the oligosaccharide yield in the laboratory method. Increasing the citric acid (catalyst) concentration increased the oligosaccharide yield for the pure sugar blend and when permeate powder was used. The utility of the laboratory method to predict oligosaccharide yields was confirmed during extrusion trials of permeate when this increased acid catalyst concentration resulted in similar oligosaccharide concentrations. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Chemical characteristics and enhanced hepatoprotective activities of Maillard reaction products derived from milk protein-sugar system.

PubMed

Oh, Nam Su; Young Lee, Ji; Lee, Hyun Ah; Joung, Jae Yeon; Shin, Yong Kook; Kim, Sae Hun; Kim, Younghoon; Lee, Kwang Won

2016-02-01

The objective of this study was to investigate the characteristics, antioxidative properties, and hepatoprotective effects of Maillard reaction products (MRP) from milk protein reacted with sugars. The MRP were obtained from milk protein, whey protein concentrates and sodium caseinate, using 2 types of sugars, lactose and glucose, by heating the mixture at 55°C for 7d in a sodium phosphate buffer (pH 7.4). Changes in the chemical modification of the milk protein were monitored by measuring the protein-bound carbonyls and PAGE protein profiles. The results showed that the amount of protein-bound carbonyls increased after Maillard reaction (MR). In addition, sodium dodecyl sulfate-PAGE analysis indicated a formation of high-molecular weight complexes through MR. The modification sites induced by MR of milk protein were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of tryptic-digested gel spots of MRP. As a result, modification and their localization in AA sequence of MRP was identified. Also, the MRP showed higher antioxidant activities than the intact milk protein, and they reduced intracellular reactive oxygen species production and inhibited the depletion of the reduced glutathione concentrations in the HepG2 cells. In particular, glucose-sodium caseinate MRP showed the highest biological activities among all MRP. Therefore, these results suggest that the MRP from milk protein reacting with sugars possess effective antioxidant activity and have a protective ability against oxidative damage. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Distinct hydroxy-radical-induced damage of 3'-uridine monophosphate in RNA: a theoretical study.

PubMed

Zhang, Ru bo; Eriksson, Leif A

2009-01-01

RNA strand scission and base release in 3'-uridine monophosphate (UMP), induced by OH radical addition to uracil, is studied at the DFT B3LYP/6-31+G(d,p) level in the gas phase and in solution. In particular, the mechanism of hydrogen-atom transfer subsequent to radical formation, from C2' on the sugar to the C6 site on the base, is explored. The barriers of (C2'-)H2'(a) abstraction by the C6 radical site range from 11.2 to 20.0 kcal mol(-1) in the gas phase and 14.1 to 21.0 kcal mol(-1) in aqueous solution, indicating that the local surrounding governs the hydrogen-abstraction reaction in a stereoselective way. The calculated N1-C1' (N1-glycosidic bond) and beta-phosphate bond strengths show that homolytic and heterolytic bond-breaking processes are largely favored in each case, respectively. The barrier for beta-phosphate bond rupture is approximately 3.2-4.0 kcal mol(-1) and is preferred by 8-12 kcal mol(-1) over N1-glycosidic bond cleavage in both the gas phase and solution. The beta-phosphate bond-rupture reactions are exothermal in the gas phase and solution, whereas N1-C1' bond-rupture reactions require both solvation and thermal corrections at 298 K to be energetically favored. The presence of the ribose 2'-OH group and its formation of low-barrier hydrogen bonds with oxygen atoms of the 3'-phosphate linkage are highly important for hydrogen transfer and the subsequent bond-breakage reactions.

Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

PubMed

Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

2015-10-23

The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of selected chemical parameters in Piemontese wines.

PubMed

Stępień, Agnieszka E; Stawarczyk, Kinga; Bilek, Maciej; Kędziora, Katarzyna M

2015-01-01

Piemontese wines are well known and valued all over the world. The most popular of them are Barolo and Barbaresco wines. However, in Poland, they are still little known and only now are being gradually introduced to a wider range of consumers. The aim of this study was to evaluate the content of inorganic anions, minerals, sugars and glycerol of Piemontese wines from micro-region Langhe, classified as DOCG ("Denominazione di Origine Controllata e Garantita", ie. controlled designation of origin guaranteed) and DOC ("Denominazione di Origine Controllata", ie. controlled designation of origin) products. Seven types of red wines and one type of white wine were tested. High Performance Ion Chromatography with conductometric detection (HPLC-CD) was used to measure the content of inorganic anions, ie. fluorides, chlorides, sulfates and phosphates. Flame atomic absorption spectrometry (F-AAS) was used to measure the content of minerals, ie. magnesium, calcium, sodium, copper, potassium, zinc and iron, while High Performance Liquid Chromatography with charged aerosol detection (HPLC-CAD) was used to measure the content of glycerol and sugars, ie. fructose, glucose and sucrose. Our studies show that although Piemontese wines are characterized by a relatively low content of minerals in comparison with the wines from other regions, they contain a lot of ingredients that have beneficial effects for human health. Moreover, we observed that the studied wines contain particularly high concentration of inorganic ions--phosphates and fluorides. Furthermore, all tested red wines show far reaching similarities in their chemical properties, which is possibly a direct consequence of using in their production locally cultivated grape varieties. Analysis of the wines from the Piemont region, classified as DOCG, DOC, confirmed that these are dry wines of a high quality.

Sucrose-Metabolizing Enzymes in Transport Tissues and Adjacent Sink Structures in Developing Citrus Fruit 1

PubMed Central

Lowell, Cadance A.; Tomlinson, Patricia T.; Koch, Karen E.

1989-01-01

Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import remains to be determined. PMID:16666942

In situ effect of CPP-ACP chewing gum upon erosive enamel loss

PubMed Central

de ALENCAR, Catarina Ribeiro Barros; de OLIVEIRA, Gabriela Cristina; MAGALHÃES, Ana Carolina; BUZALAF, Marília Afonso Rabelo; MACHADO, Maria Aparecida de Andrade Moreira; HONÓRIO, Heitor Marques; RIOS, Daniela

2017-01-01

Abstract Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is able to increase salivary calcium and phosphate levels at an acidic pH. Previous studies demonstrated that a CPP-ACP chewing gum was able to enhance the re-hardening of erosion lesions, but could not diminish enamel hardness loss. Therefore, there is no consensus regarding the effectiveness of CPP-ACP on dental erosion. Objective This in situ study investigated the ability of a CPP-ACP chewing gum in preventing erosive enamel loss. Material and Methods: During three experimental crossover phases (one phase per group) of seven days each, eight volunteers wore palatal devices with human enamel blocks. The groups were: GI – Sugar free chewing gum with CPP-ACP; GII – Conventional sugar free chewing gum; and GIII – No chewing gum (control). Erosive challenge was extraorally performed by immersion of the enamel blocks in cola drink (5 min, 4x/day). After each challenge, in groups CPP and No CPP, volunteers chewed one unit of the corresponding chewing gum for 30 minutes. Quantitative analysis of enamel loss was performed by profilometry (µm). Data were analyzed by Repeated-Measures ANOVA and Tukey’s test (p<0.05). Results The use of chewing gum (CPP and No CPP) resulted in lower erosive enamel loss compared with the control group (p<0.05). CPP-ACP chewing gum (CPP) did not improve the protection against erosive enamel loss compared with conventional chewing gum (No CPP) (p>0.05). Conclusion The CPP-ACP chewing gum was not able to enhance the anti-erosive effect of conventional chewing gum against enamel loss. PMID:28678944

Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

PubMed Central

Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

2002-01-01

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

Functional specialization in nucleotide sugar transporters occurred through differentiation of the gene cluster EamA (DUF6) before the radiation of Viridiplantae

PubMed Central

2011-01-01

Background The drug/metabolite transporter superfamily comprises a diversity of protein domain families with multiple functions including transport of nucleotide sugars. Drug/metabolite transporter domains are contained in both solute carrier families 30, 35 and 39 proteins as well as in acyl-malonyl condensing enzyme proteins. In this paper, we present an evolutionary analysis of nucleotide sugar transporters in relation to the entire superfamily of drug/metabolite transporters that considers crucial intra-protein duplication events that have shaped the transporters. We use a method that combines the strengths of hidden Markov models and maximum likelihood to find relationships between drug/metabolite transporter families, and branches within families. Results We present evidence that the triose-phosphate transporters, domain unknown function 914, uracil-diphosphate glucose-N-acetylglucosamine, and nucleotide sugar transporter families have evolved from a domain duplication event before the radiation of Viridiplantae in the EamA family (previously called domain unknown function 6). We identify previously unknown branches in the solute carrier 30, 35 and 39 protein families that emerged simultaneously as key physiological developments after the radiation of Viridiplantae, including the "35C/E" branch of EamA, which formed in the lineage of T. adhaerens (Animalia). We identify a second cluster of DMTs, called the domain unknown function 1632 cluster, which has non-cytosolic N- and C-termini, and thus appears to have been formed from a different domain duplication event. We identify a previously uncharacterized motif, G-X(6)-G, which is overrepresented in the fifth transmembrane helix of C-terminal domains. We present evidence that the family called fatty acid elongases are homologous to transporters, not enzymes as had previously been thought. Conclusions The nucleotide sugar transporters families were formed through differentiation of the gene cluster EamA (domain unknown function 6) before Viridiplantae, showing for the first time the significance of EamA. PMID:21569384

Cooperation through Competition-Dynamics and Microeconomics of a Minimal Nutrient Trade System in Arbuscular Mycorrhizal Symbiosis.

PubMed

Schott, Stephan; Valdebenito, Braulio; Bustos, Daniel; Gomez-Porras, Judith L; Sharma, Tripti; Dreyer, Ingo

2016-01-01

In arbuscular mycorrhizal (AM) symbiosis, fungi and plants exchange nutrients (sugars and phosphate, for instance) for reciprocal benefit. Until now it is not clear how this nutrient exchange system works. Here, we used computational cell biology to simulate the dynamics of a network of proton pumps and proton-coupled transporters that are upregulated during AM formation. We show that this minimal network is sufficient to describe accurately and realistically the nutrient trade system. By applying basic principles of microeconomics, we link the biophysics of transmembrane nutrient transport with the ecology of organismic interactions and straightforwardly explain macroscopic scenarios of the relations between plant and AM fungus. This computational cell biology study allows drawing far reaching hypotheses about the mechanism and the regulation of nutrient exchange and proposes that the "cooperation" between plant and fungus can be in fact the result of a competition between both for the same resources in the tiny periarbuscular space. The minimal model presented here may serve as benchmark to evaluate in future the performance of more complex models of AM nutrient exchange. As a first step toward this goal, we included SWEET sugar transporters in the model and show that their co-occurrence with proton-coupled sugar transporters results in a futile carbon cycle at the plant plasma membrane proposing that two different pathways for the same substrate should not be active at the same time.

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair.

PubMed

Bhat, Javaid Y; Thieulin-Pardo, Gabriel; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO 2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO 2 molecule and binding of a Mg 2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

Gas-phase Reactivity of meta-Benzyne Analogs Toward Small Oligonucleotides of Differing Lengths

NASA Astrophysics Data System (ADS)

Widjaja, Fanny; Max, Joann P.; Jin, Zhicheng; Nash, John J.; Kenttämaa, Hilkka I.

2017-07-01

The gas-phase reactivity of two aromatic carbon-centered σ,σ-biradicals ( meta-benzyne analogs) and a related monoradical towards small oligonucleotides of differing lengths was investigated in a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer coupled with laser-induced acoustic desorption (LIAD). The mono- and biradicals were positively charged to allow for manipulation in the mass spectrometer. The oligonucleotides were evaporated into the gas phase as intact neutral molecules by using LIAD. One of the biradicals was found to be unreactive. The reactive biradical reacts with dinucleoside phosphates and trinucleoside diphosphates mainly by addition to a nucleobase moiety followed by cleavage of the glycosidic bond, leading to a nucleobase radical (e.g., base-H) abstraction. In some instances, after the initial cleavage, the unquenched radical site of the biradical abstracts a hydrogen atom from the neutral fragment, which results in a net nucleobase abstraction. In sharp contrast, the related monoradical mainly undergoes facile hydrogen atom abstraction from the sugar moiety. As the size of the oligonucleotides increases, the rate of hydrogen atom abstraction from the sugar moiety by the monoradical was found to increase due to the presence of more hydrogen atom donor sites, and it is the only reaction observed for tetranucleoside triphosphates. Hence, the monoradical only attacks sugar moieties in these substrates. The biradical also shows significant attack at the sugar moiety for tetranucleoside triphosphates. This drastic change in reactivity indicates that the size of the oligonucleotides plays a key role in the outcome of these reactions. This finding is attributed to more compact conformations in the gas phase for the tetranucleoside triphosphates than for the smaller oligonucleotides, which result from stronger stabilizing interactions between the nucleobases.

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Rodriguez, Jr., Miguel; ...

2017-01-10

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of themore » metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derived sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.« less

Kr-86 Ion-Beam Irradiation of Hydrated DNA: Free Radical and Unaltered Base Yields

PubMed Central

Becker, David; Adhikary, Amitava; Tetteh, Smedley T.; Bull, Arthur W.; Sevilla, Michael D.

2012-01-01

This work reports an ESR and product analysis investigation of Kr-86 ion-beam irradiation of hydrated DNA at 77 K. The irradiation results in the formation and trapping of both base radicals and sugar phosphate radicals (DNA backbone radicals). The absolute yields (G, μmol/J) of the base radicals are smaller than the yields found in similarly prepared γ-irradiated DNA samples, and the relative yields of backbone radicals relative to base radicals are much higher than that found in γ-irradiated samples. From these results, we have elaborated our radiation chemical model of the track structure for ion-beam irradiated DNA as it applies to krypton ion-beams. The base radicals, which are trapped as ion radicals or reversibly protonated or deprotonated ion radicals, are formed almost entirely in the track penumbra, a region in which radiation chemical effects are similar to those found in γ-irradiated samples. By comparing the yields of base radicals in ion-beam samples to the yields of the same radicals in γ-irradiated samples, the partition of energy between the low-LET region (penumbra) and the core is experimentally determined. The neutral sugar and other backbone radicals, which are not as susceptible to recombination as are ion radicals, are formed largely in the track core. The backbone radicals show a linear dose response up to very high doses. Unaltered base release yields in Kr-86 irradiated hydrated DNA are equal to sugar radical yields within experimental error limits, consistent with radiation-chemical processes in which all base release originates with sugar radicals. Two phosphorus-centered radicals from fragmentation of the DNA backbone are found in low yields. PMID:23106211

Kr-86 ion-beam irradiation of hydrated DNA: free radical and unaltered base yields.

PubMed

Becker, David; Adhikary, Amitava; Tetteh, Smedley T; Bull, Arthur W; Sevilla, Michael D

2012-12-01

This work reports an ESR and product analysis investigation of Kr-86 ion-beam irradiation of hydrated DNA at 77 K. The irradiation results in the formation and trapping of both base radicals and sugar phosphate radicals (DNA backbone radicals). The absolute yields (G, μmol/J) of the base radicals are smaller than the yields found in similarly prepared γ-irradiated DNA samples, and the relative yields of backbone radicals relative to base radicals are much higher than that found in γ-irradiated samples. From these results, we have elaborated our radiation chemical model of the track structure for ion-beam irradiated DNA as it applies to krypton ion-beams. The base radicals, which are trapped as ion radicals or reversibly protonated or deprotonated ion radicals, are formed almost entirely in the track penumbra, a region in which radiation chemical effects are similar to those found in γ-irradiated samples. By comparing the yields of base radicals in ion-beam samples to the yields of the same radicals in γ-irradiated samples, the partition of energy between the low-LET region (penumbra) and the core is experimentally determined. The neutral sugar and other backbone radicals, which are not as susceptible to recombination as are ion radicals, are formed largely in the track core. The backbone radicals show a linear dose response up to very high doses. Unaltered base release yields in Kr-86 irradiated hydrated DNA are equal to sugar radical yields within experimental error limits, consistent with radiation-chemical processes in which all base release originates with sugar radicals. Two phosphorus-centered radicals from fragmentation of the DNA backbone are found in low yields.

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Rodriguez, Jr., Miguel

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of themore » metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derived sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.« less

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

PubMed

Zhu, Zhiguang; Zhang, Y-H Percival

2017-01-01

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm -2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Rate of Glycolate Formation During Photosynthesis at High pH 1

PubMed Central

Orth, Gertrude M.; Tolbert, N. E.; Jimenez, Eduardo

1966-01-01

The products of C14O2 fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C14 was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C14 was found in glycolate products and only a trace of C14 was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C14 was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C14 accumulated in phosphoglycerate. C14 labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C14 was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle. Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC14O3 was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C14 accumulated in glycolate. PMID:16656223

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

PubMed Central

Aleman, Lorenzo; Ortega, Jose Luis; Martinez-Grimes, Martha; Seger, Mark; Holguin, Francisco Omar; Uribe, Diana J.; Garcia-Ibilcieta, David

2013-01-01

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in Vmax and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules. PMID:19898977

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michalska, Karolina; Cuff, Marianne E.; Structural Biology Center, Biosciences Division, Argonne National Laboratory

The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as amore » phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.« less

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

PubMed Central

Liu, Lixia; Zhang, Lei; Tang, Wei; Gu, Yang; Hua, Qiang; Yang, Sheng; Jiang, Weihong

2012-01-01

Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter−1. To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation. PMID:22865845

Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development▿

PubMed Central

Greig, Kylie T.; Antonchuk, Jennifer; Metcalf, Donald; Morgan, Phillip O.; Krebs, Danielle L.; Zhang, Jian-Guo; Hacking, Douglas F.; Bode, Lars; Robb, Lorraine; Kranz, Christian; de Graaf, Carolyn; Bahlo, Melanie; Nicola, Nicos A.; Nutt, Stephen L.; Freeze, Hudson H.; Alexander, Warren S.; Hilton, Douglas J.; Kile, Benjamin T.

2007-01-01

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity. PMID:17548465

Prebiotic chemistry and nucleic acid replication

NASA Technical Reports Server (NTRS)

Orgel, L. E.; Lohrmann, R.

1974-01-01

Recent work is reviewed on some reactions that could have occurred on the primitive earth and that could have played a part in the evolution of a self-replicating system. The transition from the primitive atmosphere to the simplest replicating molecules is considered in four stages: (1) the formation of a 'prebiotic soup' of organic precursors, including the purine and pyrimidine bases and the pentose sugars; (2) the condensation of these precursors and inorganic phosphate to form monomeric nucleotides and activated nucleotide derivatives; (3) the polymerization of nucleotide derivatives to oligonucleotides; and (4) the complementary replication of oligonucleotides in a template-directed process that depends on Watson-Crick base pairing.

Glutathione-related enzymes and the eye.

PubMed

Ganea, Elena; Harding, John J

2006-01-01

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.

Understanding the mechanism of DNA deactivation in ion therapy of cancer cells: hydrogen peroxide action*

NASA Astrophysics Data System (ADS)

Piatnytskyi, Dmytro V.; Zdorevskyi, Oleksiy O.; Perepelytsya, Sergiy M.; Volkov, Sergey N.

2015-11-01

Changes in the medium of biological cells under ion beam irradiation has been considered as a possible cause of cell function disruption in the living body. The interaction of hydrogen peroxide, a long-lived molecular product of water radiolysis, with active sites of DNA macromolecule was studied, and the formation of stable DNA-peroxide complexes was considered. The phosphate groups of the macromolecule backbone were picked out among the atomic groups of DNA double helix as a probable target for interaction with hydrogen peroxide molecules. Complexes consisting of combinations including: the DNA phosphate group, H2O2 and H2O molecules, and Na+ counterion, were considered. The counterions have been taken into consideration insofar as under the natural conditions they neutralise DNA sugar-phosphate backbone. The energy of the complexes have been determined by considering the electrostatic and the Van der Waals interactions within the framework of atom-atom potential functions. As a result, the stability of various configurations of molecular complexes was estimated. It was shown that DNA phosphate groups and counterions can form stable complexes with hydrogen peroxide molecules, which are as stable as the complexes with water molecules. It has been demonstrated that the formation of stable complexes of H2O2-Na+-PO4- may be detected experimentally by observing specific vibrations in the low-frequency Raman spectra. The interaction of H2O2 molecule with phosphate group of the double helix backbone can disrupt DNA biological function and induce the deactivation of the cell genetic apparatus. Thus, the production of hydrogen peroxide molecules in the nucleus of living cells can be considered as an additional mechanism by which high-energy ion beams destroy tumour cells during ion beam therapy. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

Isolation and identification of a novel bacterium, Lactobacillus sakei subsp. dgh strain 5, and optimization of growth condition for highest antagonistic activity.

PubMed

Tashakor, Amin; Hosseinzadehdehkordi, Mahshid; Emruzi, Zeynab; Gholami, Dariush

2017-05-01

In the present study, we isolated Lactobacillus sakei strain DGH5 from raw beef meat. This bacterium plays an inhibitory effect against food-spoiling bacteria and food-borne pathogens, including Listeria monocytogenes, a gram-positive and pathogenic bacterium. Lactobacillus sakei strain DGH5 was identified through both phenotypical and biochemical tests accompanied with 16S rRNA sequence analysis. Among all the sources of carbon, nitrogen and phosphorous forms, we selected the most potent compounds to optimize the condition for the highest antagonistic activity. Among the sugars, polygalacturonic acid demonstrated to improve the antagonistic activity. Ammonium nitrate demonstrated to be suitable nitrogen sources. Amongst phosphorous sources, disodium hydrogen phosphate had the greatest antagonistic effect. According to Taguchi's orthogonal array, temperature, disodium hydrogen phosphate and soy Peptone had significant effect on antagonistic activity. Furthermore, mean comparisons showed that the optimum conditions achieved at pH 6.0, 25 °C temperature, 1.5% (w/v) Na 2 HPO 4 and 0.5% (w/v) peptone. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quinovosamycins: new tunicamycin-type antibiotics in which the α, β-1″,11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

PubMed

Price, Neil Pj; Labeda, David P; Naumann, Todd A; Vermillion, Karl E; Bowman, Michael J; Berhow, Mark A; Metcalf, William W; Bischoff, Kenneth M

2016-08-01

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.

Physicochemical characterization of mozzarella cheese wheys and stretchwaters in comparison with several other sweet wheys.

PubMed

Gernigon, G; Piot, M; Beaucher, E; Jeantet, R; Schuck, P

2009-11-01

To better understand the origins of the problems occurring during mozzarella cheese whey concentration, lactose crystallization, and spray-drying steps, a physicochemical characterization was achieved. For this purpose, mozzarella cheese wheys were sampled and their content in different compounds such as total nitrogen, noncasein nitrogen, nonprotein nitrogen, lactate, citrate, chloride, sulfate, phosphate anions, calcium, magnesium, potassium, sodium cations, and the sugars glucose and galactose were measured. In a second step, the results were compared with the corresponding content in cheddar cheese wheys, raclette cheese wheys, soft cheese wheys, and Swiss-type cheese wheys. At the end of this survey, it was shown that mozzarella cheese wheys were more concentrated in lactate and in minerals--especially phosphate, calcium, and magnesium--than the other cheese wheys and that they contained galactose. These constituents are known to be hygroscopic. Complementary surveys are now necessary to compare the hygroscopicity of galactose and lactate and discover whether the amounts of these compounds found in mozzarella cheese wheys are a factor in the problems encountered during the concentration, lactose crystallization, and spray-drying steps.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ambudkar, S.V.; Sonna, L.A.; Maloney, P.C.

Phosphate:2-deoxyglucose 6-phosphate (Pi:2DG6P) antiport was extracted from Streptococcus lactis or Staphylococcus aureus with 1.1% octylglucoside in the presence of 0.37% E. coli lipid and reconstituted by detergent dilution. Because previous work suggested inactivation at an early stage, the authors introduced protein stabilants during solubilization. When 20% glycerol was used, proteoliposomes showed a 20-fold increase in /sup 32/Pi transport. This enhanced recovery required phospholipid plus glycerol, and was found only when both were added together with the detergent. Glycerol protection yielded proteoliposomes in which antiporters retained their normal kinetic properties, and Pi exchange by the streptococcal example gave a maximal ratemore » (200-400 nmol/min per mg protein) and a turnover number (30-50/s) which suggested that inactivation had been avoided. Further study showed that 20% glycerol could be replaced by equally high concentrations of compounds classified as osmolytes polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose) and certain amino acids (glycine, proline, but not valine). The authors suggest that osmolytes may be used to fully stabilize chemiosmotic transporters during reconstitution.« less

A nucleobase-centered coarse-grained representation for structure prediction of RNA motifs.

PubMed

Poblete, Simón; Bottaro, Sandro; Bussi, Giovanni

2018-02-28

We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the $\\mathcal {E}$SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base-phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation.

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes.

PubMed

Ciudad, C J; Massagué, J; Salavert, A; Guinovart, J J

1980-03-20

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.

Sensory profile, soluble sugars, organic acids, and mineral content in milk- and soy-juice based beverages.

PubMed

Andrés, Víctor; Tenorio, M Dolores; Villanueva, M José

2015-04-15

The juice industry has undergone a continuous innovation to satisfy the increasing healthy food demand by developing, among others, beverages based on fruits and milk or soybeans. The comparison among the sensory attributes between nineteen commercial mixed beverages showed significant differences in colour, sweetness, acidity, and consistency. Sucrose and citric acid were found in large proportion due to their natural presence or their addition. Potassium was the major macromineral (148-941 mg/L), especially in soy beverages. The low concentration of sodium in soy drinks is a healthy characteristic. The profile of inorganic anions has been included for the first time. Sulphate (39-278 mg/L) and phosphate (51-428 mg/L) were the predominant anions. High correlations were found between the percentage of fruit and consistency, fructose, malic acid, potassium and phosphate content (r(2)>0.790). Based on the data obtained, these beverages show pleasant organoleptic characteristics and constitute a good source of essential nutrients for regular consumers. Copyright © 2014 Elsevier Ltd. All rights reserved.

Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts 1

PubMed Central

Robinson, Simon P.; Walker, David A.

1980-01-01

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of 14C-labeled products during photosynthesis. In the dark, CO2 fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis. PMID:16661305

Experimental investigation of ionic liquid pretreatment of sugarcane bagasse with 1,3-dimethylimadazolium dimethyl phosphate.

PubMed

Bahrani, Samaneh; Raeissi, Sona; Sarshar, Mohammad

2015-06-01

In this study, an imidazolium-based ionic liquid (IL), 1,3-dimethylimidazolium dimethyl phosphate ([Mmim][DMP]), was applied for pretreating sugarcane bagasse to produce bioethanol. The main goal of this study was to investigate the feasibility of bagasse pretreatment with this IL, and to verify the effect of different operational parameters on the pretreatment process. Results indicated that temperature and duration of IL-pretreatment have optimum values. Within the range investigated, a maximum fermentable sugar conversion of 70.38% was achieved with this IL at 120°C and 120min. The corresponding value was 28.65% for the untreated biomass. The main cause for the observed enhancement in enzymatic hydrolysis was the reduction of cellulose crystallinity in the IL-pretreated biomass, as compared to the untreated sample, because it resulted in higher accessibility of the enzymes to the biomass after pretreatment. Moreover, the results indicated that aqueous [Mmim][DMP] mixtures are not as effective for pretreatment as the pure IL. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro nonenzymatic glycation of guanosine 5'-triphosphate by dihydroxyacetone phosphate.

PubMed

Li, Yuyuan; Cohenford, Menashi A; Dutta, Udayan; Dain, Joel A

2008-11-01

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.

Application of chitosan/polyacrylamide nanofibres for removal of chromate and phosphate in water

NASA Astrophysics Data System (ADS)

Nthumbi, Richard M.; Catherine Ngila, J.; Moodley, Brenda; Kindness, Andrew; Petrik, Leslie

Water pollution is an intractable environmental problem in South Africa. Management of the water resource is vital in order to address the water scarcity issues. Research on remediation of contaminated water has focused mainly on the removal of heavy metals such as Pb, Cd, Zn, Hg and Cu and neglected other inorganic pollutants. In this work we focus on the removal of anions, namely chromate and phosphate. Chromium is extensively used in the textile, leather and metallurgy industries and contaminates surface water and groundwater when inadequately treated industrial effluents are discharged. Chromium has been associated with irregular sugar metabolism, nosebleeds and ulcers, and it is also carcinogenic. The phosphate ion is an essential micronutrient responsible for healthy plant growth. However, excess phosphate intake stimulates rapid growth of photosynthetic algae and cyanobacteria, resulting in eutrophication. This phenomenon (algal bloom) causes other organisms to die due to reduced oxygen in the water. In order to offer remediation measures, this study reports the use of electrospun nanofibres for the removal of chromate and phosphate anions. Adsorption experiments were carried out using nanofibres electrospun from chitosan and polyacrylamide polymer blends, cross-linked with glutaraldehyde. Quantification of chromium was done using ICP-OES while UV-Vis spectrophotometry was used for the determination of phosphates. Batch adsorption experiments were done to determine optimum adsorption parameters such as pH, contact time, temperature and initial analyte concentration. Removal of the ions using a flow-adsorption technique through a micro-column was performed. The experimental data obtained were analysed using Langmuir and Freundlich models to study the adsorption mechanisms. The nanofibres had an adsorption capacity for Cr(VI) and PO43- of 0.26 mg g-1 and 392 mg g-1, respectively, and removal efficiencies of 93% and 97.4%, in the same order, in synthetic water samples and environmental water samples. It was observed that both chromium(VI) and phosphate adsorption followed pseudo-second-order kinetics. During the regeneration process, it was established that Cr(VI) was reduced to Cr(III) at the surface of the sorbent. This reaction offers the advantage of reducing the toxicity of chromium(VI) in water. The results of this work have potential applications in the removal of these anions in contaminated drinking water thus improving its quality for human consumption.

Impact Chemistry and the Origin of Life

NASA Astrophysics Data System (ADS)

Melosh, H. J.; Pasek, M.

2012-12-01

Most discussions of global environmental effects of large impacts focus on changes deleterious to extant life. However, impacts may also produce changes that enhance or even create conditions beneficial to the origin of life. Many other authors have discussed impact delivery of organic molecules, and some have shown the shock synthesis of prebiotic molecules such as amino acids during impact. Our past work on the chemistry of impacts demonstrated that strong chemical reduction occurs in impact melt ejecta (spherules and melt droplets; tektites). Here we focus on the element phosphorus (P), whose role is crucial in biology as the backbone of DNA and RNA, and in metabolic biochemical energy transfer. Pasek previously showed that reduced P readily enters into interesting biological compounds with organic molecules in aqueous solution, and that these reduced P compounds may generate structures similar to sugar phosphates, which are critical to life as we know it. In this talk we argue that impact reduction of P transforms terrestrial and meteoritic phosphates bearing an oxidation state of +5 to the lower redox states of +3 (phosphites) and 0 as an alloy with metal (phosphides). We base this argument on studies of fulgurites—glasses formed by cloud-to-ground lightning—that bear phosphides and phosphites as major carriers of P. Fulgurite chemistry frequently parallels that of impact glasses. Additionally, thermodynamic calculations show that separation of an O-rich vapor from a melt readily results in the transformation of phosphate to phosphites and metal phosphides. These results are confirmed by the presence of metal phosphides within tektites. The impact reduction of phosphates followed by global dispersal of reduced P in the form of glassy droplets likely played a major role in the origin of life on Earth and perhaps on other young planets.

Glucosamine Inhibits Decidualization of Human Endometrial Stromal Cells and Decreases Litter Sizes in Mice1

PubMed Central

Tsai, Jui-He; Schulte, Maureen; O'Neill, Kathleen; Chi, Maggie M.-Y.; Frolova, Antonina I.; Moley, Kelle H.

2013-01-01

ABSTRACT Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 μg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive. PMID:23718985

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Effects of dietary cis and trans unsaturated and saturated fatty acids on the glucose metabolites and enzymes of rats.

PubMed

Bernal, Claudio A; Rovira, Jordi; Colandré, María E; Cussó, Roser; Cadefau, Joan A

2006-05-01

The aim of the present study was to examine whether the level of dietary cis fatty acid (cFA), or the isomers (trans or cis) and/or the saturation of the fatty acids at high dietary fat levels altered the intracellular glucose metabolites and certain regulatory enzyme activities in the skeletal muscle and liver of rats. The animals were fed for 30 d on either a recommended control diet (7 % cFA, w/w) or a high-fat diet (20 % fatty acids, w/w). The high-fat diet was enriched with either cFA, trans fatty acid (tFA), a moderate proportion of saturated fatty acid (MSFA), or a high proportion of saturated fatty acid (HSFA). The most striking findings were observed in the gastrocnemius muscle with a HSFA diet. There was a significant increase in glucose-6-phosphate (306 %), glucose-1-phosphate (245 %), fructose-6-phosphate (400 %), fructose-1,6-bisphosphate (86 %), glyceraldehyde-3-phosphate (38 %), pyruvate (341 %), lactate (325 %), citrate (79 %) and the bisphosphorylated sugars as compared with the cFA diet. These changes were paralleled by an increase in muscle triacylglycerol content (49 %) and a decrease in glucose (39 %). In addition, the amount of cFA and the other types of fatty acid (i.e. tFA and MSFA) led to no great differences in glucose metabolism as compared with the respective control group. These data support the hypothesis that glucose changes induced by a HSFA diet are a multifaceted abnormality. Glucose and lactate transport and intracellular glucose metabolism could be the key biochemical defects involved in this detrimental effect on glucose metabolism.

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

PubMed

van Rooijen, R J; Dechering, K J; Niek, C; Wilmink, J; de Vos, W M

1993-02-01

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

Metabolic Flux Analysis during the Exponential Growth Phase of Saccharomyces cerevisiae in Wine Fermentations

PubMed Central

Quirós, Manuel; Martínez-Moreno, Rubén; Albiol, Joan; Morales, Pilar; Vázquez-Lima, Felícitas; Barreiro-Vázquez, Antonio; Ferrer, Pau; Gonzalez, Ramon

2013-01-01

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations. PMID:23967264

Cooperation through Competition—Dynamics and Microeconomics of a Minimal Nutrient Trade System in Arbuscular Mycorrhizal Symbiosis

PubMed Central

Schott, Stephan; Valdebenito, Braulio; Bustos, Daniel; Gomez-Porras, Judith L.; Sharma, Tripti; Dreyer, Ingo

2016-01-01

In arbuscular mycorrhizal (AM) symbiosis, fungi and plants exchange nutrients (sugars and phosphate, for instance) for reciprocal benefit. Until now it is not clear how this nutrient exchange system works. Here, we used computational cell biology to simulate the dynamics of a network of proton pumps and proton-coupled transporters that are upregulated during AM formation. We show that this minimal network is sufficient to describe accurately and realistically the nutrient trade system. By applying basic principles of microeconomics, we link the biophysics of transmembrane nutrient transport with the ecology of organismic interactions and straightforwardly explain macroscopic scenarios of the relations between plant and AM fungus. This computational cell biology study allows drawing far reaching hypotheses about the mechanism and the regulation of nutrient exchange and proposes that the “cooperation” between plant and fungus can be in fact the result of a competition between both for the same resources in the tiny periarbuscular space. The minimal model presented here may serve as benchmark to evaluate in future the performance of more complex models of AM nutrient exchange. As a first step toward this goal, we included SWEET sugar transporters in the model and show that their co-occurrence with proton-coupled sugar transporters results in a futile carbon cycle at the plant plasma membrane proposing that two different pathways for the same substrate should not be active at the same time. PMID:27446142

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair

PubMed Central

Bhat, Javaid Y.; Thieulin-Pardo, Gabriel; Hartl, F. Ulrich; Hayer-Hartl, Manajit

2017-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO2 molecule and binding of a Mg2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are “misfire” products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins. PMID:28443288

Identification of mannitol as compatible solute in Gluconobacter oxydans.

PubMed

Zahid, Nageena; Schweiger, Paul; Galinski, Erwin; Deppenmeier, Uwe

2015-07-01

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100 mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600 mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10 mM) and 300 mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Scenedesmus obliquus in poultry wastewater bioremediation.

PubMed

Oliveira, Ana Cristina; Barata, Ana; Batista, Ana P; Gouveia, Luísa

2018-06-18

Wastewater biological treatment with microalgae can be an effective technology, removing nutrients and other contaminants while reducing chemical oxygen demand. This can be particularly interesting for the meat producing industry which produces large volumes of wastewater from the slaughtering of animals and cleaning of their facilities. The main purpose of this research was the treatment of poultry wastewater using Scenedesmus obliquus in an economical and environmentally sustainable way. Two wastewaters were collected from a Portuguese poultry slaughterhouse (poultry raw - PR and poultry flocculated - PF) and the bioremediation was evaluated. The performance of microalga biomass growth and biochemical composition were assessed for two illumination sources (fluorescent vs LEDs). S. obliquus achieved positive results when grown in highly contaminated agro-industrial wastewater from the poultry industry, independently of the light source. The wastewater bioremediation revealed results higher than 97% for both ammonium and phosphate removal efficiency, for a cultivation time of 13 days. The saponifiable matter obtained from the biomass of the microalga cultures was, on average, 11% and 27% (m/m alga ) with PR and PF wastewater, respectively. In opposition, higher sugar content was obtained from microalgae biomass grown in PR wastewater (average 34% m/m alga ) in comparison to PF wastewater (average 23% m/m alga ), independently of the illumination source. Therefore, biomass obtained with PR wastewater will be more appropriate as a raw material for bioethanol/biohydrogen production (higher sugar content) while biomass produced in PF wastewater will have a similar potential as feedstock for both biodiesel and bioethanol/biohydrogen production (similar lipid and sugar content).

Evaluation of the fermentation of high gravity thick sugar beet juice worts for efficient bioethanol production

PubMed Central

2013-01-01

Background Sugar beet and intermediates of sugar beet processing are considered to be very attractive feedstock for ethanol production due to their content of fermentable sugars. In particular, the processing of the intermediates into ethanol is considerably facilitated because it does not require pretreatment or enzymatic treatment in contrast to production from starch raw materials. Moreover, the advantage of thick juice is high solid substance and saccharose content which eliminates problems with the storability of this feedstock. Results The objective of this study were to investigate bioethanol production from thick juice worts and the effects of their concentration, the type of mineral supplement, as well as the dose of yeast inoculum on fermentation dynamics and ethanol yield. The obtained results show that to ensure efficient ethanolic fermentation of high gravity thick juice worts, one needs to use a yeast strain with high ethanol tolerance and a large amount of inoculum. The highest ethanol yield (94.9 ± 2.8% of the theoretical yield) and sugars intake of 96.5 ± 2.9% were obtained after the fermentation of wort with an extract content of 250 g/kg supplemented with diammonium hydrogen phosphate (0.3 g/L of wort) and inoculated with 2 g of Ethanol Red dry yeast per L of wort. An increase in extract content in the fermentation medium from 250 g/L to 280 g/kg resulted in decreased efficiency of the process. Also the distillates originating from worts with an extract content of 250 g/kg were characterized by lower acetaldehyde concentration than those obtained from worts with an extract content of 280 g/kg. Conclusions Under the favorable conditions determined in our experiments, 38.9 ± 1.2 L of 100% (v/v) ethyl alcohol can be produced from 100 kg of thick juice. The obtained results show that the selection of process conditions and the yeast for the fermentation of worts with a higher sugar content can improve the economic performance of the alcohol-distilling industry due to more efficient ethanol production, reduced consumption of cooling water, and energy for ethanol distillation, as well as a decreased volume of fermentation stillage. PMID:24206573

Evaluation of the fermentation of high gravity thick sugar beet juice worts for efficient bioethanol production.

PubMed

Dziugan, Piotr; Balcerek, Maria; Pielech-Przybylska, Katarzyna; Patelski, Piotr

2013-11-08

Sugar beet and intermediates of sugar beet processing are considered to be very attractive feedstock for ethanol production due to their content of fermentable sugars. In particular, the processing of the intermediates into ethanol is considerably facilitated because it does not require pretreatment or enzymatic treatment in contrast to production from starch raw materials. Moreover, the advantage of thick juice is high solid substance and saccharose content which eliminates problems with the storability of this feedstock. The objective of this study were to investigate bioethanol production from thick juice worts and the effects of their concentration, the type of mineral supplement, as well as the dose of yeast inoculum on fermentation dynamics and ethanol yield.The obtained results show that to ensure efficient ethanolic fermentation of high gravity thick juice worts, one needs to use a yeast strain with high ethanol tolerance and a large amount of inoculum. The highest ethanol yield (94.9 ± 2.8% of the theoretical yield) and sugars intake of 96.5 ± 2.9% were obtained after the fermentation of wort with an extract content of 250 g/kg supplemented with diammonium hydrogen phosphate (0.3 g/L of wort) and inoculated with 2 g of Ethanol Red dry yeast per L of wort. An increase in extract content in the fermentation medium from 250 g/L to 280 g/kg resulted in decreased efficiency of the process. Also the distillates originating from worts with an extract content of 250 g/kg were characterized by lower acetaldehyde concentration than those obtained from worts with an extract content of 280 g/kg. Under the favorable conditions determined in our experiments, 38.9 ± 1.2 L of 100% (v/v) ethyl alcohol can be produced from 100 kg of thick juice. The obtained results show that the selection of process conditions and the yeast for the fermentation of worts with a higher sugar content can improve the economic performance of the alcohol-distilling industry due to more efficient ethanol production, reduced consumption of cooling water, and energy for ethanol distillation, as well as a decreased volume of fermentation stillage.

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose.

PubMed

Lawford, Hugh G; Rousseau, Joyce D

2002-01-01

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

PubMed

Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

PubMed Central

Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

Expression Profiles of the Trehalose-6-Phosphate Synthase Gene Associated With Thermal Stress in Ostrinia furnacalis (Lepidoptera: Crambidae)

PubMed Central

Jin, Tingting; Gao, Yulin; He, Kanglai

2018-01-01

Abstract Trehalose is the major blood sugar in insects. Physiological significance of this compound has been extensively reported. Trehalose-6-phosphate synthase (TPS) is an important enzyme in the trehalose biosynthesis pathway. Full-length cDNAs of TPS (Of tps) and its alternative splicing isoform (Of tps_isoformI) were cloned from the Asian corn borer (ACB), Ostrinia furnacalis (Guenée; Lepidoptera: Crambidae) larvae. The Of tps and Of tps_isoformI transcripts were 2913 and 1689 bp long, contained 2529 and 1293 bp open reading frames encoding proteins of 842 and 430 amino acids with a molecular mass of 94.4 and 48.6 kDa, respectively. Transcriptional profiling and response to thermal stress of Of tps gene were determined by quantitative real-time PCR showing that the Of tps was predominantly expressed in the larval fat body, significantly enhanced during molting and transformation; and thermal stress also induced Of tps expression. Gene structure analysis is indicating that one TPS domain and one trehalose-6-phosphate phosphatase (TPP) domain were located at the N- and C-termini of Of        TPS, respectively, while only the TPS domain was detected in OfTPS_isoformI. Three-dimensional modeling and heterologous expression were developed to predict the putative functions of OfTPS and Of   TPS_isoformI. We infer that the expression of Of tps gene is thermally induced and might be crucial for larvae survival.

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium.

PubMed

Smiri, Moêz; Chaoui, Abdelilah; El Ferjani, Ezzedine

2009-02-15

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

Engineering of N-acetylglucosamine metabolism for improved antibiotic production in Streptomyces coelicolor A3(2) and an unsuspected role of NagA in glucosamine metabolism.

PubMed

Świątek, Magdalena A; Urem, Mia; Tenconi, Elodie; Rigali, Sébastien; van Wezel, Gilles P

2012-01-01

N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

[Chemicals added to cigarettes and their effects on tobacco dependence].

PubMed

Gonseth, S; Cornuz, J

2009-07-01

This paper summarizes data on the factors involved in addiction and dependence to cigarettes. Nicotine has been intensively studied by the tobacco industry, for instance for its addictive effect at the lowest possible rates. The addition of diammonium phosphate and urea produces an alcalinization of the pH of cigarette smoke, and promotes the absorption and the trans-membrane passage of nicotine. The taste, the smell of smoke, and the visual aspect of the pack of cigarettes are also sensory components that promote addiction. Finally, menthol, sugar, cocoa and liquorice added to cigarettes also play a role in dependence and addiction to cigarettes by, for instance, making an anesthetic effect on the airways.

Piezoresistive cantilever array sensor for consolidated bioprocess monitoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seonghwan Sam; Rahman, Touhidur; Senesac, Larry R

2009-01-01

Cellulolytic microbes occur in diverse natural niches and are being screened for industrial modification and utility. A microbe for Consolidated bioprocessing (CBP) development can rapidly degrade pure cellulose and then ferment the resulting sugars into fuels. To identify and screen for novel microbes for CBP, we have developed a piezoresistive cantilever array sensor which is capable of simultaneous monitoring of glucose and ethanol concentration changes in a phosphate buffer solution. 4-mercaptophenylboronic acid (4-MPBA) and polyethyleneglycol (PEG)-thiol are employed to functionalize each piezoresistive cantilever for glucose and ethanol sensing, respectively. Successful concentration measurements of glucose and ethanol with minimal interferences aremore » obtained with our cantilever array sensor.« less

Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo.

PubMed

Takahashi, N; Washio, J

2011-12-01

Dental caries is initiated by demineralization of the tooth surface through acid production from sugar by plaque biofilm. Fluoride and xylitol have been used worldwide as caries-preventive reagents, based on in vitro-proven inhibitory mechanisms on bacterial acid production. We attempted to confirm the inhibitory mechanisms of fluoride and xylitol in vivo by performing metabolome analysis on the central carbon metabolism in supragingival plaque using the combination of capillary electrophoresis and a time-of-flight mass spectrometer. Fluoride (225 and 900 ppm F(-)) inhibited lactate production from 10% glucose by 34% and 46%, respectively, along with the increase in 3-phosphoglycerate and the decrease in phosphoenolpyruvate in the EMP pathway in supragingival plaque. These results confirmed that fluoride inhibited bacterial enolase in the EMP pathway and subsequently repressed acid production in vivo. In contrast, 10% xylitol had no effect on acid production and the metabolome profile in supragingival plaque, although xylitol 5-phosphate was produced. These results suggest that xylitol is not an inhibitor of plaque acid production but rather a non-fermentative sugar alcohol. Metabolome analyses of plaque biofilm can be applied for monitoring the efficacy of dietary components and medicines for plaque biofilm, leading to the development of effective plaque control.

3DNALandscapes: a database for exploring the conformational features of DNA.

PubMed

Zheng, Guohui; Colasanti, Andrew V; Lu, Xiang-Jun; Olson, Wilma K

2010-01-01

3DNALandscapes, located at: http://3DNAscapes.rutgers.edu, is a new database for exploring the conformational features of DNA. In contrast to most structural databases, which archive the Cartesian coordinates and/or derived parameters and images for individual structures, 3DNALandscapes enables searches of conformational information across multiple structures. The database contains a wide variety of structural parameters and molecular images, computed with the 3DNA software package and known to be useful for characterizing and understanding the sequence-dependent spatial arrangements of the DNA sugar-phosphate backbone, sugar-base side groups, base pairs, base-pair steps, groove structure, etc. The data comprise all DNA-containing structures--both free and bound to proteins, drugs and other ligands--currently available in the Protein Data Bank. The web interface allows the user to link, report, plot and analyze this information from numerous perspectives and thereby gain insight into DNA conformation, deformability and interactions in different sequence and structural contexts. The data accumulated from known, well-resolved DNA structures can serve as useful benchmarks for the analysis and simulation of new structures. The collective data can also help to understand how DNA deforms in response to proteins and other molecules and undergoes conformational rearrangements.

In response to 'Can sugars be produced from fatty acids? A test case for pathway analysis tools'.

PubMed

Faust, Karoline; Croes, Didier; van Helden, Jacques

2009-12-01

In their article entitled 'Can sugars be produced from fatty acids? A test case for pathway analysis tools' de Figueiredo and co-authors assess the performance of three pathway prediction tools (METATOOL, PathFinding and Pathway Hunter Tool) using the synthesis of glucose-6-phosphate (G6P) from acetyl-CoA in humans as a test case. We think that this article is biased for three reasons: (i) the metabolic networks used as input for the respective tools were of very different sizes; (ii) the 'assessment' is restricted to two study cases; (iii) developers are inherently more skilled to use their own tools than those developed by other people. We extended the analyses led by de Figueiredo and clearly show that the apparent superior performance of their tool (METATOOL) is partly due to the differences in input network sizes. We also see a conceptual problem in the comparison of tools that serve different purposes. In our opinion, metabolic path finding and elementary mode analysis are answering different biological questions, and should be considered as complementary rather than competitive approaches. Supplementary data are available at Bioinformatics online.

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

PubMed

Yang, Jiangang; Zhu, Yueming; Men, Yan; Sun, Shangshang; Zeng, Yan; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

2016-12-21

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenfield, N.J.; McKenzie, M.A.; Adebodun, F.

/sup 13/C NMR and /sup 31/P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-(1-/sup 13/C)- or D-(6-/sup 13/C)glucose as substrates, the major metabolic products identified from /sup 13/C NMR spectra were (2-/sup 13/C)ethanol, (3-/sup 13/C)alanine, and C/sub 1/- and C/sub 6/-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C/sub 6/- than from C/sub 1/-labeled glucose; (ii) C/sub 1/-labeledmore » glucose yielded 19% C/sub 6/-labeled trehalose, while C/sub 6/-labeled glucose yielded only 4% C/sub 1/-labeled trehalose; (iii) a substantial transfer of /sup 13/C from C/sub 2/-labeled glucose to the C/sub 2/-position of ethanol. /sup 31/P NMR spectra showed millimolar levels of intracellular inorganic phosphate (P/sub i/), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic P/sub i/ and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase in the rate of ethanol production; (ii) a 38% increase in the rate of alanine production; (iii) a 27% increase in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO/sub 2/ and glycogen, as previously shown.« less

Nucleic acid reactivity: challenges for next-generation semiempirical quantum models.

PubMed

Huang, Ming; Giese, Timothy J; York, Darrin M

2015-07-05

Semiempirical quantum models are routinely used to study mechanisms of RNA catalysis and phosphoryl transfer reactions using combined quantum mechanical (QM)/molecular mechanical methods. Herein, we provide a broad assessment of the performance of existing semiempirical quantum models to describe nucleic acid structure and reactivity to quantify their limitations and guide the development of next-generation quantum models with improved accuracy. Neglect of diatomic differential overlap and self-consistent density-functional tight-binding semiempirical models are evaluated against high-level QM benchmark calculations for seven biologically important datasets. The datasets include: proton affinities, polarizabilities, nucleobase dimer interactions, dimethyl phosphate anion, nucleoside sugar and glycosidic torsion conformations, and RNA phosphoryl transfer model reactions. As an additional baseline, comparisons are made with several commonly used density-functional models, including M062X and B3LYP (in some cases with dispersion corrections). The results show that, among the semiempirical models examined, the AM1/d-PhoT model is the most robust at predicting proton affinities. AM1/d-PhoT and DFTB3-3ob/OPhyd reproduce the MP2 potential energy surfaces of 6 associative RNA phosphoryl transfer model reactions reasonably well. Further, a recently developed linear-scaling "modified divide-and-conquer" model exhibits the most accurate results for binding energies of both hydrogen bonded and stacked nucleobase dimers. The semiempirical models considered here are shown to underestimate the isotropic polarizabilities of neutral molecules by approximately 30%. The semiempirical models also fail to adequately describe torsion profiles for the dimethyl phosphate anion, the nucleoside sugar ring puckers, and the rotations about the nucleoside glycosidic bond. The modeling of pentavalent phosphorus, particularly with thio substitutions often used experimentally as mechanistic probes, was problematic for all of the models considered. Analysis of the strengths and weakness of the models suggests that the creation of robust next-generation models should emphasize the improvement of relative conformational energies and barriers, and nonbonded interactions. © 2015 Wiley Periodicals, Inc.

Nucleic acid reactivity : challenges for next-generation semiempirical quantum models

PubMed Central

Huang, Ming; Giese, Timothy J.; York, Darrin M.

2016-01-01

Semiempirical quantum models are routinely used to study mechanisms of RNA catalysis and phosphoryl transfer reactions using combined quantum mechanical/molecular mechanical methods. Herein, we provide a broad assessment of the performance of existing semiempirical quantum models to describe nucleic acid structure and reactivity in order to quantify their limitations and guide the development of next-generation quantum models with improved accuracy. Neglect of diatomic diffierential overlap (NDDO) and self-consistent density-functional tight-binding (SCC-DFTB) semiempirical models are evaluated against high-level quantum mechanical benchmark calculations for seven biologically important data sets. The data sets include: proton affinities, polarizabilities, nucleobase dimer interactions, dimethyl phosphate anion, nucleoside sugar and glycosidic torsion conformations, and RNA phosphoryl transfer model reactions. As an additional baseline, comparisons are made with several commonly used density-functional models, including M062X and B3LYP (in some cases with dispersion corrections). The results show that, among the semiempirical models examined, the AM1/d-PhoT model is the most robust at predicting proton affinities. AM1/d-PhoT and DFTB3-3ob/OPhyd reproduce the MP2 potential energy surfaces of 6 associative RNA phosphoryl transfer model reactions reasonably well. Further, a recently developed linear-scaling “modified divide-and-conquer” model exhibits the most accurate results for binding energies of both hydrogen bonded and stacked nucleobase dimers. The semiempirical models considered here are shown to underestimate the isotropic polarizabilities of neutral molecules by approximately 30%. The semiempirical models also fail to adequately describe torsion profiles within the dimethyl phosphate anion, the nucleoside sugar ring puckers, and the rotations about the nucleoside glycosidic bond. The modeling of pentavalent phosphorus, particularly with thio substitutions often used experimentally as mechanistic probes, was problematic for all of the models considered. Analysis of the strengths and weakness of the models suggest that the creation of robust next-generation models should emphasize the improvement of relative conformational energies and barriers, and nonbond interactions. PMID:25943338

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.

PubMed

Jacobsen, Jacob H; Frigaard, Niels-Ulrik

2014-01-01

D-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L(-1) and a production rate of 0.15 g mannitol L(-1) day(-1). This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria. © 2013 International Metabolic Engineering Society Published by International Metabolic Engineering Society All rights reserved.

From the Primitive Atmosphere to the Prebiotic Soup to the Pre-RNA World

NASA Technical Reports Server (NTRS)

Miller, Stanley L.

1996-01-01

Organic compounds would have been produced in an earth's atmosphere that was reducing. The soup would contain amino and hydroxy acids, together with smaller amounts of purines and pyrimidines. The presence' of sugars is less likely, although they can be produced by the formose reaction from formaldehyde. However, the prebiotic synthesis of RNA has not been demonstrated. One problem is that ribose is not produced selectively over other pentoses and hexoses, except under special conditions. The second problem is that ribose is unstable, with a half-life at pH7 and 100 C of 73 minutes (44 years at 0 C). Other sugars are similarly unstable. Another problem is that there is no efficient prebiotic synthesis of polyphosphates, nor the glycosidic bond of nucleosides. This suggests that there may have been an informational macromolecule that preceded RNA. The RNA world refers to the time when RNA carried both the genetic information and the catalytic activity, and was subsequently converted to the DNA/protein world when protein synthesis began. Preceeding the RNA world was the Pre-RNA world, where a backbone different from ribose phosphate was used, and the bases may have been different from adenine, uracil, guanine and cytosine. We have shown recently that cytosine and uracil can be synthesized efficiently under prebiotic conditions using a dried lagoon model instead of the usual dilute ocean hypothesis. In addition, we have shown that uracil adds formaldehyde efficiently to give 5- hydroxymethyl uracil, which in turn adds various nucleophiles to give uracil analogs of most of the amino acids that occur in proteins. For example, the ammonia, guanidine and imidazole adducts from the analogs of lysine, arginine and histidine. This suggests that the catalytic potential of RNA may have been much more extensive than previously assumed. The major problem is finding out what was the precursor to the ribose phosphate backbone. This will be the key to developing prebiotic self-replicating systems.

The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose.

PubMed

Yonekura, Madoka; Aoki, Naohiro; Hirose, Tatsuro; Onai, Kiyoshi; Ishiura, Masahiro; Okamura, Masaki; Ohsugi, Ryu; Ohto, Chikara

2013-01-01

Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes.

Final Report for research on The Glucose 6-Phosphate Shunt Around the Calvin-Benson Cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharkey, Thomas D.

In this research, photosynthetic carbon metabolism was studied to identify mechanisms by which plants store energy from sunlight as carbon compounds, especially sugars. Conditions were identified in which carbon appeared to flow backwards from outside the photosynthetic compartment (chloroplast) back into it. A specific gene product was manipulated to make the flow bigger or smaller. Preventing the flow (by eliminating the gene) had little effect on plant growth but increasing the flow, by overexpressing the gene, caused the plants to become extremely sensitive to changes in light. Plants with the gene overexpressed had high rates of cyclic electron flow, themore » photosynthetic electron transport pathway that occurs when plants need more of the energy molecule ATP. These and other observations led us to conclude that a metabolic pathway that is normally turned off because it is counter-productive during photosynthesis, in fact occurs at about 10% of the rate of normal photosynthesis. This creates an inefficiency but may stabilize photosynthesis allowing it to cope with the very large and rapid changes that leaves experience such as the hundred-fold changes in light intensity that can occur in seconds on a partly cloudy day. We also concluded that the back flow of carbon into chloroplasts could be important at high rates of photosynthesis allowing increased rates of starch synthesis. Starch synthesis allows plants to store sugars during the day for use at night. At high rates of photosynthesis starch synthesis becomes very important to protect against end-product inhibition of photosynthesis. This research identified two metabolic pathways that extend the primary carbon fixation pathway called the Calvin-Benson cycle. These pathway extensions are now called the cytosolic bypass and the glucose 6-phosphate shunt. This improvement in our understanding of carbon metabolism of photosynthesis will guide efforts to increase photosynthesis to increase production of food, fuel, and fiber.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Egli, Martin; Pallan, Pradeep S.; Pattanayek, Rekha

An experimental rationalization of the structure type encountered in DNA and RNA by systematically investigating the chemical and physical properties of alternative nucleic acids has identified systems with a variety of sugar-phosphate backbones that are capable of Watson-Crick base pairing and in some cases cross-pairing with the natural nucleic acids. The earliest among the model systems tested to date, (4{prime} {yields} 6{prime})-linked oligo(2{prime},3{prime}-dideoxy-{beta}-d-glucopyranosyl)nucleotides or homo-DNA, shows stable self-pairing, but the pairing rules for the four natural bases are not the same as those in DNA. However, a complete interpretation and understanding of the properties of the hexapyranosyl (4{prime} {yields} 6{prime})more » family of nucleic acids has been impeded until now by the lack of detailed 3D-structural data. We have determined the crystal structure of a homo-DNA octamer. It reveals a weakly twisted right-handed duplex with a strong inclination between the hexose-phosphate backbones and base-pair axes, and highly irregular values for helical rise and twist at individual base steps. The structure allows a rationalization of the inability of allo-, altro-, and glucopyranosyl-based oligonucleotides to form stable pairing systems.« less

Identification and Levels of 2′-Carboxyarabinitol in Leaves 1

PubMed Central

Moore, Brandon d.; Sharkey, Thomas D.; Kobza, John; Seemann, Jeffrey R.

1992-01-01

2′-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C3, C4, and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants. PMID:16669072

Sucrose metabolism gene families and their biological functions

PubMed Central

Jiang, Shu-Ye; Chi, Yun-Hua; Wang, Ji-Zhou; Zhou, Jun-Xia; Cheng, Yan-Song; Zhang, Bao-Lan; Ma, Ali; Vanitha, Jeevanandam; Ramachandran, Srinivasan

2015-01-01

Sucrose, as the main product of photosynthesis, plays crucial roles in plant development. Although studies on general metabolism pathway were well documented, less information is available on the genome-wide identification of these genes, their expansion and evolutionary history as well as their biological functions. We focused on four sucrose metabolism related gene families including sucrose synthase, sucrose phosphate synthase, sucrose phosphate phosphatase and UDP-glucose pyrophosphorylase. These gene families exhibited different expansion and evolutionary history as their host genomes experienced differentiated rates of the whole genome duplication, tandem and segmental duplication, or mobile element mediated gene gain and loss. They were evolutionarily conserved under purifying selection among species and expression divergence played important roles for gene survival after expansion. However, we have detected recent positive selection during intra-species divergence. Overexpression of 15 sorghum genes in Arabidopsis revealed their roles in biomass accumulation, flowering time control, seed germination and response to high salinity and sugar stresses. Our studies uncovered the molecular mechanisms of gene expansion and evolution and also provided new insight into the role of positive selection in intra-species divergence. Overexpression data revealed novel biological functions of these genes in flowering time control and seed germination under normal and stress conditions. PMID:26616172

Double differential cross sections for proton induced electron emission from molecular analogues of DNA constituents for energies in the Bragg peak region

NASA Astrophysics Data System (ADS)

Rudek, Benedikt; Bennett, Daniel; Bug, Marion U.; Wang, Mingjie; Baek, Woon Yong; Buhr, Ticia; Hilgers, Gerhard; Champion, Christophe; Rabus, Hans

2016-09-01

For track structure simulations in the Bragg peak region, measured electron emission cross sections of DNA constituents are required as input for developing parameterized model functions representing the scattering probabilities. In the present work, double differential cross sections were measured for the electron emission from vapor-phase pyrimidine, tetrahydrofuran, and trimethyl phosphate that are structural analogues to the base, the sugar, and the phosphate residue of the DNA, respectively. The range of proton energies was from 75 keV to 135 keV, the angles ranged from 15° to 135°, and the electron energies were measured from 10 eV to 200 eV. Single differential and total electron emission cross sections are derived by integration over angle and electron energy and compared to the semi-empirical Hansen-Kocbach-Stolterfoht (HKS) model and a quantum mechanical calculation employing the first Born approximation with corrected boundary conditions (CB1). The CB1 provides the best prediction of double and single differential cross section, while total cross sections can be fitted with semi-empirical models. The cross sections of the three samples are proportional to their total number of valence electrons.

A nucleobase-centered coarse-grained representation for structure prediction of RNA motifs

PubMed Central

Poblete, Simón; Bottaro, Sandro; Bussi, Giovanni

2018-01-01

Abstract We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mathcal {E}$\\end{document}SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base–phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation. PMID:29272539

Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity†

PubMed Central

Zaragoza, Oscar; Blazquez, Miguel A.; Gancedo, Carlos

1998-01-01

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 106 CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation. PMID:9683476

Disruption of the Candida albicans TPS1 gene encoding trehalose-6-phosphate synthase impairs formation of hyphae and decreases infectivity.

PubMed

Zaragoza, O; Blazquez, M A; Gancedo, C

1998-08-01

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30 degreesC was indistinguishable from that of the wild type. However, at 42 degreesC it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37 degreesC, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42 degreesC, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10(6) CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.

Batch production of Pyranose 2-oxidase from Trametes versicolor (ATCC 11235) in medium with a lignocellulosic substrate and enzymatic bleaching of cotton fabrics.

PubMed

Pazarlioglu, Nurdan Kasikara; Erden, Emre; Ucar, M Cigdem; Akkaya, Alper; Sariisik, A Merih

2012-04-01

The aim of this work was to determine new, different and low-cost substrates that can be used for enzyme production from the white rot fungus Trametes versicolor (ATCC 11235) by taking advantage of the broad substrate specificity of pyranose 2-oxidase. In this report, we investigated the production of pyranose 2-oxidase from T. versicolor (ATCC 11235) using ten different agricultural residues such as clover straw, almond shells, hazelnut cobs, grass and others. Pyranose 2-oxidase activity was determined as 2.332 U/g at the 9th day in a submerged culture containing clover straw and tap water shaken at 150 rpm and 26°C, and the optimum clover straw concentration was determined to be 12 g/l. The effects of different glucose, nitrogen and phosphate sources on the production of pyranose 2-oxidase were studied in the clover straw medium. Analyses of biomass, protein, reduced sugar and nitrogen concentrations were also monitored in a clover straw medium that did not contain carbon or nitrogen and phosphate sources under the parameters determined. The produced pyranose 2-oxidase was used for improving the properties of cotton fabrics.

Efficient Synthesis and Bioactivity of Novel Triazole Derivatives.

PubMed

Hu, Boyang; Zhao, Hanqing; Chen, Zili; Xu, Chen; Zhao, Jianzhuang; Zhao, Wenting

2018-03-21

Triazole pesticides are organic nitrogen-containing heterocyclic compounds, which contain 1,2,3-triazole ring. In order to develop potential glucosamine-6-phosphate synthase (GlmS) inhibitor fungicides, forty compounds of triazole derivatives were synthesized in an efficient way, thirty nine of them were new compounds. The structures of all the compounds were confirmed by high resolution mass spectrometer (HRMS), ¹H-NMR and 13 C-NMR. The fungicidal activities results based on means of mycelium growth rate method indicated that some of the compounds exhibited good fungicidal activities against P. CapasiciLeonian , Sclerotinia sclerotiorum (Lib.) de Bary, Pyricularia oryzae Cav. and Fusarium oxysporum Schl. F.sp. vasinfectum (Atk.) Snyd. & Hans. at the concentration of 50 µg/mL, especially the inhibitory rates of compounds 1-d and 1-f were over 80%. At the same time, the preliminary studies based on the Elson-Morgan method indicated that the compounds exhibited some inhibitory activity toward glucosamine-6-phosphate synthase (GlmS). These compounds will be further studied as potential antifungal lead compounds. The structure-activity relationships (SAR) were discussed in terms of the effects of the substituents on both the benzene and the sugar ring.

Photoinduced catalytic synthesis of biologically important metabolites from formaldehyde and ammonia under plausible "prebiotic" conditions

NASA Astrophysics Data System (ADS)

Delidovich, I. V.; Taran, O. P.; Simonov, A. N.; Matvienko, L. G.; Parmon, V. N.

2011-08-01

The article analyzes new and previously reported data on several catalytic and photochemical processes yielding biologically important molecules. UV-irradiation of formaldehyde aqueous solution yields acetaldehyde, glyoxal, glycolaldehyde and glyceraldehyde, which can serve as precursors of more complex biochemically relevant compounds. Photolysis of aqueous solution of acetaldehyde and ammonium nitrate results in formation of alanine and pyruvic acid. Dehydration of glyceraldehyde catalyzed by zeolite HZSM-5-17 yields pyruvaldehyde. Monosaccharides are formed in the course of the phosphate-catalyzed aldol condensation reactions of glycolaldehyde, glyceraldehyde and formaldehyde. The possibility of the direct synthesis of tetroses, keto- and aldo-pentoses from pure formaldehyde due to the combination of the photochemical production of glycolahyde and phosphate-catalyzed carbohydrate chain growth is demonstrated. Erythrulose and 3-pentulose are the main products of such combined synthesis with selectivity up to 10%. Biologically relevant aldotetroses, aldo- and ketopentoses are more resistant to the photochemical destruction owing to the stabilization in hemiacetal cyclic forms. They are formed as products of isomerization of erythrulose and 3-pentulose. The conjugation of the concerned reactions results in a plausible route to the formation of sugars, amino and organic acids from formaldehyde and ammonia under presumed 'prebiotic' conditions.

Conformation-dependent restraints for polynucleotides: I. Clustering of the geometry of the phosphodiester group

PubMed Central

Kowiel, Marcin; Brzezinski, Dariusz; Jaskolski, Mariusz

2016-01-01

The refinement of macromolecular structures is usually aided by prior stereochemical knowledge in the form of geometrical restraints. Such restraints are also used for the flexible sugar-phosphate backbones of nucleic acids. However, recent highly accurate structural studies of DNA suggest that the phosphate bond angles may have inadequate description in the existing stereochemical dictionaries. In this paper, we analyze the bonding deformations of the phosphodiester groups in the Cambridge Structural Database, cluster the studied fragments into six conformation-related categories and propose a revised set of restraints for the O-P-O bond angles and distances. The proposed restraints have been positively validated against data from the Nucleic Acid Database and an ultrahigh-resolution Z-DNA structure in the Protein Data Bank. Additionally, the manual classification of PO4 geometry is compared with geometrical clusters automatically discovered by machine learning methods. The machine learning cluster analysis provides useful insights and a practical example for general applications of clustering algorithms for automatic discovery of hidden patterns of molecular geometry. Finally, we describe the implementation and application of a public-domain web server for automatic generation of the proposed restraints. PMID:27521371

Effect of dilution rate and nutrients addition on the fermentative capability and synthesis of aromatic compounds of two indigenous strains of Saccharomyces cerevisiae in continuous cultures fed with Agave tequilana juice.

PubMed

Morán-Marroquín, G A; Córdova, J; Valle-Rodríguez, J O; Estarrón-Espinosa, M; Díaz-Montaño, D M

2011-11-15

Knowledge of physiological behavior of indigenous tequila yeast used in fermentation process is still limited. Yeasts have significant impact on the productivity fermentation process as well as the sensorial characteristics of the alcoholic beverage. For these reasons a better knowledge of the physiological and metabolic features of these yeasts is required. The effects of dilution rate, nitrogen and phosphorus source addition and micro-aeration on growth, fermentation and synthesis of volatile compounds of two native Saccharomyces cerevisiae strains, cultured in continuous fed with Agave tequilana juice were studied. For S1 and S2 strains, maximal concentrations of biomass, ethanol, consumed sugars, alcohols and esters were obtained at 0.04 h⁻¹. Those concentrations quickly decreased as D increased. For S. cerevisiae S1 cultures (at D=0.08 h⁻¹) supplemented with ammonium phosphate (AP) from 1 to 4 g/L, concentrations of residual sugars decreased from 29.42 to 17.60 g/L and ethanol increased from 29.63 to 40.08 g/L, respectively. The S1 culture supplemented with AP was then micro-aerated from 0 to 0.02 vvm, improving all the kinetics parameters: biomass, ethanol and glycerol concentrations increased from 5.66, 40.08 and 3.11 g/L to 8.04, 45.91 and 4.88 g/L; residual sugars decreased from 17.67 g/L to 4.48 g/L; and rates of productions of biomass and ethanol, and consumption of sugars increased from 0.45, 3.21 and 7.33 g/L·h to 0.64, 3.67 and 8.38 g/L·h, respectively. Concentrations of volatile compounds were also influenced by the micro-aeration rate. Ester and alcohol concentrations were higher, in none aerated and in aerated cultures respectively. Copyright © 2011. Published by Elsevier B.V.

Arbuscular Mycorrhizal Fungus Enhances Lateral Root Formation in Poncirus trifoliata (L.) as Revealed by RNA-Seq Analysis

PubMed Central

Chen, Weili; Li, Juan; Zhu, Honghui; Xu, Pengyang; Chen, Jiezhong; Yao, Qing

2017-01-01

Arbuscular mycorrhizal fungi (AMF) establish symbiosis with most terrestrial plants, and greatly regulate lateral root (LR) formation. Phosphorus (P), sugar, and plant hormones are proposed being involved in this regulation, however, no global evidence regarding these factors is available so far, especially in woody plants. In this study, we inoculated trifoliate orange seedlings (Poncirus trifoliata L. Raf) with an AMF isolate, Rhizophagus irregularis BGC JX04B. After 4 months of growth, LR formation was characterized, and sugar contents in roots were determined. RNA-Seq analysis was performed to obtain the transcriptomes of LR root tips from non-mycorrhizal and mycorrhizal seedlings. Quantitative real time PCR (qRT-PCR) of selected genes was also conducted for validation. The results showed that AMF significantly increased LR number, as well as plant biomass and shoot P concentration. The contents of glucose and fructose in primary root, and sucrose content in LR were also increased. A total of 909 differentially expressed genes (DEGs) were identified in response to AMF inoculation, and qRT-PCR validated the transcriptomic data. The numbers of DEGs related to P, sugar, and plant hormones were 31, 32, and 25, respectively. For P metabolism, the most up-regulated DEGs mainly encoded phosphate transporter, and the most down-regulated DEGs encoded acid phosphatase. For sugar metabolism, the most up-regulated DEGs encoded polygalacturonase and chitinase. For plant hormones, the most up-regulated DEGs were related to auxin signaling, and the most down-regulated DEGs were related to ethylene signaling. PLS-SEM analysis indicates that P metabolism was the most important pathway by which AMF regulates LR formation in this study. These data reveal the changes of genome-wide gene expression in responses to AMF inoculation in trifoliate orange and provide a solid basis for the future identification and characterization of key genes involved in LR formation induced by AMF. PMID:29238356

Arbuscular Mycorrhizal Fungus Enhances Lateral Root Formation in Poncirus trifoliata (L.) as Revealed by RNA-Seq Analysis.

PubMed

Chen, Weili; Li, Juan; Zhu, Honghui; Xu, Pengyang; Chen, Jiezhong; Yao, Qing

2017-01-01

Arbuscular mycorrhizal fungi (AMF) establish symbiosis with most terrestrial plants, and greatly regulate lateral root (LR) formation. Phosphorus (P), sugar, and plant hormones are proposed being involved in this regulation, however, no global evidence regarding these factors is available so far, especially in woody plants. In this study, we inoculated trifoliate orange seedlings ( Poncirus trifoliata L. Raf) with an AMF isolate, Rhizophagus irregularis BGC JX04B. After 4 months of growth, LR formation was characterized, and sugar contents in roots were determined. RNA-Seq analysis was performed to obtain the transcriptomes of LR root tips from non-mycorrhizal and mycorrhizal seedlings. Quantitative real time PCR (qRT-PCR) of selected genes was also conducted for validation. The results showed that AMF significantly increased LR number, as well as plant biomass and shoot P concentration. The contents of glucose and fructose in primary root, and sucrose content in LR were also increased. A total of 909 differentially expressed genes (DEGs) were identified in response to AMF inoculation, and qRT-PCR validated the transcriptomic data. The numbers of DEGs related to P, sugar, and plant hormones were 31, 32, and 25, respectively. For P metabolism, the most up-regulated DEGs mainly encoded phosphate transporter, and the most down-regulated DEGs encoded acid phosphatase. For sugar metabolism, the most up-regulated DEGs encoded polygalacturonase and chitinase. For plant hormones, the most up-regulated DEGs were related to auxin signaling, and the most down-regulated DEGs were related to ethylene signaling. PLS-SEM analysis indicates that P metabolism was the most important pathway by which AMF regulates LR formation in this study. These data reveal the changes of genome-wide gene expression in responses to AMF inoculation in trifoliate orange and provide a solid basis for the future identification and characterization of key genes involved in LR formation induced by AMF.

Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

PubMed

Senyuva, Hamide Z; Cimen, Dilek; Gilbert, John

2009-01-01

The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pişmaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes.

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass.

PubMed

Poudel, Suresh; Giannone, Richard J; Rodriguez, Miguel; Raman, Babu; Martin, Madhavi Z; Engle, Nancy L; Mielenz, Jonathan R; Nookaew, Intawat; Brown, Steven D; Tschaplinski, Timothy J; Ussery, David; Hettich, Robert L

2017-01-01

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe's growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum's cellular membrane as the culture progresses. This is undoubtedly a response to the gradual accumulation of lignocellulose-derived inhibitory compounds as the organism deconstructs the switchgrass to access the embedded cellulose. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in various enzymes, including those involved in the interconversion of branched amino acids valine, leucine, and isoleucine to iso- and anteiso-fatty acid precursors. Additionally, the metabolic accumulation of hemicellulose-derived sugars and sugar alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism/pentose phosphate pathway indicates that C. thermocellum shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux in response to C5 sugar metabolites that increase during lignocellulose deconstruction. Integrated omic platforms provided complementary systems biological information that highlight C. thermocellum 's specific response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. These additional viewpoints allowed us to fully realize the level to which the organism adapts to an increasingly challenging culture environment-information that will prove critical to C. thermocellum 's industrial efficacy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John -Paul; Klesmith, Justin R.; Whitehead, Timothy A.

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium andmore » solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Furthermore, greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.« less

Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal R.; Swaminathan S.; Burley, S. K.

2012-01-01

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptidemore » chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.« less

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

DOE PAGES

Khosravi, Claire; Battaglia, Evy; Kun, Roland S.; ...

2018-03-22

Background: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involvedmore » in regulating preferential use of different carbon catabolic pathways. Results: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Conclusion: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.« less

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khosravi, Claire; Battaglia, Evy; Kun, Roland S.

Background: Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involvedmore » in regulating preferential use of different carbon catabolic pathways. Results: Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Conclusion: Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses.« less

A Simpler Nucleic Acid

NASA Technical Reports Server (NTRS)

Orgel, Leslie

2000-01-01

It has been supposed that for a nucleic acid analog to pair with RNA it must, like RNA, have a backbone with at least a sixatom repeat; a shorter backbone presumably would not stretch far enough to bind RNA properly. The Eschenmoser group has shown, however, that this first impression is incorrect.As they report in their new paper, Eschenmoser and co-workers ( I ) have now synthesized a substantial number of these polymers, which are called (L)-a-threofuranosyl oligonucleotides or TNAs. They are composed of bases linked to a threose sugar-phosphate backbone, with phosphodiester bonds connecting the nucleotides. The investigators discovered that pairs of complementary TNAs do indeed form stable Watson-Crick double helices and, perhaps more importantly, that TNAs form stable double helices with complementary RNAs and DNAs.

Identification of a Novel N-Acetylmuramic Acid Transporter in Tannerella forsythia

PubMed Central

Ruscitto, Angela; Hottmann, Isabel; Stafford, Graham P.; Schäffer, Christina

2016-01-01

ABSTRACT Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia coli MurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans, but not individually, rescued the inability of an E. coli mutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE In this study, we report the identification of a novel transporter for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia. It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique to T. forsythia, it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylum Bacteroidetes. PMID:27601356

Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp.

PubMed

Bonin, Patricia; Groisillier, Agnès; Raimbault, Alice; Guibert, Anaïs; Boyen, Catherine; Tonon, Thierry

2015-09-01

The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of low abundant trehalose-6-phosphate and related metabolites in Medicago truncatula by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry.

PubMed

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.

Metabolomic Effects of Xylitol and Fluoride on Plaque Biofilm in Vivo

PubMed Central

Takahashi, N.; Washio, J.

2011-01-01

Dental caries is initiated by demineralization of the tooth surface through acid production from sugar by plaque biofilm. Fluoride and xylitol have been used worldwide as caries-preventive reagents, based on in vitro-proven inhibitory mechanisms on bacterial acid production. We attempted to confirm the inhibitory mechanisms of fluoride and xylitol in vivo by performing metabolome analysis on the central carbon metabolism in supragingival plaque using the combination of capillary electrophoresis and a time-of-flight mass spectrometer. Fluoride (225 and 900 ppm F−) inhibited lactate production from 10% glucose by 34% and 46%, respectively, along with the increase in 3-phosphoglycerate and the decrease in phosphoenolpyruvate in the EMP pathway in supragingival plaque. These results confirmed that fluoride inhibited bacterial enolase in the EMP pathway and subsequently repressed acid production in vivo. In contrast, 10% xylitol had no effect on acid production and the metabolome profile in supragingival plaque, although xylitol 5-phosphate was produced. These results suggest that xylitol is not an inhibitor of plaque acid production but rather a non-fermentative sugar alcohol. Metabolome analyses of plaque biofilm can be applied for monitoring the efficacy of dietary components and medicines for plaque biofilm, leading to the development of effective plaque control. PMID:21940519

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

PubMed

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-02-25

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

Galactokinase promiscuity: a question of flexibility?

PubMed

McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

2016-02-01

Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. © 2016 Authors; published by Portland Press Limited.

X-ray-structure of a cytidylyl-3',5'-adenosine-proflavine complex: a self-paired parallel-chain double helical dimer with an intercalated acridine dye.

PubMed Central

Westhof, E; Sundaralingam, M

1980-01-01

The non-self-complementary dinucleoside monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base-paired parallel-chain dimer with an intercalated proflavine. The dimer complex possesses a right-handed helical twist. The dimer helix has an irregular girth with a neutral adenine-adenine (A-A) pair, hydrogen-bonded through the N6 and N7 sites (C1'...C1' separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the base N3 sites (Cl'...Cl' separation of 9.59 A). The torsion angles of the sugar-phosphate backbone are within their most preferred ranges and the sugar puckering sequence (5' leads to 3') is C3'-endo, C2'-endo. There is also a second proflavine molecule sandwiched between CpA dimers on the 21-axis. Both proflavines are necessarily disordered, being on dyad axis, and this suggests possible insights into the dynamics of intercalation of planar drugs. This structure shows that intercalation of planar drugs in nucleic acids may not be restricted to antiparallel complementary Watson-Crick pairing regions and provides additional mechanisms for acridine mutagenesis. PMID:6929524

Identification and functional analysis of the gene cluster for fructan utilization in Prevotella intermedia.

PubMed

Fuse, Haruka; Fukamachi, Haruka; Inoue, Mitsuko; Igarashi, Takeshi

2013-02-25

Fructanase enzymes hydrolyze the β-2,6 and β-2,1 linkages of levan and inulin fructans, respectively. We analyzed the influence of fructan on the growth of Prevotella intermedia. The growth of P. intermedia was enhanced by addition of inulin, implying that P. intermedia could also use inulin. Based on this finding, we identified and analyzed the genes encoding a putative fructanase (FruA), sugar transporter (FruB), and fructokinase (FruK) in the genome of strain ATCC25611. Transcript analysis by RT-PCR showed that the fruABK genes were co-transcribed as a single mRNA and semi-quantitative analysis confirmed that the fruA gene was induced in response to fructose and inulin. Recombinant FruA and FruK were purified and characterized biochemically. FruA strongly hydrolyzed inulin, with slight degradation of levan via an exo-type mechanism, revealing that FruA is an exo-β-d-fructanase. FruK converted fructose to fructose-6-phosphate in the presence of ATP, confirming that FruK is an ATP-dependent fructokinase. These results suggest that P. intermedia can utilize fructan as a carbon source for growth, and that the fructanase, sugar transporter, and fructokinase proteins we identified are involved in this fructan utilization. Copyright © 2012 Elsevier B.V. All rights reserved.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

PubMed Central

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-01-01

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

1H-NMR metabolomic profiling reveals a distinct metabolic recovery response in shoots and roots of temporarily drought-stressed sugar beets

PubMed Central

Maucourt, Mickaël; Deborde, Catherine; Moing, Annick; Gibon, Yves; Goldbach, Heiner E.; Wimmer, Monika A.

2018-01-01

Yield formation in regions with intermittent drought periods depends on the plant’s ability to recover after cessation of the stress. The present work assessed differences in metabolic recovery of leaves and roots of drought-stressed sugar beets with high temporal resolution. Plants were subjected to drought for 13 days, and rewatered for 12 days. At one to two-day intervals, plant material was harvested for untargeted 1H-NMR metabolomic profiling, targeted analyses of hexose-phosphates, starch, amino acids, nitrate and proteins, and physiological measurements including relative water content, osmotic potential, electrolyte leakage and malondialdehyde concentrations. Drought triggered changes in primary metabolism, especially increases in amino acids in both organs, but leaves and roots responded with different dynamics to rewatering. After a transient normalization of most metabolites within 8 days, a second accumulation of amino acids in leaves might indicate a stress imprint beneficial in upcoming drought events. Repair mechanisms seemed important during initial recovery and occurred at the expense of growth for at least 12 days. These results indicate that organ specific metabolic recovery responses might be related to distinct functions and concomitant disparate stress levels in above- and belowground organs. With respect to metabolism, recovery was not simply a reversal of the stress responses. PMID:29738573

Oxidation and Assimilation of Carbohydrates by Micrococcus sodonensis1

PubMed Central

Perry, Jerome J.; Evans, James B.

1966-01-01

Perry, Jerome J. (North Carolina State University, Raleigh), and James B. Evans. Oxidation and assimilation of carbohydrates by Micrococcus sodonensis. J. Bacteriol. 91:33–38. 1966.—Micrococcus sodonensis is a biotin-requiring strict aerobe that cannot utilize carbohydrates as sole sources of carbon and energy. However, addition of mannose, glucose, sucrose, or maltose to a medium on which the organism can grow resulted in an increase in total growth. M. sodonensis oxidized these sugars without induction, thus indicating the presence of constitutive enzymes for their transport, activation, and metabolism. Under appropriate nonproliferating cell conditions, glucose was readily incorporated into essential constituents of the cell. When glucose-1-C14 and glucose-6-C14 were oxidized by nonproliferating cells, the label was found in both the protein and nucleic acid fractions of the cell. The respiratory quotients of cells oxidizing glucose in saline and in phosphate buffer indicated assimilation of sugar carbon in buffer and virtually no assimilation in saline. The ability of M. sodonensis to completely oxidize glucose and to grow on intermediates of glucose oxidation but not on glucose suggests that glucose may suppress or repress some reaction(s) necessary for growth, and that growth substrates either derepress or circumvent this block. PMID:5903100

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

PubMed

Drake, Andrew C; Lee, Youngjoo; Burgess, Emma M; Karlsson, Jens O M; Eroglu, Ali; Higgins, Adam Z

2018-01-01

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.

Comparative metabolic responses and adaptive strategies of wheat (Triticum aestivum) to salt and alkali stress.

PubMed

Guo, Rui; Yang, Zongze; Li, Feng; Yan, Changrong; Zhong, Xiuli; Liu, Qi; Xia, Xu; Li, Haoru; Zhao, Long

2015-07-07

It is well known that salinization (high-pH) has been considered as a major environmental threat to agricultural systems. The aim of this study was to investigate the differences between salt stress and alkali stress in metabolic profiles and nutrient accumulation of wheat; these parameters were also evaluated to determine the physiological adaptive mechanisms by which wheat tolerates alkali stress. The harmful effect of alkali stress on the growth and photosynthesis of wheat were stronger than those of salt stress. High-pH of alkali stress induced the most of phosphate and metal ions to precipitate; as a result, the availability of nutrients significantly declined. Under alkali stress, Ca sharply increased in roots, however, it decreased under salt stress. In addition, we detected the 75 metabolites that were different among the treatments according to GC-MS analysis, including organic acids, amino acids, sugars/polyols and others. The metabolic data showed salt stress and alkali stress caused different metabolic shifts; alkali stress has a stronger injurious effect on the distribution and accumulation of metabolites than salt stress. These outcomes correspond to specific detrimental effects of a highly pH environment. Ca had a significant positive correlation with alkali tolerates, and increasing Ca concentration can immediately trigger SOS Na exclusion system and reduce the Na injury. Salt stress caused metabolic shifts toward gluconeogenesis with increased sugars to avoid osmotic stress; energy in roots and active synthesis in leaves were needed by wheat to develop salt tolerance. Alkali stress (at high pH) significantly inhibited photosynthetic rate; thus, sugar production was reduced, N metabolism was limited, amino acid production was reduced, and glycolysis was inhibited.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer

PubMed Central

Burgess, Emma M.; Karlsson, Jens O. M.; Eroglu, Ali

2018-01-01

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants. PMID:29304068

A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation.

PubMed

Bera, Aloke K; Ho, Nancy W Y; Khan, Aftab; Sedlak, Miroslav

2011-05-01

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.

Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xi; Goh, Ee-Been; Beller, Harry R.

Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our mostmore » efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C 6 and C 5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.« less

In situ effect of a CPP-ACP chewing gum on enamel erosion associated or not with abrasion.

PubMed

de Oliveira, Andressa Feitosa Bezerra; de Oliveira Diniz, Luciana Vilar; Forte, Franklin Delano Soares; Sampaio, Fabio Correia; Ccahuana-Vásquez, Renzo Alberto; Tochukwu Amaechi, Bennett

2017-01-01

The purpose of this study is to analyze the in situ effect of a casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP) chewing gum on human enamel erosion lesion associated or not with abrasion. A three-way crossover study of 7 days was conducted involving 10 volunteers subjected to the same protocol: (G1) CPP-ACP sugar-free chewing gum, (G2) regular sugar-free chewing gum without CPP-ACP, and (G3) saliva-no chewing gum. An abrasion test was included in each phase. A 3D non-contact profilometry measurement of lesion depth and surface roughness was obtained of sound and eroded surfaces. A salivary calcium concentration was determined for all volunteers. ANOVA followed by Tukey's test were used with a p < 0.05. The enamel depth and the enamel surface roughness of the CPP-ACP gum group were significantly lower than the others (ANOVA, p < 0.05). No significant differences were observed between the treatments when associated with abrasion (p > 0.05). A positive and significant correlation was seen between the lesion depth and enamel surface roughness for GI (r = 0.87, p = 0.00) and GIII (r = 0.79, p = 0.00) groups. The estimated total calcium presented in the saliva after the chewed CPP-ACP gum showed no statistical significance between the mean absorbance values at the different time collections (p > 0.05). It is demonstrated that the incorporation of the CPP-ACP into a sugar-free gum significantly increased the remineralization/protection of eroded enamel surface. The CPP-ACP added to gum may be a suitable alternative vehicle, to deliver calcium ions to saliva and therefore protecting enamel.

A mathematical model of the influence of salivary urea on the pH of fasted dental plaque and on the changes occurring during a cariogenic challenge.

PubMed

Dibdin, G H; Dawes, C

1998-01-01

Urea diffusing from saliva into dental plaque is converted to ammonia and carbon dioxide by bacterial ureases. The influence of normal salivary urea levels on the pH of fasted plaque and on the depth and duration of a Stephan curve is uncertain. A numerical model which simulates a cariogenic challenge (a 10% sucrose rinse alone or one followed by use of chewing-gum with or without sugar) was modified to include salivary urea levels from 0 to 30 mmol/l. It incorporated: site-dependent exchange between bulk saliva and plaque surfaces via a salivary film; sugar and urea diffusion into plaque; pH-dependent rates of acid formation and urea breakdown; diffusion and dissociation of end-products and other buffers (acetate, lactate, phosphate, ammonia and carbonate); diffusion of protons and other ions; equilibration with fixed and mobile buffers; and charge-coupling between ionic flows. The Km (2.12 mmol/l) and Vmax (0.11 micromol urea/min/mg dry weight) values for urease activity and the pH dependence of Vmax were taken from the literature. From the results, it is predicted that urea concentrations normally present in saliva (3-5 mmol/l) will increase the pH at the base of a 0.5-mm-thick fasted plaque by up to 1 pH unit, and raise the pH minimum after a sucrose rinse or sugar-containing chewing-gum by at least half a pH unit. The results suggest that plaque cariogenicity may be inversely related to salivary urea concentrations, not only when the latter are elevated because of disease, but even when they are in the normal range.

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism

PubMed Central

Nardozza, Simona; MacRae, Elspeth A.; Sulpice, Ronan; Clearwater, Michael J.

2013-01-01

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species. PMID:24058160

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

PubMed

Nardozza, Simona; Boldingh, Helen L; Osorio, Sonia; Höhne, Melanie; Wohlers, Mark; Gleave, Andrew P; MacRae, Elspeth A; Richardson, Annette C; Atkinson, Ross G; Sulpice, Ronan; Fernie, Alisdair R; Clearwater, Michael J

2013-11-01

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production

DOE PAGES

Wang, Xi; Goh, Ee-Been; Beller, Harry R.

2018-01-27

Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our mostmore » efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C 6 and C 5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.« less

Dibasic Ammonium Phosphate Application Enhances Aromatic Compound Concentration in Bog Bilberry Syrup Wine.

PubMed

Wang, Shao-Yang; Li, Yi-Qing; Li, Teng; Yang, Hang-Yu; Ren, Jie; Zhang, Bo-Lin; Zhu, Bao-Qing

2016-12-29

A nitrogen deficiency always causes bog bilberry syrup wine to have a poor sensory feature. This study investigated the effect of nitrogen source addition on volatile compounds during bog bilberry syrup wine fermentation. The syrup was supplemented with 60, 90, 120 or 150 mg/L dibasic ammonium phosphate (DAP) before fermentation. Results showed that an increase of DAP amounts accelerated fermentation rate, increased alcohol content, and decreased sugar level. Total phenol and total flavonoid content were also enhanced with the increase of DAP amounts. A total of 91 volatile compounds were detected in the wine and their concentrations were significantly enhanced with the increase of DAP. Ethyl acetate, isoamyl acetate, phenethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, isobutanol, isoamyl alcohol, levo -2,3-butanediol, 2-phenylethanol, meso -2,3-butanediol, isobutyric acid, hexanoic acid, and octanoic acid exhibited a significant increase of their odor activity value (OAV) with the increase of DAP amounts. Bog bilberry syrup wine possessed fruity, fatty, and caramel flavors as its major aroma, whereas a balsamic note was the least present. The increase of DAP amounts significantly improved the global aroma attributes, thereby indicating that DAP supplementation could promote wine fermentation performance and enhance the sensory quality of bog bilberry syrup wine.

Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold-induced sweetening and apical dominance of potato (Solanum tuberosum L.) tubers.

PubMed

Liu, Tengfei; Fang, Hui; Liu, Jun; Reid, Stephen; Hou, Juan; Zhou, Tingting; Tian, Zhendong; Song, Botao; Xie, Conghua

2017-12-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme that functions in producing energy and supplying intermediates for cellular metabolism. Recent researches indicate that GAPDHs have multiple functions beside glycolysis. However, little information is available for functions of GAPDHs in potato. Here, we identified 4 putative cytosolic GAPDH genes in potato genome and demonstrated that the StGAPC1, StGAPC2, and StGAPC3, which are constitutively expressed in potato tissues and cold inducible in tubers, encode active cytosolic GAPDHs. Cosuppression of these 3 GAPC genes resulted in low tuber GAPDH activity, consequently the accumulation of reducing sugars in cold stored tubers by altering the tuber metabolite pool sizes favoring the sucrose pathway. Furthermore, GAPCs-silenced tubers exhibited a loss of apical dominance dependent on cell death of tuber apical bud meristem (TAB-meristem). It was also confirmed that StGAPC1, StGAPC2, and StGAPC3 interacted with the autophagy-related protein 3 (ATG3), implying that the occurrence of cell death in TAB-meristem could be induced by ATG3 associated events. Collectively, the present research evidences first that the GAPC genes play crucial roles in diverse physiological and developmental processes in potato tubers. © 2017 John Wiley & Sons Ltd.

Phytotoxic effects of Sicyos deppei (Cucurbitaceae) in germinating tomato seeds.

PubMed

Lara-Núñez, Aurora; Sánchez-Nieto, Sobeida; Luisa Anaya, Ana; Cruz-Ortega, Rocio

2009-06-01

The phytotoxic effect of allelochemicals is referred to as allelochemical stress and it is considered a biotic stress. Sicyos deppei G. Don (Cucurbitaceae) is an allelopathic weed that causes phytotoxicity in Lycopersicon esculentum, delaying seed germination and severely inhibiting radicle growth. This paper reports in in vitro conditions, the effects of the aqueous leachate of S. deppei-throughout tomato germination times-on (1) the dynamics of starch and sugars metabolism, (2) activity and expression of the cell wall enzymes involved in endosperm weakening that allows the protrusion of the radicle, and (3) whether abscisic acid (ABA) is involved in this altered metabolic processes. Results showed that S. deppei leachate on tomato seed germination mainly caused: (1) delay in starch degradation as well as in sucrose hydrolysis; (2) lower activity of sucrose phosphate synthase, cell wall invertase, and alpha-amylase; being sucrose phosphate synthase (SPS) gene expression down-regulated, and the last two up regulated; (3) also, lower activity of endo beta-mannanase, beta-1,3 glucanase, alpha-galactosidase, and exo-polygalacturonase with altered gene expression; and (4) higher content of ABA during all times of germination. The phytotoxic effect of S. deppei aqueous leachate is because of the sum of many metabolic processes affected during tomato seed germination that finally is evidenced by a strong inhibition of radicle growth.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Stacking interactions and DNA intercalation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Dr. Shen; Cooper, Valentino R; Thonhauser, Prof. Timo

2009-01-01

The relationship between stacking interactions and the intercalation of proflavine and ellipticine within DNA is investigated using a nonempirical van der Waals density functional for the correlation energy. Our results, employing a binary stack model, highlight fundamental, qualitative differences between base-pair base-pair interactions and that of the stacked intercalator base pair system. Most notable result is the paucity of torque which so distinctively defines the Twist of DNA. Surprisingly, this model, when combined with a constraint on the twist of the surrounding base-pair steps to match the observed unwinding of the sugar-phosphate backbone, was sufficient for explaining the experimentally observedmore » proflavine intercalator configuration. Our extensive mapping of the potential energy surface of base-pair intercalator interactions can provide valuable information for future nonempirical studies of DNA intercalation dynamics.« less

Electrostatic contribution to twist rigidity of DNA.

PubMed

Mohammad-Rafiee, Farshid; Golestanian, Ramin

2004-06-01

The electrostatic contribution to the twist rigidity of DNA is studied, and it is shown that the Coulomb self-energy of the double-helical sugar-phosphate backbone makes a considerable contribution-the electrostatic twist rigidity of DNA is found to be C(elec) approximately 5 nm, which makes up about 7% of its total twist rigidity ( C(DNA) approximately 75 nm). The electrostatic twist rigidity is found, however, to depend only weakly on the salt concentration, because of a competition between two different screening mechanisms: (1) Debye screening by the salt ions in the bulk, and (2) structural screening by the periodic charge distribution along the backbone of the helical polyelectrolyte. It is found that, depending on the parameters, the electrostatic contribution to the twist rigidity could stabilize or destabilize the structure of a helical polyelectrolyte.

Feedback Inhibition of Starch Degradation in Arabidopsis Leaves Mediated by Trehalose 6-Phosphate1[W][OPEN

PubMed Central

Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

2013-01-01

Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. PMID:24043444

Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism

PubMed Central

Shaik, Shahnoor S.; Obata, Toshihiro; Hebelstrup, Kim H.; Schwahn, Kevin; Fernie, Alisdair R.; Mateiu, Ramona V.; Blennow, Andreas

2016-01-01

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD. PMID:26891365

Invertebrate Trehalose-6-Phosphate Synthase Gene: Genetic Architecture, Biochemistry, Physiological Function, and Potential Applications.

PubMed

Tang, Bin; Wang, Su; Wang, Shi-Gui; Wang, Hui-Juan; Zhang, Jia-Yong; Cui, Shuai-Ying

2018-01-01

The non-reducing disaccharide trehalose is widely distributed among various organisms. It plays a crucial role as an instant source of energy, being the major blood sugar in insects. In addition, it helps countering abiotic stresses. Trehalose synthesis in insects and other invertebrates is thought to occur via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. In many insects, the TPP gene has not been identified, whereas multiple TPS genes that encode proteins harboring TPS/OtsA and TPP/OtsB conserved domains have been found and cloned in the same species. The function of the TPS gene in insects and other invertebrates has not been reviewed in depth, and the available information is quite fragmented. The present review discusses the current understanding of the trehalose synthesis pathway, TPS genetic architecture, biochemistry, physiological function, and potential sensitivity to insecticides. We note the variability in the number of TPS genes in different invertebrate species, consider whether trehalose synthesis may rely only on the TPS gene, and discuss the results of in vitro TPS overexpression experiment. Tissue expression profile and developmental characteristics of the TPS gene indicate that it is important in energy production, growth and development, metamorphosis, stress recovery, chitin synthesis, insect flight, and other biological processes. We highlight the molecular and biochemical properties of insect TPS that make it a suitable target of potential pest control inhibitors. The application of trehalose synthesis inhibitors is a promising direction in insect pest control because vertebrates do not synthesize trehalose; therefore, TPS inhibitors would be relatively safe for humans and higher animals, making them ideal insecticidal agents without off-target effects.

Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides.

PubMed

Wang, Yanan; Zhang, Sufang; Zhu, Zhiwei; Shen, Hongwei; Lin, Xinping; Jin, Xiang; Jiao, Xiang; Zhao, Zongbao Kent

2018-01-01

Lipid accumulation by oleaginous microorganisms is of great scientific interest and biotechnological potential. While nitrogen limitation has been routinely employed, low-cost raw materials usually contain rich nitrogenous components, thus preventing from efficient lipid production. Inorganic phosphate (Pi) limitation has been found sufficient to promote conversion of sugars into lipids, yet the molecular basis of cellular response to Pi limitation and concurrent lipid accumulation remains elusive. Here, we performed multi-omic analyses of the oleaginous yeast Rhodosporidium toruloides to shield lights on Pi-limitation-induced lipid accumulation. Samples were prepared under Pi-limited as well as Pi-repleted chemostat conditions, and subjected to analysis at the transcriptomic, proteomic, and metabolomic levels. In total, 7970 genes, 4212 proteins, and 123 metabolites were identified. Results showed that Pi limitation facilitates up-regulation of Pi-associated metabolism, RNA degradation, and triacylglycerol biosynthesis while down-regulation of ribosome biosynthesis and tricarboxylic acid cycle. Pi limitation leads to dephosphorylation of adenosine monophosphate and the allosteric activator of isocitrate dehydrogenase key to lipid biosynthesis. It was found that NADPH, the key cofactor for fatty acid biosynthesis, is limited due to reduced flux through the pentose phosphate pathway and transhydrogenation cycle and that this can be overcome by over-expression of an endogenous malic enzyme. These phenomena are found distinctive from those under nitrogen limitation. Our data suggest that Pi limitation activates Pi-related metabolism, RNA degradation, and TAG biosynthesis while inhibits ribosome biosynthesis and TCA cycle, leading to enhanced carbon fluxes into lipids. The information greatly enriches our understanding on microbial oleaginicity and Pi-related metabolism. Importantly, systems data may facilitate designing advanced cell factories for production of lipids and related oleochemicals.

Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves 1

PubMed Central

Klepper, Lowell; Flesher, Donna; Hageman, R. H.

1971-01-01

An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO3 (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals. By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase. It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction. This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue. The level of nitrate reductase activity obtained with the in vitro assay is higher (2.5- to 20-fold) than with the in vivo assay for most plant species. The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species. The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted. PMID:16657841

Feedback inhibition of starch degradation in Arabidopsis leaves mediated by trehalose 6-phosphate.

PubMed

Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

2013-11-01

Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.

Computer Folding of RNA Tetraloops: Identification of Key Force Field Deficiencies.

PubMed

Kührová, Petra; Best, Robert B; Bottaro, Sandro; Bussi, Giovanni; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

2016-09-13

The computer-aided folding of biomolecules, particularly RNAs, is one of the most difficult challenges in computational structural biology. RNA tetraloops are fundamental RNA motifs playing key roles in RNA folding and RNA-RNA and RNA-protein interactions. Although state-of-the-art Molecular Dynamics (MD) force fields correctly describe the native state of these tetraloops as a stable free-energy basin on the microsecond time scale, enhanced sampling techniques reveal that the native state is not the global free energy minimum, suggesting yet unidentified significant imbalances in the force fields. Here, we tested our ability to fold the RNA tetraloops in various force fields and simulation settings. We employed three different enhanced sampling techniques, namely, temperature replica exchange MD (T-REMD), replica exchange with solute tempering (REST2), and well-tempered metadynamics (WT-MetaD). We aimed to separate problems caused by limited sampling from those due to force-field inaccuracies. We found that none of the contemporary force fields is able to correctly describe folding of the 5'-GAGA-3' tetraloop over a range of simulation conditions. We thus aimed to identify which terms of the force field are responsible for this poor description of TL folding. We showed that at least two different imbalances contribute to this behavior, namely, overstabilization of base-phosphate and/or sugar-phosphate interactions and underestimated stability of the hydrogen bonding interaction in base pairing. The first artifact stabilizes the unfolded ensemble, while the second one destabilizes the folded state. The former problem might be partially alleviated by reparametrization of the van der Waals parameters of the phosphate oxygens suggested by Case et al., while in order to overcome the latter effect we suggest local potentials to better capture hydrogen bonding interactions.

Invertebrate Trehalose-6-Phosphate Synthase Gene: Genetic Architecture, Biochemistry, Physiological Function, and Potential Applications

PubMed Central

Tang, Bin; Wang, Su; Wang, Shi-Gui; Wang, Hui-Juan; Zhang, Jia-Yong; Cui, Shuai-Ying

2018-01-01

The non-reducing disaccharide trehalose is widely distributed among various organisms. It plays a crucial role as an instant source of energy, being the major blood sugar in insects. In addition, it helps countering abiotic stresses. Trehalose synthesis in insects and other invertebrates is thought to occur via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. In many insects, the TPP gene has not been identified, whereas multiple TPS genes that encode proteins harboring TPS/OtsA and TPP/OtsB conserved domains have been found and cloned in the same species. The function of the TPS gene in insects and other invertebrates has not been reviewed in depth, and the available information is quite fragmented. The present review discusses the current understanding of the trehalose synthesis pathway, TPS genetic architecture, biochemistry, physiological function, and potential sensitivity to insecticides. We note the variability in the number of TPS genes in different invertebrate species, consider whether trehalose synthesis may rely only on the TPS gene, and discuss the results of in vitro TPS overexpression experiment. Tissue expression profile and developmental characteristics of the TPS gene indicate that it is important in energy production, growth and development, metamorphosis, stress recovery, chitin synthesis, insect flight, and other biological processes. We highlight the molecular and biochemical properties of insect TPS that make it a suitable target of potential pest control inhibitors. The application of trehalose synthesis inhibitors is a promising direction in insect pest control because vertebrates do not synthesize trehalose; therefore, TPS inhibitors would be relatively safe for humans and higher animals, making them ideal insecticidal agents without off-target effects. PMID:29445344

Glycerol-3-phosphate-induced catabolite repression in Escherichia coli.

PubMed

Eppler, Tanja; Postma, Pieter; Schütz, Alexandra; Völker, Uwe; Boos, Winfried

2002-06-01

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA(Glc), as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA(Glc) phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA(Glc). Isopropyl-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA(Glc). A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

PubMed

Yang, Jiangang; Zhu, Yueming; Li, Jitao; Men, Yan; Sun, Yuanxia; Ma, Yanhe

2015-01-01

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. © 2014 Wiley Periodicals, Inc.

Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Hasunuma, Tomohisa; Matsuda, Mami; Kato, Yuichi; Vavricka, Christopher John; Kondo, Akihiko

2018-05-27

Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13 C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO 2 , leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO 3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO 2 capture and utilization. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

PubMed

Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Fujimoto, Yuki; Furuie, Sumie; Yamamura, Ken-Ichi; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Moriyama, Mitsuaki; Sinasac, David S; Yamamoto, Takashi; Furukawa, Tatsuhiko; Kobayashi, Keiko

2017-04-01

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of glycation on α-crystallin structure and chaperone-like function

PubMed Central

Kumar, P. Anil; Kumar, M. Satish; Reddy, G. Bhanuprakash

2007-01-01

The chaperone-like activity of α-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of α-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of α-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of α-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of α-crystallin. Modification of α-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [Nϵ-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of α-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of α-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, α-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of α-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describes the effects of glycation on the structure and chaperone-like activity of α-crystallin. PMID:17696877

SU-E-T-494: Influence of Proton Track-Cell Nucleus Incidence Angle On Relative Biological Effectiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pater, P; Backstrom, G; Enger, S

2015-06-15

Purpose: To explain a Monte Carlo (MC) simulation artifact whereby differences in relative biological effectiveness (RBE) in the induction of initial double strand breaks are observed as a function of the proton track incidence angles in a geometric cell nucleus model. Secondly, to offer an alternative isotropic irradiation procedure to mitigate this effect. Methods: MC tracks of 1 MeV protons were generated in an event-by-event mode. They were overlaid on a cylindrical model of a cell nucleus containing 6×109 nucleotide base pairs. The tracks incidence angle θ with respect to the cell nucleus’s axis was varied in 10 degrees intervals,more » each time generating one hundred fractions of ∼2 Gy. Strand breaks were scored in the modeled DNA sugar-phosphate groups and further sub-classified into single or double strand breaks (ssbs or dsbs). For each angle, an RBE for the induction of initial dsbs with reference to Co-60 was calculated. Results: Our results show significant angular dependencies of RBE, with maximum values for incidence angles parallel to the nucleus central axis. Further examination shows that the higher cross-sections for the creation of dsbs is due to the preferential alignment of tracks with geometrical sub-parts of the cell nucleus model, especially the nucleosomes containing the sugar-phosphate groups. To alleviate the impact of this simulation artifact, an average RBE was calculated with a procedure based on a weighted sampling of the angular data. Conclusion: This work demonstrates a possible numerical artifact in estimated RBE if the influence of the particle incidence angle is not correctly taken into account. A correction procedure is presented to better conform the simulations to real-life experimental conditions. We would like to acknowledge support from the Fonds de recherche du Quebec Sante (FRQS), from the CREATE Medical Physics Research Training Network grant (number 432290) of NSERC, support from NSERC under grants RGPIN 397711-11 and RGPIN-2014-06475 and support from the CIHR under grants MOP-114910, MOP-136774 and MOP-102550.« less

Catalytic analysis of APOBEC3G involving real-time NMR spectroscopy reveals nucleic acid determinants for deamination.

PubMed

Kamba, Keisuke; Nagata, Takashi; Katahira, Masato

2015-01-01

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

PubMed Central

2014-01-01

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. PMID:24725382

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

NASA Astrophysics Data System (ADS)

van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

Geometrical correlations in the nucleosomal DNA conformation and the role of the covalent bonds rigidity

PubMed Central

Ghorbani, Maryam; Mohammad-Rafiee, Farshid

2011-01-01

We develop a simple elastic model to study the conformation of DNA in the nucleosome core particle. In this model, the changes in the energy of the covalent bonds that connect the base pairs of each strand of the DNA double helix, as well as the lateral displacements and the rotation of adjacent base pairs are considered. We show that because of the rigidity of the covalent bonds in the sugar-phosphate backbones, the base pair parameters are highly correlated, especially, strong twist-roll-slide correlation in the conformation of the nucleosomal DNA is vividly observed in the calculated results. This simple model succeeds to account for the detailed features of the structure of the nucleosomal DNA, particularly, its more important base pair parameters, roll and slide, in good agreement with the experimental results. PMID:20972223

Mannitol and Mannitol Dehydrogenases in Conidia of Aspergillus oryzae

PubMed Central

Horikoshi, Koki; Iida, Shigeji; Ikeda, Yonosuke

1965-01-01

Horikoshi, Koki (The Institute of Physical and Chemical Research, Tokyo, Japan), Shigeji Iida, and Yonosuke Ikeda. Mannitol and mannitol dehydrogenases in conidia of Aspergillus oryzae. J. Bacteriol. 89:326–330. 1965.—A sugar alcohol was isolated from the conidia of Aspergillus oryzae and identified as d-mannitol. Two types of d-mannitol dehydrogenases, nicotinamide adenine dinucleotide phosphate-linked and nicotinamide adenine dinucleotide-linked, were found in the conidia. Substrate specificities, pH optima, Michaelis-Menton constants, and the effects of inhibitors were studied. d-Mannitol was converted to fructose by the dehydrogenases. Synthesis of d-mannitol dehydrogenases was not observed during germination; the content of d-mannitol decreased at an early stage of germination. It was assumed, therefore, that d-mannitol might be used as the source of endogenous respiration and provide energy for the germination. PMID:14255698

Novel fermentation: the production of R(-)-1,2-propanediol and acetol by Clostridium thermosaccharolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cameron, D.C.; Cooney, C.L.

1986-07-01

Three strains of Clostridium thermosaccharolyticum were found that produce R(-)-1,2-propanediol from a variety of sugars, including D-glucose and D-xylose. The fermentation of glucose by strain HG-8 (ATCC 31960) gave 7.9 g/l of R(-)-1,2-propanediol with a best yield of 0.27 g/g glucose and an enantiomeric excess of greater than 99%. Acetol accumulated to 1.47 g/l. Product formation was not affected by phosphate concentrations up to 113 mM. A possible pathway to these products involves a variation of the methylglyoxal bypass. Methylglyoxal is reduced to acetol, which is further reduced to %(-)-1,2-propanediol. This fermentation provides a unique route to R(-)-1,2-propanediol and acetolmore » from inexpensive, readily available substrates.« less

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

PubMed Central

Senderek, Jan; Müller, Juliane S.; Dusl, Marina; Strom, Tim M.; Guergueltcheva, Velina; Diepolder, Irmgard; Laval, Steven H.; Maxwell, Susan; Cossins, Judy; Krause, Sabine; Muelas, Nuria; Vilchez, Juan J.; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P.; Steinlein, Ortrud K.; Schlotter, Beate; Schoser, Benedikt; Kirschner, Janbernd; Herrmann, Ralf; Voit, Thomas; Oldfors, Anders; Lindbergh, Christopher; Urtizberea, Andoni; von der Hagen, Maja; Hübner, Angela; Palace, Jacqueline; Bushby, Kate; Straub, Volker; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2011-01-01

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general. PMID:21310273

Organic Analysis in Miller Range 090657 and Buckley Island 10933 CR2 Chondrites: Part 1 In-Situ Observation of Carbonaceous Material

NASA Technical Reports Server (NTRS)

Cao, T.; Nakamura-Messenger, K.; Berger, E. L.; Burton, A. S.; Messenger, S.; Clemett, S. J.

2016-01-01

Primitive carbonaceous chondrites contain a wide variety of organic material, ranging from soluble discrete molecules to insoluble unstructured kerogen-like component as well as structured nano-globules of macromolecular carbon. The relationship between the soluble organic molecules, macromolecular organic material, and host minerals are poorly understood. Due to the differences in extractability of soluble and insoluble organic materials, the analysis methods for each differ and are often performed independently. The combination of soluble and insoluble analyses, when performed concurrently, can provide a wider understanding on spatial distribution, and elemental, structural and isotopic composition of organic material in primitive meteorites. Furthermore, they can provide broader perspective on how extraterrestrial organic ma-terials potentially contributed to the synthesis of life's essential compounds such as amino acids, sugar acids, activated phosphates and nucleobases.

Microbial, Physicochemical, and Sensory Characteristics of Quality Grade 2 Beef Enhanced by Injection of Pineapple Concentrate and Honey

PubMed Central

Yoon, Ji Won; Lee, Da Gyeom; Lee, Hyun Jung; Choe, Juhui; Jung, Samooel; Jo, Cheorun

2017-01-01

This study investigated the effect of injecting pineapple concentrate and honey into low marbled beef in order to enhance its sensory qualities, particularly tenderness and flavor, without compromising its fresh appearance. Beef loin was injected with a solution of 6.0% pineapple concentrate, 2.5% honey, 0.5% monosodium L-glutamate, 0.5% phosphate, and 0.3% salt (w/w) to 120% (w/w) of initial meat weight and stored for 14 d. Non-injected beef loin served as a control. Total aerobic bacterial counts, surface meat color, shear force, reducing sugar content, and sensory evaluation of the beef were analyzed at 0.5, 7, and 14 d of storage. Injection did not affect the total aerobic bacterial counts or color of the beef. However, injection increased the stability of meat color, compared with that of the control, during storage. The shear force value was significantly lower in the injected beef than that in the control. The injected beef had a significantly higher reducing sugar content compared with that of the control. In sensory evaluation, tenderness, juiciness, flavor, and overall acceptance of the injected beef were significantly higher than those of the control at 0.5 d. In conclusion, injection of pineapple concentrate and honey can improve the sensory qualities of low marbled beef, during short storage periods, without changing the fresh appearance of the beef. PMID:28943761

Suppression of Wolffia arrhiza growth by brassinazole, an inhibitor of brassinosteroid biosynthesis and its restoration by endogenous 24-epibrassinolide.

PubMed

Bajguz, Andrzej; Asami, Tadao

2005-08-01

The effect of the brassinosteroid (BR) 24-epibrassinolide (epiBL; 10(-13)-10(-6)M) on growth and levels of chlorophylls, carotenoids, sugars and protein in Wolffia arrhiza after 7 days of cultivation is reported. Application of epiBL to W. arrhiza cultures stimulates the growth and increases the content of photosynthetic pigments, sugar and protein. The greatest effect of epiBL is observed at a concentration of 10(-9)M. We tested the action of Brz2001, a specific BR biosynthesis inhibitor, in the range of 10(-6)-10(-4)M. Addition of Brz2001 to W. arrhiza cultures inhibits their growth after 7 days of cultivation. The inhibition of growth could be reversed by the addition of epiBL. Moreover, there was not complete recovery to the level of control, especially at 5 x 10(-5)-10(-4)M Brz2001. The effects of treatment with 10(-9)M epiBL mixed with a mevalonate pathway inhibitor (mevinolin), or a 2-methylerythritol 4-phosphate pathway inhibitor (clomazone), were also investigated. Mevinolin did not inhibit growth of W. arrhiza after 7 days of cultivation. However, clomazone did. Addition of epiBL overcame this inhibition. These results suggest that the mevalonate pathway may not function well in W. arrhiza and that biosynthesis of BRs through the non-mevalonate pathway in W. arrhiza could be possible.

The influence of 1-triacontanol on the growth, flowering, and quality of potted Bougainvillea plants (Bougainvillea glabra var. "Elizabeth Angus") under natural conditions.

PubMed

Khandaker, Mohammad Moneruzzaman; Faruq, Golam; Rahman, M Motior; Sofian-Azirun, M; Boyce, Amru Nasrulhaq

2013-01-01

Selected physiological and biochemical parameters were monitored at the vegetative and reproductive growth stages in potted Bougainvillea plants treated with five different concentrations of TRIA. Advanced flowering, flower bud number, and blooming rate increased significantly with 0.5 and 1.0 mg/L TRIA treatments. Similarly, photosynthetic rate, pigment content, quantum yield, and stomatal conductance increased significantly with 2.5, 1.0, and 5.0 mg/L TRIA treatments. Higher levels of N, P, and K, as well as increased total soluble solids (TSS) and higher sugar and protein contents, were recorded in treated plants. Furthermore, 46% more flowers, a 1.5-fold increase in bract weight, increased longevity, and 40% less leaf abscission were recorded following 2.5 mg/L TRIA treatment. Phenol and flavonoid contents, sucrose phosphate synthase (SPS), and antioxidant activities were also markedly increased with 2.5 and 1.0 mg/L TRIA treatments. However, ethylene production was significantly lower in the treated plants. Positive correlations were observed between leaf TSS and flowering time and flower number, between leaf sugar content and bract weight, and between net photosynthesis and bract growth and dry matter production. It can be concluded that the foliar spray of TRIA stimulates growth, enhances flowering, and improves the quality of potted Bougainvillea plants.

The Influence of 1-Triacontanol on the Growth, Flowering, and Quality of Potted Bougainvillea Plants (Bougainvillea glabra var. “Elizabeth Angus”) under Natural Conditions

PubMed Central

Faruq, Golam; Rahman, M. Motior; Sofian-Azirun, M.; Boyce, Amru Nasrulhaq

2013-01-01

Selected physiological and biochemical parameters were monitored at the vegetative and reproductive growth stages in potted Bougainvillea plants treated with five different concentrations of TRIA. Advanced flowering, flower bud number, and blooming rate increased significantly with 0.5 and 1.0 mg/L TRIA treatments. Similarly, photosynthetic rate, pigment content, quantum yield, and stomatal conductance increased significantly with 2.5, 1.0, and 5.0 mg/L TRIA treatments. Higher levels of N, P, and K, as well as increased total soluble solids (TSS) and higher sugar and protein contents, were recorded in treated plants. Furthermore, 46% more flowers, a 1.5-fold increase in bract weight, increased longevity, and 40% less leaf abscission were recorded following 2.5 mg/L TRIA treatment. Phenol and flavonoid contents, sucrose phosphate synthase (SPS), and antioxidant activities were also markedly increased with 2.5 and 1.0 mg/L TRIA treatments. However, ethylene production was significantly lower in the treated plants. Positive correlations were observed between leaf TSS and flowering time and flower number, between leaf sugar content and bract weight, and between net photosynthesis and bract growth and dry matter production. It can be concluded that the foliar spray of TRIA stimulates growth, enhances flowering, and improves the quality of potted Bougainvillea plants. PMID:23970832

Parallel increase of dolichol pathway and nucleotide pyrophosphatases in rat hepatocytes by dexamethasone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarkar, M.; Mookerjea, S.

1986-05-01

Incorporation of (/sup 14/C)-mannose to dolichol phosphate mannose, dolichol pyrophosphate oligosaccharide and N-linked glycoproteins in cultured hepatocytes was increased by dexamethasone. Nucleotide pyrophosphatases are now measured to investigate possible control of glycosylation by the nucleotide sugar pools. Dexamethasone caused about 2 fold increase of UDP-GlcNAc and GDP-Man pyrophosphatase activity which is evident as early as 4 hr and increased up to 12 hr of incubation. The K/sub m/ for UDP-GlcNAc and GDP-Man were respectively 0.43 mM and 0.47 mM in homogenate membrane and the values remained unchanged by dexamethasone treatment. However the V/sub max/ of the enzymes were increased withmore » both UDP-GlcNAc and GDP-Man. The broad pH optima of the enzymes (pH 8 to 10) indicated their alkaline nature. Mixing experiments of the cell homogenates from control and dexamethasone treated cells showed that UDP-GlcNAc and GDP-Man pyrophosphatase activities were additive which ruled out the possibility of presence of any activator or removal of any inhibitor due to dexamethasone. The parallel increase of nucleotide pyrophosphatase and dolichol linked pathway by dexamethasone does not support the possibility that stimulation of glycoprotein synthesis by dexamethasone is mediated by transfer of nucleotide sugars towards dolichol saccharides.« less

Soft drinks and in vitro dental erosion.

PubMed

Gravelle, Brent L; Hagen Ii, Ted W; Mayhew, Susan L; Crumpton, Brooks; Sanders, Tyler; Horne, Victoria

2015-01-01

The purpose of this investigation was to determine to what extent the in vitro exposure of healthy teeth to various commonly consumed carbonated soft drinks may precipitate dental erosion. Forty-two healthy, extracted, previously unerupted human molars were weighed prior to, during, and after suspension in various sugared and diet or zero-calorie carbonated beverages for 20 days; the specimens were stored at room temperature while being stirred at 275 rpm. The percentage decrease in tooth weight from before to after exposure represented the weight loss due to enamel erosion; values in the experimental groups varied from 3.22% to 44.52% after 20 days' exposure. Data were subjected to analysis of variance and post hoc Scheffe testing at a level of α = 0.05. Nonsugared drinks (diet and zero-calorie) as a whole were more erosive than sugared beverages. A significant positive correlation was found between the amount of titratable acid and percentage of tooth erosion, while a significant negative correlation was revealed between the beverage pH and percentage of tooth erosion. No significant correlations were found between calcium or phosphate ion concentrations and the amount of erosion. It appears that enamel erosion is dependent on not only the beverage flow rate, pH, and amount of titratable acid, but also whether the soft drink is of the diet or zero-calorie variety, which reflects the type of artificial sweetener present.

Properties of novel bone hemostat prepared using sugar-modified hydroxyapatite, phosphoryl oligosaccharides of calcium and thermoplastic resin

NASA Astrophysics Data System (ADS)

Mimira, Tokio; Umeda, Tomohiro; Musha, Yoshiro; Itatani, Kiyoshi

2013-12-01

A novel hemostatic agent was prepared using phosphoryl oligosaccharides of calcium (POs-Ca), hydroxyapatite (Ca10(PO4)6(OH)2; HAp) obtained by the hydrolysis of POs-Ca or sugar-containing HAp (s-HAp; 60.3 mass% calcium-deficient HAp and 39.5 mass% organic materials, Ca/P ratio = 1.56) and thermoplastic resin (the mixture of random copolymer of ethylene oxide/propylene oxide (EPO) and polyethylene oxide (EO); EPO : EO : water = 25 : 15 : 60 (mass ratio); 25EPO-15EO). The gel formed by mixing 25EPO-15EO with water (25EPO-15EO/water mass ratio: 0.20) was flash frozen at -80°C, freeze-dried at -50°C for 15 h and then ground using mixer. The consistency conditions of hemostats mixed with POs-Ca or s-HAp were optimized for the practical uses. The mean stanching times of hemostats were: s-HAp/25EPO-15EO (8.2 h; s-HAp/25EPO-15EO = 0.20) > 25EPO-15EO (5.3 h) > POs-Ca/25EPO-15EO (4.7 h; POs-Ca/25EPO-15EO = 0.20). The gentamicin, a typical antibiotic agent, loaded s-HAp/25EPO-15EO composite hemostat showed the steady state releasing in phosphate buffered saline till 10 h immersion at 37.0°C.

Sweet taste transduction in hamster: role of protein kinases.

PubMed

Varkevisser, B; Kinnamon, S C

2000-05-01

Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweeteners stimulate production of inositol 1,4, 5-tris-phosphate (IP(3)) and diacylglycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K(+) conductance, but the intermediate steps in the transduction pathways have not been examined. In this study, the loose-patch recording technique was used to examine the role of protein kinases and other downstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from approximately 35% of fungiform taste buds in response to sucrose (200 mM) or NC-00274-01 (NC-01, 200 microM), a synthetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PDBu (10 microM). In all sweet-responsive taste buds tested (n = 11), PDBu elicited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmaleimide I (0.15 microM) resulted in inhibition of the NC-01 response by approximately 75%, whereas the response to sucrose either increased or remained unchanged. These data suggest that activation of PKC is required for the transduction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined in the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 microM). Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transduction of sugars, but may play a modulatory role in both pathways, such as adaptation of the response. We also examined whether Ca(2+)-calmodulin dependent cAMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, by examining responses to sucrose and synthetic sweeteners in the presence of the CaM-PDE inhibitor W-7 (100 microM). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchanged. These data suggest that the pathways for sugars and sweeteners are negatively coupled; the Ca(2+) that is released from intracellular stores during stimulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis

PubMed Central

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-01-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis. Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis. Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis. However, these results require verification in further studies. PMID:28962162

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

PubMed Central

Chavarría, Max; Kleijn, Roelco J.; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

2012-01-01

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. PMID:22434849

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

PubMed

Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

2016-07-01

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis.

PubMed

Liu, Wenlan; Sun, Zhirong; Qu, Jixu; Yang, Chunning; Zhang, Xiaomin; Wei, Xinxin

2017-09-01

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis . Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis . Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis . However, these results require verification in further studies.

Microbially induced selective flotation of sphalerite from galena using mineral-adapted strains of Bacillus megaterium.

PubMed

Vasanthakumar, B; Ravishankar, H; Subramanian, S

2013-12-01

The selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using cells and extracellular secretions of Bacillus megaterium after adaptation to the chosen minerals. The extracellular secretions obtained after thermolysis of bacterial cells adapted to sphalerite yield the highest flotation recovery of sphalerite with a selectivity index value of 24.5, in comparison to the other cellular and extra-cellular bio-reagents studied. The protein profile for the unadapted and mineral-adapted cells has been found to differ distinctly, attesting to variation in the yield and nature of extra-cellular polymeric substances (EPS). The changes induced in the bacterial cell wall components after adaptation to sphalerite or galena with respect to the contents of phosphate, uronic acid and acetylated sugars of B. megaterium have been quantified. The role of the dissolved metal ions from the minerals as well as that of the constituents of extracellular secretions in modulating the surface charge of the bacterial cells as well as the minerals under study has been confirmed using various enzymatic treatments of the bacterial cells. It has been demonstrated that the induction of additional molecular weight protein fractions as well as the higher amount of extracellular proteins and phosphate secreted after adaptation to sphalerite vis-à-vis galena are contributory factors for the selective separation of sphalerite from galena. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats.

PubMed

Shen, Szu-Chuan; Cheng, Fang-Chi; Wu, Ning-Jung

2008-11-01

This study investigated the effect of aqueous and ethanol soluble solid extracts of guava (Psidium guajava Linn.) leaves on hypoglycemia and glucose metabolism in type 2 diabetic rats. Low-dose streptozotocin (STZ) and nicotinamide were injected into Sprague-Dawley (SD) rats to induce type 2 diabetes. Acute and long-term feeding tests were carried out, and an oral glucose tolerance test (OGTT) to follow the changes in plasma glucose and insulin levels was performed to evaluate the antihyperglycemic effect of guava leaf extracts in diabetic rats.The results of acute and long-term feeding tests showed a significant reduction in the blood sugar level in diabetic rats fed with either the aqueous or ethanol extract of guava leaves (p < 0.05). Long-term administration of guava leaf extracts increased the plasma insulin level and glucose utilization in diabetic rats. The results also indicated that the activities of hepatic hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase in diabetic rats fed with aqueous extracts were higher than in the normal diabetic group (p < 0.05). On the other hand, diabetic rats treated with the ethanol extract raised the activities of hepatic hexokinase and glucose-6-phosphate dehydrogenase (p < 0.05) only. The experiments provided evidence to support the antihyperglycemic effect of guava leaf extract and the health function of guava leaves against type 2 diabetes.

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

PubMed

González-Mula, Almudena; Lang, Julien; Grandclément, Catherine; Naquin, Delphine; Ahmar, Mohammed; Soulère, Laurent; Queneau, Yves; Dessaux, Yves; Faure, Denis

2018-07-01

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

PubMed

Creek, Darren J; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J; Chokkathukalam, Achuthanunni; Weidt, Stefan K; Burgess, Karl E V; Breitling, Rainer; Watson, David G; Bringaud, Frédéric; Barrett, Michael P

2015-03-01

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Prebiological evolution and the metabolic origins of life.

PubMed

Pratt, Andrew J

2011-01-01

The chemoton model of cells posits three subsystems: metabolism, compartmentalization, and information. A specific model for the prebiological evolution of a reproducing system with rudimentary versions of these three interdependent subsystems is presented. This is based on the initial emergence and reproduction of autocatalytic networks in hydrothermal microcompartments containing iron sulfide. The driving force for life was catalysis of the dissipation of the intrinsic redox gradient of the planet. The codependence of life on iron and phosphate provides chemical constraints on the ordering of prebiological evolution. The initial protometabolism was based on positive feedback loops associated with in situ carbon fixation in which the initial protometabolites modified the catalytic capacity and mobility of metal-based catalysts, especially iron-sulfur centers. A number of selection mechanisms, including catalytic efficiency and specificity, hydrolytic stability, and selective solubilization, are proposed as key determinants for autocatalytic reproduction exploited in protometabolic evolution. This evolutionary process led from autocatalytic networks within preexisting compartments to discrete, reproducing, mobile vesicular protocells with the capacity to use soluble sugar phosphates and hence the opportunity to develop nucleic acids. Fidelity of information transfer in the reproduction of these increasingly complex autocatalytic networks is a key selection pressure in prebiological evolution that eventually leads to the selection of nucleic acids as a digital information subsystem and hence the emergence of fully functional chemotons capable of Darwinian evolution.

Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.

2003-12-01

Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in themore » optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans although B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. For successful production of ethanol from pyruvate, both pyruvate decarboxylase (PDC) and alcohol dehydrogenase (AHD) need to be produced at optimal levels in these biocatalysts. A plasmid containing the S. ventriculi pdc gene and the adh gene from geobacillus stearothermophilus was constructed using plasmid pWH1520 that was successfully used for expression of pdc in B. megaterium. The resulting portable ethanol (PET) plasmid, pJAM423, was transformed into B. megaterium. After xylose induction, a significant fraction of cell cytoplasm was composed of the S. ventriculi PDC and G. stearothermophilus ADH proteins. In preliminary experiments, the amount of ethanol produced by b. megaterium with plasmid pJAM423 was about twice (20 mM) of the bacterium without the plasmid. These results show that the PET operon is functional in B. megaterium but high level ethanol production needs further genetic and metabolic engineering. A genetic transfer system for the second generation biocatalysts needs to be developed for transferring the plasmid pJAM423 and its derivatives for engineering these organisms for ethanol production from biomass derived sugars and cellulose to ethanol. One of the new biocatalysts, strain P4-102B was found to be transformable with plasmids and the method for introducing plasmid pJAM423 into this strain and expression of the encoded DNA is being optimized. These new second generation biocatalysts have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource for production of fuels and chemicals.« less

Source/sink interactions underpin crop yield: the case for trehalose 6-phosphate/SnRK1 in improvement of wheat

PubMed Central

Lawlor, David W.; Paul, Matthew J.

2014-01-01

Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving the non-reducing disaccharide trehalose (TRE). TRE is at small concentrations in mesophytes such as Arabidopsis thaliana and Triticum aestivum, excluding a role in osmoregulation once suggested for it. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and more important role of the pathway in regulation of many processes in development, growth, and photosynthesis. It has now been established that rather than TRE, it is trehalose 6-phosphate (T6P) which has such profound effects. T6P is the intermediary in TRE synthesis formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase. The concentration of T6P is determined both by the rate of synthesis, which depends on the sucrose concentration, and also by the rate of breakdown by trehalose-6-phosphate phosphatase which produces TRE. Changing T6P concentrations by genetically modifying the enzymes of synthesis and breakdown has altered photosynthesis, sugar metabolism, growth, and development which affect responses to, and recovery from, environmental factors. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. T6P inhibits the activity of SnRK1, which de-represses genes encoding proteins involved in anabolism. Consequently, a large concentration of sucrose increases T6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolic activity. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing grains in cereal plants, is achieved. This review briefly summarizes the factors determining, and limiting, yield of wheat (particularly mass/grain which is highly conserved) and considers how T6P/SnRK1 might function to determine grain yield and might be altered to increase them. Increasing the potential rate of filling and mass/grain are ways in which total crop yield could be increased with good husbandry which maintains crop assimilation Cereal yields globally are not increasing, despite the greater production required to meet human demand. Careful targeting of T6P is showing much promise for optimization of source/sink for yield improvement and offers yet further possibilities for increasing sink demand and grain size in wheat. PMID:25202319

GlpR is a direct transcriptional repressor of fructose metabolic genes in Haloferax volcanii.

PubMed

Martin, Jonathan H; Rawls, Katie Sherwood; Chan, Jou Chin; Hwang, Sungmin; Martinez-Pastor, Mar; McMillan, Lana J; Prunetti, Laurence; Schmid, Amy K; Maupin-Furlow, Julie A

2018-06-18

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon Haloferax volcanii , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase ( pfkB ) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that Hfx. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators. IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species. Copyright © 2018 American Society for Microbiology.

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

PubMed Central

Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

2012-01-01

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

RCrane: semi-automated RNA model building.

PubMed

Keating, Kevin S; Pyle, Anna Marie

2012-08-01

RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.

Four highly pseudosymmetric and/or twinned structures of d(CGCGCG) 2 extend the repertoire of crystal structures of Z-DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Zhipu; Dauter, Zbigniew; Gilski, Miroslaw

DNA oligomer duplexes containing alternating cytosines and guanines in their sequences tend to form left-handed helices of the Z-DNA type, with the sugar and phosphate backbone in a zigzag conformation and a helical repeat of two successive nucleotides. Z-DNA duplexes usually crystallize as hexagonally arranged parallel helical tubes, with various relative orientations and translation of neighboring duplexes. Four novel high-resolution crystal structures of d(CGCGCG) 2duplexes are described here. They are characterized by a high degree of pseudosymmetry and/or twinning, with three or four independent duplexes differently oriented in a monoclinicP2 1lattice of hexagonal metric. The various twinning criteria give somewhatmore » conflicting indications in these complicated cases of crystal pathology. The details of molecular packing in these crystal structures are compared with other known crystal forms of Z-DNA.« less

Improving formaldehyde consumption drives methanol assimilation in engineered E. coli.

PubMed

Woolston, Benjamin M; King, Jason R; Reiter, Michael; Van Hove, Bob; Stephanopoulos, Gregory

2018-06-19

Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. However, attempts to engineer Escherichia coli to metabolize methanol have achieved limited success. Here, we provide a rigorous systematic analysis of several potential pathway bottlenecks. We show that regeneration of ribulose 5-phosphate in E. coli is insufficient to sustain methanol assimilation, and overcome this by activating the sedoheptulose bisphosphatase variant of the ribulose monophosphate pathway. By leveraging the kinetic isotope effect associated with deuterated methanol as a chemical probe, we further demonstrate that under these conditions overall pathway flux is kinetically limited by methanol dehydrogenase. Finally, we identify NADH as a potent kinetic inhibitor of this enzyme. These results provide direction for future engineering strategies to improve methanol utilization, and underscore the value of chemical biology methodologies in metabolic engineering.

Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria

PubMed Central

Tsai, Yi-Chin Candace; Lapina, Maria Claribel; Bhushan, Shashi; Mueller-Cajar, Oliver

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation. PMID:26567524

Recent structural and mechanistic insights into post-translational enzymatic glycosylation.

PubMed

Hurtado-Guerrero, Ramon; Davies, Gideon J

2012-12-01

Enzymatic glycosylation of proteins, a post-transitional modification of great significance, is carried out by diverse glycosyltransferases (GTs) that harness activated sugar donors, typically nucleotide or lipid-phosphate linked species. Recent work has seen a major increase in the study of the 3D structure and reaction mechanism of these enzymes. Key advances include the dissection of the classical O-glycosylating and N-glycosylating apparatus, revealing unusual folds and hitherto unconsidered chemical mechanisms for acceptor activation. There has been considerable success in the application of kinetic isotope effects and quantum simulations to address the controversial issue of the reaction mechanism of retaining GTs. New roles for old modifications, exemplified by potential epigenetic roles for glycosylation, have been discovered and there has also been a plethora of studies into important mammalian glycosylations that play key roles in cellular biology, opening up new targets for chemical intervention approaches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Self-assembled block copolymer photonic crystal for selective fructose detection.

PubMed

Ayyub, Omar B; Ibrahim, Michael B; Briber, Robert M; Kofinas, Peter

2013-08-15

The use of one-dimensional photonic crystals fabricated from a self-assembled lamellar block copolymer as a sensitive and selective fructose sensor is investigated. The polystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) films are functionalized with 2-(bromomethyl)phenylboronic acid. The boronic acid moiety confined within the lamellar morphology can reversibly bind to sugars such as fructose, imparting the photonic properties of the PS-b-P2VP film. The films exhibit a detection limit of 500 μM in water and 1mM in phosphate buffered saline. Exposure to a 50 mM solution of fructose invokes a highly visible color change from blue to orange. The films are also able to selectively recognize and respond to fructose in competitive studies in the presence of glucose, mannose and sucrose. Copyright © 2013 Elsevier B.V. All rights reserved.

Sweetened Blood Cools Hot Tempers: Physiological Self-Control and Aggression

PubMed Central

DeWall, C. Nathan; Deckman, Timothy; Gailliot, Matthew T.; Bushman, Brad J.

2014-01-01

Aggressive and violent behaviors are restrained by self-control. Self-control consumes a lot of glucose in the brain, suggesting that low glucose and poor glucose metabolism are linked to aggression and violence. Four studies tested this hypothesis. Study 1 found that participants who consumed a glucose beverage behaved less aggressively than did participants who consumed a placebo beverage. Study 2 found an indirect relationship between diabetes (a disorder marked by low glucose levels and poor glucose metabolism) and aggressiveness through low self-control. Study 3 found that states with high diabetes rates also had high violent crime rates. Study 4 found that countries with high rates of glucose-6-phosphate dehydrogenase deficiency (a metabolic disorder related to low glucose levels) also had higher killings rates, both war related and non-war related. All four studies suggest that a spoonful of sugar helps aggressive and violent behaviors go down. PMID:21064166

Eyes can See What Mind Can’t see

DOE Office of Scientific and Technical Information (OSTI.GOV)

Irfan, Ahsan, E-mail: irfanahsanrajpoot@gmail.com; Zulfiqar, Arif; Ali, Ghani

A 55 year old Caucasian female with a history of Iron deficiency anemia with hemoglobin of 6.1 gm/dl was admitted from her hematologist office for a Venofer infusion. She had profound confusion on presentation. Physical examination was pertinent for fever of 101οF, and laboratory data pertinent for elevated blood sugars of 590 mg/dl, leukocytosis of 10.3 K/UL with bandemia of 13%, elevated AST 170 U/L, ALT 184 U/L, and normal alkaline phosphate and total bilirubin. She was subsequently found to have Strep. Viridian bacteremia and CT scan of Chestabdomen- pelvis revealed multiple hepatosplenic masses consistent with malignancy or abscesses. Livermore » biopsy showed benign liver cells with a background of acute and chronic inflammatory cells and abscess (*image shown). She was treated with long term course of Ceftriaxone via PICC line.« less

Mechanisms for radiation damage in DNA. Progress report, January 1, 1980-December 31, 1980

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sevilla, M D

1980-09-01

In this project several mechanisms are proposed for radiation damage to DNA constituents and DNA, and a series of experiments detailed utilizing electron spin resonance spectrometry to test the proposed mechanisms. Under current investigation are irradiated systems of DNA constituents which may shed light on indirect effects. In addition, studies of radiation effects on lipids have been undertaken which will shed light on the only other proposed site for cell kill, the membrane. Studies completed during the past year are: (1) ..pi.. cations produced in DNA bases by attack of oxidizing radicals; (2) INDO studies of radicals produced in peptidesmore » and carboxylic acid model compounds; (3) electron reactions with carboxylic acids, ketones and aldehydes; and (4) ..gamma..-irradiation of esters and triglycerides. Progress has been made this year in a study of radicals generated in model compounds for the sugar-phosphate backbone.« less

The influence of ionic strength on DNA diffusion in gel networks

NASA Astrophysics Data System (ADS)

Fu, Yuanxi; Jee, Ah-Young; Kim, Hyeong-Ju; Granick, Steve

Cations are known to reduce the rigidity of the DNA molecules by screening the negative charge along the sugar phosphate backbone. This was established by optical tweezer pulling experiment of immobilized DNA strands. However, little is known regarding the influence of ions on the motion of DNA molecules as they thread through network meshes. We imaged in real time the Brownian diffusion of fluorescent labeled lambda-DNA in an agarose gel network in the presence of salt with monovalent or multivalent cations. Each movie was analyzed using home-written program to yield a trajectory of center of the mass and the accompanying history of the shape fluctuations. One preliminary finding is that ionic strength has a profound influence on the slope of the trace of mean square displacement (MSD) versus time. The influence of ionic strength on DNA diffusion in gel networks.

[Indirect and repeated electromagnetic irradiation of extremely high freguency of bacteria Escherichia coli].

PubMed

Isakhanian, V; Trchunian, A

2005-01-01

It has been shown that separate irradiation of distilled water and tris-phosphate buffer containing some inorganic ions, with Escherichia coli K12 grown in anaerobic conditions upon fermentation of sugar (glucose) with "noise" electromagnetic radiation of extremely high frequencies (53.5-68 gHz) or millimeter waves (wavelength of 3 to 8 mm) with low flux capacity (0.01 mW) for 10, 30 and 60 min caused opposite effects, changing the growth of these bacteria. The irradiation of water has a bactericide effect, whereas the irradiation of the buffer stimulates bacterial growth although the buffer itself inhibits the growth. These results point out the role of water in the bactericide action of "noise" electromagnetic radiation of extremely high frequencies, and confirm the significance of membranotropic effects. The bactericide action disappeared after repeated irradiation for 10 and 30 min with 2-h intervals. This indicates the operation of some compensatory mechanisms in bacteria.

[Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

PubMed

Zhang, Xiaomei; Dou, Wenfang; Xu, Hongyu; Xu, Zhenghong

2010-10-01

Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

The 3D model: explaining densification and deformation mechanisms by using 3D parameter plots.

PubMed

Picker, Katharina M

2004-04-01

The aim of the study was to analyze very differently deforming materials using 3D parameter plots and consequently to gain deeper insights into the densification and deformation process described with the 3D model in order to define an ideal tableting excipient. The excipients used were dicalcium phosphate dihydrate (DCPD), sodium chloride (NaCl), microcrystalline cellulose (MCC), xylitol, mannitol, alpha-lactose monohydrate, maltose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (NaCMC), cellulose acetate (CAC), maize starch, potato starch, pregelatinized starch, and maltodextrine. All of the materials were tableted to graded maximum relative densities (rhorel, max) using an eccentric tableting machine. The data which resulted, namely force, displacement, and time, were analyzed by the application of 3D modeling. Different particle size fractions of DCPD, CAC, and MCC were analyzed in addition. Brittle deforming materials such as DCPD exhibited a completely different 3D parameter plot, with low time plasticity, d, and low pressure plasticity, e, and a strong decrease in omega values when densification increased, in contrast to the plastically deforming MCC, which had much higher d, e, and omega values. e and omega values changed only slightly when densification increased for MCC. NaCl showed less of a decrease in omega values than DCPD did, and the d and e values were between those of MCC and DCPD. The sugar alcohols, xylitol and mannitol, behaved in a similar fashion to sodium chloride. This is also valid for the crystalline sugars, alpha-lactose monohydrate, and maltose. However, the sugars are more brittle than the sugar alcohols. The cellulose derivatives, HPMC, NaCMC, and CAC, are as plastic as MCC, however, their elasticity depends on substitution indicated by lower (more elastic) or higher (less elastic) omega values. The native starches, maize starch and potato starch, are very elastic, and pregelatinized starch and maltodextrine are less elastic and exhibited higher omega values. Deformation behavior as shown in 3D parameter plots depends on particle size for polymers such as CAC and MCC; however, it does not depend on particle size for brittle materials such as DCPD. An ideally deforming tableting excipient should exhibit high e, d, and omega values with a constant ratio of e and omega at increasing densification.

Studies on preparation of medium fat liquid dairy whitener from buffalo milk employing ultrafiltration process.

PubMed

Khatkar, Sunil Kumar; Gupta, Vijay Kumar; Khatkar, Anju Boora

2014-09-01

A study was conducted to develop good quality medium fat liquid dairy whitener from buffalo milk employing ultrafiltration (UF) process. The buffalo skim milk was UF concentrated to 4.05 to 4.18 (23.63 ± 0.30 % TS) fold and standardized to 10 % fat (on Dry Matter Basis) (i.e. formulation) and homogenized at 175.76 kg/cm(2). The addition of 0.4 % mixture of monosodium and disodium phosphate (2:1 w/w) improved the heat stability of homogenized formulation to an optimum of 66 min. The bland flavour of homogenized formulation with added 0.4 % mixture of monosodium phosphate and disodium phosphate (2:1 w/w) and 18 % sugar (on DMB) (i.e. medium fat liquid dairy whitener) was improved significantly (P < 0.01) with the addition of 0.2 % potassium chloride, but heat stability of medium fat liquid dairy whitener got reduced substantially (i.e. 19 min). With subsequent heat treatment to 85 °C for 5 min, heat stability of medium fat liquid dairy whitener got improved to reasonable level of 27 min. Whitening ability in terms of L* value of medium fat liquid dairy whitener in both tea and coffee was significantly (P < 0.01) better when homogenized at 175.76 kg/cm(2) vis-à-vis 140.61 kg/cm(2). Standardized medium fat liquid dairy whitener had significantly (P < 0.01) greater protein content (i.e. approximately 2.43 times) compared to market dairy whitener samples. At 2 % solids level, standardized medium fat liquid dairy whitener in tea/coffee fetched significantly (P < 0.01) better sensory attributes and instrumental whitening ability compared to market sample at 3 % solids level. There could be clear 33 % solids quantity saving in case of developed product compared to market dairy whitener sample.

Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Mitochondrial Physiology in the Major Arbovirus Vector Aedes aegypti: Substrate Preferences and Sexual Differences Define Respiratory Capacity and Superoxide Production

PubMed Central

Soares, Juliana B. R. Correa; Gaviraghi, Alessandro; Oliveira, Marcus F.

2015-01-01

Adult females of Aedes aegypti are facultative blood sucking insects and vectors of Dengue and yellow fever viruses. Insect dispersal plays a central role in disease transmission and the extremely high energy demand posed by flight is accomplished by a very efficient oxidative phosphorylation process, which take place within flight muscle mitochondria. These organelles play a central role in energy metabolism, interconnecting nutrient oxidation to ATP synthesis, but also represent an important site of cellular superoxide production. Given the importance of mitochondria to cell physiology, and the potential contributions of this organelle for A. aegypti biology and vectorial capacity, here, we conducted a systematic assessment of mitochondrial physiology in flight muscle of young adult A. aegypti fed exclusively with sugar. This was carried out by determining the activities of mitochondrial enzymes, the substrate preferences to sustain respiration, the mitochondrial bioenergetic efficiency and capacity, in both mitochondria-enriched preparations and mechanically permeabilized flight muscle in both sexes. We also determined the substrates preferences to promote mitochondrial superoxide generation and the main sites where it is produced within this organelle. We observed that respiration in A. aegypti mitochondria was essentially driven by complex I and glycerol 3 phosphate dehydrogenase substrates, which promoted distinct mitochondrial bioenergetic capacities, but with preserved efficiencies. Respiration mediated by proline oxidation in female mitochondria was strikingly higher than in males. Mitochondrial superoxide production was essentially mediated through proline and glycerol 3 phosphate oxidation, which took place at sites other than complex I. Finally, differences in mitochondrial superoxide production among sexes were only observed in male oxidizing glycerol 3 phosphate, exhibiting higher rates than in female. Together, these data represent a significant step towards the understanding of fundamental mitochondrial processes in A. aegypti, with potential implications for its physiology and vectorial capacity. PMID:25803027

Modelling studies determing the mode of action of anthelmintics inhibiting in vitro trehalose-6-phosphate phosphatase (TPP) of Anisakis simplex s.l.

PubMed

Łopieńska-Biernat, Elżbieta; Molcan, Tomasz; Paukszto, Łukasz; Jastrzębski, Jan Paweł; Myszczyński, Kamil

2018-01-01

The trehalose-6-phosphate phosphatase (TPP) enzyme is involved in the synthesis of trehalose, the main sugar in the energy metabolism of nematodes. TPP is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. The presence of conserved active sites of TPP in closely related nematodes and its absence in humans makes it a promising target for antiparasitic drugs. In the present study, homology modeling, molecular docking and MD simulation techniques were used to explore the structure and dynamics of TPP. In the active site, a magnesium ion is stabilized by 3 coordinate bonds formed by D 189 , D 191 and D 400 . The key amino acids involved in ligand binding by the enzyme are C 198 , Y 201 ,T 357 , D 191 and Y 197 . This study relied on docking to select potential inhibitors of TPP which were tested in vitro for sensitivity to anthelmintic drugs such as levamisole and ivermectin targeting Anisakis simplex. The higher toxicity of LEV than IVM was demonstrated after 96 h, 30% of larvae were motile in cultures with 100 μg/ml of LEV and 1000 μg/ml of IVM. We identified drug combination of LEV-IVM against in vitro A. simplex as agonistic effect (CI = 1.1). Levamisole appeared to be a more effective drug which inhibited enzyme activity after 48 h and expression of mRNA after 96 h at a concentration of 10 μg/ml. This preliminary study predicted the structure of TPP, and the results of an in vitro experiment involving A. simplex will contribute to the development of effective inhibitors with potential antiparasitic activity in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Comparative proteomic and metabolomic analyses reveal mechanisms of improved cold stress tolerance in bermudagrass (Cynodon dactylon (L.) Pers.) by exogenous calcium.

PubMed

Shi, Haitao; Ye, Tiantian; Zhong, Bao; Liu, Xun; Chan, Zhulong

2014-11-01

As an important second messenger, calcium is involved in plant cold stress response, including chilling (<20 °C) and freezing (<0 °C). In this study, exogenous application of calcium chloride (CaCl2 ) improved both chilling and freezing stress tolerances, while ethylene glycol-bis-(β-aminoethyl) ether-N,N,N,N-tetraacetic acid (EGTA) reversed CaCl2 effects in bermudagrass (Cynodon dactylon (L.) Pers.). Physiological analyses showed that CaCl2 treatment alleviated the reactive oxygen species (ROS) burst and cell damage triggered by chilling stress, via activating antioxidant enzymes, non-enzymatic glutathione antioxidant pool, while EGTA treatment had the opposite effects. Additionally, comparative proteomic analysis identified 51 differentially expressed proteins that were enriched in redox, tricarboxylicacid cycle, glycolysis, photosynthesis, oxidative pentose phosphate pathway, and amino acid metabolisms. Consistently, 42 metabolites including amino acids, organic acids, sugars, and sugar alcohols were regulated by CaCl2 treatment under control and cold stress conditions, further confirming the common modulation of CaCl2 treatment in carbon metabolites and amino acid metabolism. Taken together, this study reported first evidence of the essential and protective roles of endogenous and exogenous calcium in bermudagrass response to cold stress, partially via activation of the antioxidants and modulation of several differentially expressed proteins and metabolic homeostasis in the process of cold acclimation. © 2014 Institute of Botany, Chinese Academy of Sciences.

Contrasting Changes Caused by Drought and Submergence Stresses in Bermudagrass (Cynodon dactylon)

PubMed Central

Ye, Tiantian; Shi, Haitao; Wang, Yanping; Chan, Zhulong

2015-01-01

In this study, we investigated the mechanisms by which bermudagrass withstands the drought and submergence stresses through physiological, proteomic and metabolomic approaches. The results showed that significant physiological changes were observed after drought treatment, while only slight changes after submergence treatment, including compatible solute contents, ROS levels and antioxidant enzyme activities. Proteomics results showed that 81 proteins regulated by drought or submergence treatment were identified by MALDI-TOF-MS. Among them, 76 proteins were modulated by drought stress with 46 increased abundance and 30 decreased abundance. Forty-five showed abundance changes after submergence treatment with 10 increased and 35 decreased. Pathway enrichment analysis revealed that pathways of amino acid metabolism and mitochondrial electron transport/ATP synthesis were only enriched by drought treatment, while other pathways including photosynthesis, biodegradation of xenobiotics, oxidative pentose phosphate, glycolysis and redox were commonly over-represented after both drought and submergence treatments. Metabolomic analysis indicated that most of the metabolites were up-regulated by drought stress, while 34 of 40 metabolites contents exhibited down-regulation or no significant changes when exposed to submergence stress, including sugars and sugar alcohols. These data indicated that drought stress extensively promoted photosynthesis and redox metabolisms while submergence stress caused declined metabolisms and dormancy in Cynodon dactylon. Taken together, the quiescence strategy with retarded growth might allow bermudagrass to be adaptive to long-term submerged environment, while activation of photosynthesis and redox, and accumulation of compatible solutes and molecular chaperones increased bermudagrass tolerance to drought stress. PMID:26617615

Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

DOE PAGES

Linger, Jeffrey G.; Hobdey, Sarah E.; Franden, Mary Ann; ...

2016-02-02

Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, wemore » engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Furthermore, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.« less

Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

DOE Office of Scientific and Technical Information (OSTI.GOV)

Linger, Jeffrey G.; Hobdey, Sarah E.; Franden, Mary Ann

Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, wemore » engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Furthermore, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.« less

Physiological and Proteomic Analyses of Saccharum spp. Grown under Salt Stress

PubMed Central

Murad, Aline Melro; Molinari, Hugo Bruno Correa; Magalhães, Beatriz Simas; Franco, Augusto Cesar; Takahashi, Frederico Scherr Caldeira; de Oliveira-, Nelson Gomes; Franco, Octávio Luiz; Quirino, Betania Ferraz

2014-01-01

Sugarcane (Saccharum spp.) is the world most productive sugar producing crop, making an understanding of its stress physiology key to increasing both sugar and ethanol production. To understand the behavior and salt tolerance mechanisms of sugarcane, two cultivars commonly used in Brazilian agriculture, RB867515 and RB855536, were submitted to salt stress for 48 days. Physiological parameters including net photosynthesis, water potential, dry root and shoot mass and malondialdehyde (MDA) content of leaves were determined. Control plants of the two cultivars showed similar values for most traits apart from higher root dry mass in RB867515. Both cultivars behaved similarly during salt stress, except for MDA levels for which there was a delay in the response for cultivar RB867515. Analysis of leaf macro- and micronutrients concentrations was performed and the concentration of Mn2+ increased on day 48 for both cultivars. In parallel, to observe the effects of salt stress on protein levels in leaves of the RB867515 cultivar, two-dimensional gel electrophoresis followed by MS analysis was performed. Four proteins were differentially expressed between control and salt-treated plants. Fructose 1,6-bisphosphate aldolase was down-regulated, a germin-like protein and glyceraldehyde 3-phosphate dehydrogenase showed increased expression levels under salt stress, and heat-shock protein 70 was expressed only in salt-treated plants. These proteins are involved in energy metabolism and defense-related responses and we suggest that they may be involved in protection mechanisms against salt stress in sugarcane. PMID:24893295

Metabolomic Profiling of Soybeans (Glycine max L.) Reveals the Importance of Sugar and Nitrogen Metabolism under Drought and Heat Stress

PubMed Central

Das, Aayudh; Rushton, Paul J.; Rohila, Jai S.

2017-01-01

Soybean is an important crop that is continually threatened by abiotic stresses, especially drought and heat stress. At molecular levels, reduced yields due to drought and heat stress can be seen as a result of alterations in metabolic homeostasis of vegetative tissues. At present an incomplete understanding of abiotic stress-associated metabolism and identification of associated metabolites remains a major gap in soybean stress research. A study with a goal to profile leaf metabolites under control conditions (28/24 °C), drought [28/24 °C, 10% volumetric water content (VWC)], and heat stress (43/35 °C) was conducted in a controlled environment. Analyses of non-targeted metabolomic data showed that in response to drought and heat stress, key metabolites (carbohydrates, amino acids, lipids, cofactors, nucleotides, peptides and secondary metabolites) were differentially accumulated in soybean leaves. The metabolites for various cellular processes, such as glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, and starch biosynthesis, that regulate carbohydrate metabolism, amino acid metabolism, peptide metabolism, and purine and pyrimidine biosynthesis, were found to be affected by drought as well as heat stress. Computationally based regulatory networks predicted additional compounds that address the possibility of other metabolites and metabolic pathways that could also be important for soybean under drought and heat stress conditions. Metabolomic profiling demonstrated that in soybeans, keeping up with sugar and nitrogen metabolism is of prime significance, along with phytochemical metabolism under drought and heat stress conditions. PMID:28587097

The role of Tre6P and SnRK1 in maize early kernel development and events leading to stress-induced kernel abortion.

PubMed

Bledsoe, Samuel W; Henry, Clémence; Griffiths, Cara A; Paul, Matthew J; Feil, Regina; Lunn, John E; Stitt, Mark; Lagrimini, L Mark

2017-04-12

Drought stress during flowering is a major contributor to yield loss in maize. Genetic and biotechnological improvement in yield sustainability requires an understanding of the mechanisms underpinning yield loss. Sucrose starvation has been proposed as the cause for kernel abortion; however, potential targets for genetic improvement have not been identified. Field and greenhouse drought studies with maize are expensive and it can be difficult to reproduce results; therefore, an in vitro kernel culture method is presented as a proxy for drought stress occurring at the time of flowering in maize (3 days after pollination). This method is used to focus on the effects of drought on kernel metabolism, and the role of trehalose 6-phosphate (Tre6P) and the sucrose non-fermenting-1-related kinase (SnRK1) as potential regulators of this response. A precipitous drop in Tre6P is observed during the first two hours after removing the kernels from the plant, and the resulting changes in transcript abundance are indicative of an activation of SnRK1, and an immediate shift from anabolism to catabolism. Once Tre6P levels are depleted to below 1 nmol∙g -1 FW in the kernel, SnRK1 remained active throughout the 96 h experiment, regardless of the presence or absence of sucrose in the medium. Recovery on sucrose enriched medium results in the restoration of sucrose synthesis and glycolysis. Biosynthetic processes including the citric acid cycle and protein and starch synthesis are inhibited by excision, and do not recover even after the re-addition of sucrose. It is also observed that excision induces the transcription of the sugar transporters SUT1 and SWEET1, the sucrose hydrolyzing enzymes CELL WALL INVERTASE 2 (INCW2) and SUCROSE SYNTHASE 1 (SUSY1), the class II TREHALOSE PHOSPHATE SYNTHASES (TPS), TREHALASE (TRE), and TREHALOSE PHOSPHATE PHOSPHATASE (ZmTPPA.3), previously shown to enhance drought tolerance (Nuccio et al., Nat Biotechnol (October 2014):1-13, 2015). The impact of kernel excision from the ear triggers a cascade of events starting with the precipitous drop in Tre6P levels. It is proposed that the removal of Tre6P suppression of SnRK1 activity results in transcription of putative SnRK1 target genes, and the metabolic transition from biosynthesis to catabolism. This highlights the importance of Tre6P in the metabolic response to starvation. We also present evidence that sugars can mediate the activation of SnRK1. The precipitous drop in Tre6P corresponds to a large increase in transcription of ZmTPPA.3, indicating that this specific enzyme may be responsible for the de-phosphorylation of Tre6P. The high levels of Tre6P in the immature embryo are likely important for preventing kernel abortion.

Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

PubMed Central

Han, Bingying; Fu, Lili; Zhang, Dan; He, Xiuquan; Chen, Qiang; Peng, Ming; Zhang, Jiaming

2016-01-01

Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz) is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD). Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis). All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW), and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS), 10 trehalose-6-phosphate phosphatases (TPP), and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4) that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1) in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose under normal conditions. MeTPS1 was then transformed into tobacco (Nicotiana benthamiana). Results indicated that transgenic tobacco lines accumulated significant level of trehalose and possessed improved drought stress tolerance. In conclusion, cassava accumulated significantly high amount of trehalose under normal conditions due to multiplied trehalose biosynthesis gene families and constant expression of the active MeTPS1 gene. High levels of trehalose subsequently contributed to high drought stress tolerance. PMID:27420056

Comparison of the effect of two sugar-substituted chewing gums on different caries- and gingivitis-related variables: a double-blind, randomized, controlled clinical trial.

PubMed

Martínez-Pabón, María C; Duque-Agudelo, Lucas; Díaz-Gil, Juan D; Isaza-Guzmán, Diana M; Tobón-Arroyave, Sergio I

2014-01-01

The aim of this study was to compare the effect of two sugar-substituted chewing gums besides toothbrushing on different clinical, microbiological, and biochemical caries- and gingivitis-related variables. The study was designed as a double-blind, randomized, controlled trial with three parallel arms. A total of 130 dental students, who volunteered after signing an informed consent, were randomly allocated to receive one of the following interventions: hexitol-sweetened gum containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), pentitol-sweetened gum containing no CPP-ACP, and control group with no gum. Subjects within the experimental groups chewed two gum pellets for 20 min three times a day after meals. The daily consumption level of both polyols was 6.0 g. Clinical examinations and salivary samplings were conducted at baseline and after 30 days of gum use. Pre- and post-intervention stimulated whole saliva samples were quantified for calcium/phosphate ionic concentration, total facultative bacterial load, Streptococcus mutans/Lactobacillus spp. counts, and Gram-negative percentage. A statistically significant reduction in visible plaque score was displayed in the hexitol/CPP-ACP gum group after the intervention when compared with baseline, but the order of the effect was in the same order as the differences between the groups at baseline. A similar tendency was seen in both the pentitol/non-CPP-ACP gum and control groups regarding total salivary facultative bacterial load and S. mutans count, but median values of these parameters were more significantly reduced in the pentitol/non-CPP-ACP gum group in comparison with those of the control group. Alterations of salivary Lactobacillus spp. were demonstrated only in the pentitol/non-CPP-ACP gum group. Although these findings might indicate that a 30-day protocol of daily chewing of pentitol-sweetened gum containing no CPP-ACP might have some a reducing effect on the salivary levels of facultative bacteria, S. mutans and Lactobacillus spp., there was only a marginal, if any, benefit from the chewing gums under study on some microbiological caries- and gingivitis-related variables. Taking into account that for transferring results into clinically relevant conclusions the findings need to be strong and consistent, adhering to single significant differences appears not appropriate. Hence, the clinical significance of chewing gums as an adjunctive tool for daily oral care remained questionable.

Preliminary safety assessment of C-8 xylitol monoester and xylitol phosphate esters.

PubMed

Silveira, J E P S; Pereda, M C V; Nogueira, C; Dieamant, G; Cesar, C K M; Assanome, K M; Silva, M S; Torello, C O; Queiroz, M L S; Eberlin, S

2016-02-01

Most of the cosmetic compounds with preservative properties available in the market pose some risks concerning safety, such as the possibility of causing sensitization. Due to the fact that there are few options, the proper development of new molecules with this purpose is needed. Xylitol is a natural sugar, and the antimicrobial properties of xylitol-derived compounds have already been described in the literature. C-8 xylitol monoester and xylitol phosphate esters may be useful for the development of skincare products. As an initial screen for safety of chemicals, the combination of in silico methods and in vitro testing can aid in prioritizing resources in toxicological investigations while reducing the ethical and monetary costs that are related to animal and human testing. This study was designed to evaluate the safety of C-8 xylitol monoester and xylitol phosphate esters regarding carcinogenicity, mutagenicity, skin and eye irritation/corrosion and sensitization through alternative methods. For the initial safety assessment, quantitative structure-activity relationship methodology was used. The prediction of the parameters carcinogenicity/mutagenicity, skin and eye irritation/corrosion and sensitization was generated from the chemical structure. The analysis also comprised physical-chemical properties, Cramer rules, threshold of toxicological concern and Michael reaction. In silico results of candidate molecules were compared to 19 compounds with preservative properties that are available in the market. Additionally, in vitro tests (Ames test for mutagenicity, cytotoxicity and phototoxicity tests and hen's egg test--chorioallantoic membrane for irritation) were performed to complement the evaluation. In silico evaluation of both molecules presented no structural alerts related to eye and skin irritation, corrosion and sensitization, but some alerts for micronucleus and carcinogenicity were detected. However, by comparison, C-8 xylitol monoester, xylitol phosphate esters showed similar or better results than the compounds available in the market. Concerning experimental data, phototoxicity and mutagenicity results were negative. As expected for compounds with preservative activity, xylitol-derived substances presented positive result in cytotoxicity test. In hen's egg test, both molecules were irritants. Our results suggested that xylitol-derived compounds appear to be suitable candidates for preservative systems in cosmetics. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogs for the glmS Ribozyme

PubMed Central

Posakony, Jeffrey J.; Ferré-D'Amaré, Adrian R.

2013-01-01

Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactor of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogs, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz) and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogs. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. PMID:23578404

High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon.

PubMed

Zhang, Jie; Guo, Shaogui; Ren, Yi; Zhang, Haiying; Gong, Guoyi; Zhou, Ming; Wang, Guizhang; Zong, Mei; He, Hongju; Liu, Fan; Xu, Yong

2017-02-01

Chromoplast development plays a crucial role in controlling carotenoid content in watermelon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and regulation is poorly understood. More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely correlated with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the fruit carotenoid accumulation. ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription factors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Functional Analysis of the N-Acetylglucosamine Metabolic Genes of Streptomyces coelicolor and Role in Control of Development and Antibiotic Production

PubMed Central

Świątek, Magdalena A.; Tenconi, Elodie; Rigali, Sébastien

2012-01-01

N-Acetylglucosamine, the monomer of chitin, is a favored carbon and nitrogen source for streptomycetes. Its intracellular catabolism requires the combined actions of the N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase NagA and the glucosamine-6-phosphate (GlcN-6P) deaminase/isomerase NagB. GlcNAc acts as a signaling molecule in the DasR-mediated nutrient sensing system, activating development and antibiotic production under poor growth conditions (famine) and blocking these processes under rich conditions (feast). In order to understand how a single nutrient can deliver opposite information according to the nutritional context, we carried out a mutational analysis of the nag metabolic genes nagA, nagB, and nagK. Here we show that the nag genes are part of the DasR regulon in Streptomyces coelicolor, which explains their transcriptional induction by GlcNAc. Most likely as the result of the intracellular accumulation of GlcN-6P, nagB deletion mutants fail to grow in the presence of GlcNAc. This toxicity can be alleviated by the additional deletion of nagA. We recently showed that in S. coelicolor, GlcNAc is internalized as GlcNAc-6P via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Considering the relevance of GlcNAc for the control of antibiotic production, improved insight into GlcNAc metabolism in Streptomyces may provide new leads toward biotechnological applications. PMID:22194457

Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments

PubMed Central

Van Mooy, Benjamin A. S.; Rocap, Gabrielle; Fredricks, Helen F.; Evans, Colleen T.; Devol, Allan H.

2006-01-01

There is growing evidence that dissolved phosphorus can regulate planktonic production in the oceans’ subtropical gyres, yet there is little quantitative information about the biochemical fate of phosphorus in planktonic communities. We observed in the North Pacific Subtropical Gyre (NPSG) that the synthesis of membrane lipids accounted for 18–28% of the phosphate (PO43−) taken up by the total planktonic community. Paradoxically, Prochlorococcus, the cyanobacterium that dominates NPSG phytoplankton, primarily synthesizes sulfoquinovosyldiacylglycerol (SQDG), a lipid that contains sulfur and sugar instead of phosphate. In axenic cultures of Prochlorococcus, it was observed that <1% of the total PO43− uptake was incorporated into membrane lipids. Liquid chromatography/mass spectrometry of planktonic lipids in the NPSG confirmed that SQDG was the dominant membrane lipid. Furthermore, the analyses of SQDG synthesis genes from the Sargasso Sea environmental genome showed that the use of sulfolipids in subtropical gyres was confined primarily to picocyanobacteria; no sequences related to known heterotrophic bacterial SQDG lineages were found. This biochemical adaptation by Prochlorococcus must be a significant benefit to these organisms, which compete against phospholipid-rich heterotrophic bacteria for PO43−. Thus, evolution of this “sulfur-for-phosphorus” strategy set the stage for the success of picocyanobacteria in oligotrophic environments and may have been a major event in Earth’s early history when the relative availability of sulfate and PO43− were significantly different from today’s ocean. PMID:16731626

Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells

PubMed Central

Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

2014-01-01

We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation. PMID:24817874

Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

PubMed

Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

2014-01-01

We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?

PubMed

Saher, Shady; Fernández-García, Nieves; Piqueras, Abel; Hellín, Eladio; Olmos, Enrique

2005-06-01

Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.

Role of the plastidic glucose translocator in the export of starch degradation products from the chloroplasts in Arabidopsis thaliana.

PubMed

Cho, Man-Ho; Lim, Hyemin; Shin, Dong Ho; Jeon, Jong-Seong; Bhoo, Seong Hee; Park, Youn-Il; Hahn, Tae-Ryong

2011-04-01

In higher plants, the plastidic glucose translocator (pGlcT) is assumed to play a role in the export of starch degradation products, but this has not yet been studied in detail. To elucidate the role of pGlcT in the leaves of Arabidopsis thaliana, we generated single and double mutants lacking three plastidic sugar transporters, pGlcT, the triose-phosphate/phosphate translocator (TPT), and the maltose transporter (MEX1), and analyzed their growth phenotypes, photosynthetic properties and metabolite contents. In contrast to the pglct-1 and pglct-2 single mutants lacking a visible growth phenotype, the double mutants pglct-1/mex1 and tpt-2/mex1 displayed markedly inhibited plant growth. Notably, pglct-1/mex1 exhibited more severe growth retardation than that seen for the other mutants. In parallel, the most severe reductions in sucrose content and starch turnover were observed in the pglct-1/mex1 mutant. The concurrent loss of pGlcT and MEX1 also resulted in severely reduced photosynthetic activities and extreme chloroplast abnormalities. These findings suggest that pGlcT, together with MEX1, contributes significantly to the export of starch degradation products from chloroplasts in A. thaliana leaves, and that this starch-mediated pathway for photoassimilate export via pGlcT and MEX1 is essential for the growth and development of A. thaliana. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera.

PubMed

De Bruyn, Frederik; De Paepe, Brecht; Maertens, Jo; Beauprez, Joeri; De Cocker, Pieter; Mincke, Stein; Stevens, Christian; De Mey, Marjan

2015-08-01

Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source. © 2015 Wiley Periodicals, Inc.

Targeted gene disruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen.

PubMed

Wei, Yangdou; Shen, Wenyun; Dauk, Melanie; Wang, Feng; Selvaraj, Gopalan; Zou, Jitao

2004-01-02

Unidirectional transfer of nutrients from plant host to pathogen represents a most revealing aspect of the parasitic lifestyle of plant pathogens. Whereas much effort has been focused on sugars and amino acids, the identification of other significant metabolites is equally important for comprehensive characterization of metabolic interactions between plants and biotrophic fungal pathogens. Employing a strategy of targeted gene disruption, we generated a mutant strain (gpdhDelta) defective in glycerol-3-phosphate dehydrogenase in a hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae. The gpdhDelta strain had severe defects in carbon utilization as it could use neither glucose nor amino acids for sustained growth. Although the mutant mycelia were able to grow on potato dextrose agar medium, they displayed arrhythmicity in growth and failure to conidiate. The metabolic defect of gpdhDelta could be entirely ameliorated by glycerol in chemically defined minimal medium. Furthermore, glycerol was the one and only metabolite that could restore rhythmic growth and conidiation of gpdhDelta. Despite the profound defects in carbon source utilization, in planta the gpdhDelta strain exhibited normal pathogenicity, proceeded normally in its life cycle, and produced abundant conidia. Analysis of plant tissues at the peripheral zone of fungal infection sites revealed a time-dependent reduction in glycerol content. This study provides strong evidence for a role of glycerol as a significant transferred metabolite from plant to fungal pathogen.

Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-acetyltransferase from Shewanella denitrificans¶

PubMed Central

Chantigian, Daniel P.; Thoden, James B.; Holden, Hazel M.

2014-01-01

Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to inversion of configuration about the hexose C-4' carbon. PMID:24128043

Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis1[OPEN

PubMed Central

2016-01-01

Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis. PMID:27208265

Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses

PubMed Central

2012-01-01

Background In the present study, three ionic liquids, namely 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP), were used to partially dissolve rice husk, after which the cellulose were regenerated by the addition of water. The aim of the investigation is to examine the implications of the ionic liquid pretreatments on rice husk composition and structure. Results From the attenuated total reflectance Fourier transform-infrared (ATR FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) results, the regenerated cellulose were more amorphous, less crystalline, and possessed higher structural disruption compared with untreated rice husk. The major component of regenerated cellulose from [BMIM]Cl and [EMIM]DEP pretreatments was cellulose-rich material, while cellulose regenerated from [EMIM]OAc was a matrix of cellulose and lignin. Cellulose regenerated from ionic pretreatments could be saccharified via enzymatic hydrolysis, and resulted in relatively high reducing sugars yields, whereas enzymatic hydrolysis of untreated rice husk did not yield reducing sugars. Rice husk residues generated from the ionic liquid pretreatments had similar chemical composition and amorphousity to that of untreated rice husk, but with varying extent of surface disruption and swelling. Conclusions The structural architecture of the regenerated cellulose and rice husk residues showed that they could be used for subsequent fermentation or derivation of cellulosic compounds. Therefore, ionic liquid pretreatment is an alternative in the pretreatment of lignocellulosic biomass in addition to the conventional chemical pretreatments. PMID:22958710

Design, formulation and evaluation of green tea chewing gum

PubMed Central

Aslani, Abolfazl; Ghannadi, Alireza; Khalafi, Zeinab

2014-01-01

Background: The main purpose of this study is to design, formulate and evaluate the green tea gums with a suitable taste and quality in order to produce an anti-oxidant chewing gum. Materials and Methods: Fresh green tea leaves obtained from Northern Iran for extraction. Maceration is the extraction method that is used in this study. The contents of caffeine, catechin and flavonoids of the hydro alcoholic extract were measured. Various formulations of the 120 mg green tea extract chewing gums with different sweeteners, flavoring agents and various gum bases were prepared afterward release pattern, content uniformity, organoleptic results and other properties were characterized. Results: The contents of caffeine, catechin and flavonoid of the hydro alcoholic extraction were 207.32 mg/g, 130.00 mg/g and 200.82 mg/g, respectively. Release pattern of green tea chewing gum with different gum base ratios and various sweeteners in phosphate buffer were prepared. A total of 60 persons who were 20-30 years of age, participated in our panel test for organoleptic properties such as taste, stiffness, stickiness, etc., Acceptable gum was the one with the same ratio of the used rubber bases. Cinnamon selected as the preferred taste by volunteers. Combination of aspartame, sugar and maltitol has appropriate taste. The effect of various sweetener on release pattern was negligible, on the other hand rubber bases ratio variation, changed the release pattern obviously. Conclusion: The green tea chewing gum with sugar, maltitol and aspartame sweeteners and cinnamon flavor, using the same rubber bases ratio may be a desirable antioxidant product. PMID:25161989

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

PubMed Central

Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan

2015-01-01

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate. PMID:25595770

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes.

PubMed

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

PubMed Central

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement. PMID:26347861

Metabolite fingerprinting of pennycress (Thlaspi arvense L.) embryos to assess active pathways during oil synthesis

PubMed Central

Tsogtbaatar, Enkhtuul; Cocuron, Jean-Christophe; Sonera, Marcos Corchado; Alonso, Ana Paula

2015-01-01

Pennycress (Thlaspi arvense L.), a plant naturalized to North America, accumulates high levels of erucic acid in its seeds, which makes it a promising biodiesel and industrial crop. The main carbon sinks in pennycress embryos were found to be proteins, fatty acids, and cell wall, which respectively represented 38.5, 33.2, and 27.0% of the biomass at 21 days after pollination. Erucic acid reached a maximum of 36% of the total fatty acids. Together these results indicate that total oil and erucic acid contents could be increased to boost the economic competitiveness of this crop. Understanding the biochemical basis of oil synthesis in pennycress embryos is therefore timely and relevant to guide future breeding and/or metabolic engineering efforts. For this purpose, a combination of metabolomics approaches was conducted to assess the active biochemical pathways during oil synthesis. First, gas chromatography–mass spectrometry (GC-MS) profiling of intracellular metabolites highlighted three main families of compounds: organic acids, amino acids, and sugars/sugar alcohols. Secondly, these intermediates were quantified in developing pennycress embryos by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Finally, partitional clustering analysis grouped the intracellular metabolites that shared a similar pattern of accumulation over time into eight clusters. This study underlined that: (i) sucrose might be stored rather than cleaved into hexoses; (ii) glucose and glutamine would be the main sources of carbon and nitrogen, respectively; and (iii) glycolysis, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and the Calvin cycle were active in developing pennycress embryos. PMID:25711705

Radiation damage to nucleoprotein complexes in macromolecular crystallography

DOE PAGES

Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; ...

2015-01-30

Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under themore » same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N 1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.« less

Lubrication of chocolate during oral processing.

PubMed

Rodrigues, S A; Selway, N; Morgenstern, M P; Motoi, L; Stokes, J R; James, B J

2017-02-22

The structure of chocolate is drastically transformed during oral processing from a composite solid to an oil/water fluid emulsion. Using two commercial dark chocolates varying in cocoa solids content, this study develops a method to identify the factors that govern lubrication in molten chocolate and saliva's contribution to lubrication following oral processing. In addition to chocolate and its individual components, simulated boluses (molten chocolate and phosphate buffered saline), in vitro boluses (molten chocolate and whole human saliva) and ex vivo boluses (chocolate expectorated after chewing till the point of swallow) were tested. The results reveal that the lubrication of molten chocolate is strongly influenced by the presence of solid sugar particles and cocoa solids. The entrainment of particles into the contact zone between the interacting surfaces reduces friction such that the maximum friction coefficient measured for chocolate boluses is much lower than those for single-phase Newtonian fluids. The addition of whole human saliva or a substitute aqueous phase (PBS) to molten chocolate dissolves sugar and decreases the viscosity of molten chocolate so that thinner films are achieved. However, saliva is more lubricating than PBS, which results in lower friction coefficients for chocolate-saliva mixtures when compared to chocolate-PBS mixtures. A comparison of ex vivo and in vitro boluses also suggests that the quantity of saliva added and uniformity of mixing during oral processing affect bolus structure, which leads to differences in measured friction. It is hypothesized that inhomogeneous mixing in the mouth introduces large air bubbles and regions of non-emulsified fat into the ex vivo boluses, which enhance wetting and lubrication.

High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release.

PubMed

Gagnon, Susannah M L; Legg, Max S G; Sindhuwinata, Nora; Letts, James A; Johal, Asha R; Schuman, Brock; Borisova, Svetlana N; Palcic, Monica M; Peters, Thomas; Evans, Stephen V

2017-10-01

The human ABO(H) blood group A- and B-synthesizing glycosyltransferases GTA and GTB have been structurally characterized to high resolution in complex with their respective trisaccharide antigen products. These findings are particularly timely and relevant given the dearth of glycosyltransferase structures collected in complex with their saccharide reaction products. GTA and GTB utilize the same acceptor substrates, oligosaccharides terminating with α-l-Fucp-(1→2)-β-d-Galp-OR (where R is a glycolipid or glycoprotein), but use distinct UDP donor sugars, UDP-N-acetylgalactosamine and UDP-galactose, to generate the blood group A (α-l-Fucp-(1→2)[α-d-GalNAcp-(1→3)]-β-d-Galp-OR) and blood group B (α-l-Fucp-(1→2)[α-d-Galp-(1→3)]-β-d-Galp-OR) determinant structures, respectively. Structures of GTA and GTB in complex with their respective trisaccharide products reveal a conflict between the transferred sugar monosaccharide and the β-phosphate of the UDP donor. Mapping of the binding epitopes by saturation transfer difference NMR measurements yielded data consistent with the X-ray structural results. Taken together these data suggest a mechanism of product release where monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Autosomal recessive PGM3 mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

PubMed Central

Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J.; Datta, Shrimati; Lamborn, Ian T.; Jing, Huie; Kim, Emily S.; Biancalana, Matthew; Wolfe, Lynne A.; DiMaggio, Thomas; Matthews, Helen F.; Kranick, Sarah M.; Stone, Kelly D.; Holland, Steven M.; Reich, Daniel S.; Hughes, Jason D.; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F.; Freeze, Hudson H.; Su, Helen C.; Milner, Joshua D.

2014-01-01

Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T cell cytokine production were measured. Whole exome sequencing was performed to identify disease-causing mutations. Immunoblotting, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased TH2 and TH17 cytokine production by CD4+ T cells. Bacterial and viral infection susceptibility were noted along with T cell lymphopenia, particularly of CD8+ T cells, and reduced memory B cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced UDP-GlcNAc, along with decreased O- and N-linked protein glycosylation in patients’ cells. These results define a new Congenital Disorder of Glycosylation. Conclusions Autosomal recessive, hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability and hypomyelination. PMID:24589341

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: changes in residual sugars and water-soluble organic acids during ripening.

PubMed

Upreti, P; McKay, L L; Metzger, L E

2006-02-01

Cheddar cheese ripening involves the conversion of lactose to glucose and galactose or galactose-6-phosphate by starter and nonstarter lactic acid bacteria. Under ideal conditions (i.e., where bacteria grow under no stress of pH, water activity, and salt), these sugars are mainly converted to lactic acid. However, during ripening of cheese, survival and growth of bacteria occurs under the stressed condition of low pH, low water activity, and high salt content. This forces bacteria to use alternate biochemical pathways resulting in production of other organic acids. The objective of this study was to determine if the level and type of organic acids produced during ripening was influenced by calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), lactose at pressing (2.4 vs. 0.78%), and S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for organic acids (citric, orotic, pyruvic, lactic, formic, uric, acetic, propanoic, and butyric acids) and residual sugars (lactose, galactose) during 48 wk of ripening using an HPLC-based method. Different factors influenced changes in concentration of residual sugars and organic acids during ripening and are discussed in detail. Our results indicated that the largest decrease in lactose and the largest increase in lactic acid occurred between salting and d 1 of ripening. It was interesting to observe that although the lactose content in cheese was influenced by several factors (Ca and P, residual lactose, and S/M), the concentration of lactic acid was influenced only by S/M. More lactic acid was produced in low S/M treatments compared with high S/M treatments. Although surprising for Cheddar cheese, a substantial amount (0.2 to 0.4%) of galactose was observed throughout ripening in all treatments. Minor changes in the levels of citric, uric, butyric, and propanoic acids were observed during early ripening, whereas during later ripening, a substantial increase was observed. A gradual decrease in orotic acid and a gradual increase in pyruvic acid content of the cheeses were observed during 12 mo of ripening. In contrast, acetic acid did not show a particular trend, indicating its role as an intermediate in a biochemical pathway, rather than a final product.

Biomolecular damage induced by ionizing radiation: the direct and indirect effects of low-energy electrons on DNA.

PubMed

Alizadeh, Elahe; Orlando, Thomas M; Sanche, Léon

2015-04-01

Many experimental and theoretical advances have recently allowed the study of direct and indirect effects of low-energy electrons (LEEs) on DNA damage. In an effort to explain how LEEs damage the human genome, researchers have focused efforts on LEE interactions with bacterial plasmids, DNA bases, sugar analogs, phosphate groups, and longer DNA moieties. Here, we summarize the current understanding of the fundamental mechanisms involved in LEE-induced damage of DNA and complex biomolecule films. Results obtained by several laboratories on films prepared and analyzed by different methods and irradiated with different electron-beam current densities and fluencies are presented. Despite varied conditions (e.g., film thicknesses and morphologies, intrinsic water content, substrate interactions, and extrinsic atmospheric compositions), comparisons show a striking resemblance in the types of damage produced and their yield functions. The potential of controlling this damage using molecular and nanoparticle targets with high LEE yields in targeted radiation-based cancer therapies is also discussed.

Effect of electronic coupling of Watson-Crick hopping in DNA poly(dA)-poly(dT)

NASA Astrophysics Data System (ADS)

Risqi, A. M.; Yudiarsah, E.

2017-07-01

Charge transport properties of poly(dA)-poly(dT) DNA has been studied by using thigh binding Hamiltonian approach. Molecule DNA that we use consist of 32 base pair of adenine (A) and thymine (T) and backbone is consist of phosphate and sugar. The molecule DNA is contacted electrode at both ends. Charge transport in molecule DNA depend on the environment, we studied the effect of electronic coupling of Watson-Crick hopping in poly(dA)-poly(dT) DNA to transmission probability and characteristic I-V. The electronic coupling constant influence charge transport between adenine-thymine base pairs at the same site. Transmission probability is studied by using transfer matrix and scattering matrix method, and the result of transmission probability is used to calculate the characteristic I-V by using formula Landauer Buttiker. The result shows that when the electronic coupling increase then transmission probability and characteristic I-V increase slightly.

Low-energy electron collisions with biomolecules

NASA Astrophysics Data System (ADS)

Winstead, Carl; McKoy, Vincent

2012-11-01

We report recent progress in applying the Schwinger multichannel computational method to the interactions of slow electrons with biomolecules. Calculations on constituents of DNA, including nucleobases, phosphate esters, and models of the backbone sugar, have provided insight into the nature of the low-energy shape resonances, and thereby into possible sites and mechanisms for electron attachment that may lead to strand-breaking. At the same time, more approximate calculations on larger assemblies such as nucleosides and deoxyadenosine monophosphate indicate how the resonance properties of the subunits will or will not persist in DNA itself. We are pursuing a similar strategy for another major class of biomolecules, the proteins, by beginning with fixed-nuclei studies of the constituent amino acids; here we present preliminary results for the simplest amino acid, glycine. We also describe efforts directed at an improved understanding electron collisions with alcohols, which, in addition to basic scientific interest, may prove useful in the modeling of ignition and combustion within biofuel-powered engines.

Hydrogen Cyanide Polymerization: A Preferred Cosmochemical Pathway

NASA Astrophysics Data System (ADS)

Matthews, Clifford N.

In the presence of a base such as ammonia, liquid HCN polymerizes spontaneously at room temperature to a brown-black solid from which a yellow-brown powder can be extracted by water and further hydrolyzed to yield a-amino acids. Two types of structural units appear to be present in these polymeric products, stable ladder polymers with conjugated -C=N- bonds, and polyamidines, readily converted by water to polypeptides. Several kinds of investigations, including electric discharge experiments which produce HCN from methane and ammonia, give results consistent with the hypothesis that the original polypeptides on Earth were synthesized directly from such HCN polymers and water without the intervening formation of -amino acids. In the absence of water - on land - the intermediate polyamidines could have been the original condensing agents directing the synthesisis of nucleosides and nucleotides from available sugars, phosphates and nitrogen bases. Most significant would have been the parallel synthesis of polypeptides and polynucleotides arising from the dehydrating action of these polyamidines on nucleotides.

Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

PubMed Central

Robach, M C

1979-01-01

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present. PMID:44445

Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

PubMed

Robach, M C

1979-11-01

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present.

Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase.

PubMed

Bhat, Javaid Y; Miličić, Goran; Thieulin-Pardo, Gabriel; Bracher, Andreas; Maxwell, Andrew; Ciniawsky, Susanne; Mueller-Cajar, Oliver; Engen, John R; Hartl, F Ulrich; Wendler, Petra; Hayer-Hartl, Manajit

2017-09-07

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. Copyright © 2017 Elsevier Inc. All rights reserved.

Divergent prebiotic synthesis of pyrimidine and 8-oxo-purine ribonucleotides

NASA Astrophysics Data System (ADS)

Stairs, Shaun; Nikmal, Arif; Bučar, Dejan-Krešimir; Zheng, Shao-Liang; Szostak, Jack W.; Powner, Matthew W.

2017-05-01

Understanding prebiotic nucleotide synthesis is a long standing challenge thought to be essential to elucidating the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Here, we tackle this mutual incompatibility by recognizing that the 8-oxo-purines share an underlying generational parity with the pyrimidine nucleotides. We present a divergent synthesis of pyrimidine and 8-oxo-purine nucleotides starting from a common prebiotic precursor that yields the β-ribo-stereochemistry found in the sugar phosphate backbone of biological nucleic acids. The generational relationship between pyrimidine and 8-oxo-purine nucleotides suggests that 8-oxo-purine ribonucleotides may have played a key role in primordial nucleic acids prior to the emergence of the canonical nucleotides of biology.

Raman spectroscopic evaluation of DNA adducts of a platinum containing anticancer drug.

PubMed

Jangir, Deepak K; Mehrotra, Ranjana

2014-09-15

Mechanistic understanding of the interaction of drugs with their target molecules is important for better understanding of their mode of action and to improve their efficacy. Carboplatin is a platinum containing anticancer drug, used to treat different type of tumors. In the present work, we applied Raman spectroscopy to study the interaction of carboplatin with DNA at molecular level using different carboplatin-DNA molar ratios. These Raman spectroscopic results provide comprehensive understanding on the carboplatin-DNA interactions and indicate that DNA cross-linked adducts formed by carboplatin are similar to cisplatin adducts. The results indicate that guanine N7 and adenine N7 are the putative sites for carboplatin interaction. It is observed that carboplatin has some affinity toward cytosine in DNA. Phosphate sugar backbone of DNA showed conformation perturbation in DNA which were easily sensible at higher concentrations of carboplatin. Most importantly, carboplatin interaction induces intermediate A- and B-DNA conformations at the cross-linking sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Utilization of acid pre-treated coconut dregs as a substrate for production of detergent compatible lipase by Bacillus stratosphericus.

PubMed

Mohd Zin, Nur Bainun; Mohamad Yusof, Busyra; Oslan, Siti Nurbaya; Wasoh, Helmi; Tan, Joo Shun; Ariff, Arbakariya B; Halim, Murni

2017-12-01

In recent years, many efforts have been directed to explore the methods to reduce the production costs of industrial lipase by improving the yield and the use of low-cost agricultural wastes. Coconut dregs, which is a lignocellulosic by-product from coconut oil and milk processing plants, is rich in cellulose (36%) and crude fat (9%). A newly isolated Bacillus stratosphericus has been demonstrated to perform cellulose hydrolysis on coconut dregs producing fermentable sugars. The highest extracellular lipase activity of 140 U/mL has been achieved in submerged fermentation with acid pre-treated coconut dregs. The lipase was found to be active over a wide range of temperatures and pHs. The activity of lipase can be generally increased by the presence of detergent ingredients such as Tween-80, cetyltrimethylammonium bromide, hydrogen peroxide and phosphate per sulphate. The great compatibility of lipase in commercial detergents has also underlined its potential as an additive ingredient in biodetergent formulations.

Biosynthesis of nitrogen-containing natural products, C7N aminocyclitols and bis-indoles, from actinomycetes.

PubMed

Asamizu, Shumpei

2017-05-01

Actinomycetes are a major source of bioactive natural products with important pharmaceutical properties. Understanding the natural enzymatic assembly of complex small molecules is important for rational metabolic pathway design to produce "artificial" natural products in bacterial cells. This review will highlight current research on the biosynthetic mechanisms of two classes of nitrogen-containing natural products, C 7 N aminocyclitols and bis-indoles. Validamycin A is a member of C 7 N aminocyclitol natural products from Streptomyces hygroscopicus. Here, two important biosynthetic steps, pseudoglycosyltranferase-catalyzed C-N bond formation, and C 7 -sugar phosphate cyclase-catalyzed divergent carbasugar formation, will be reviewed. In addition, the bis-indolic natural products indolocarbazole, staurosporine from Streptomyces sp. TP-A0274, and rearranged bis-indole violacein from Chromobacterium violaceum are reviewed including the oxidative course of the assembly pathway for the bis-indolic scaffold. The identified biosynthesis mechanisms will be useful to generating new biocatalytic tools and bioactive compounds.

Cannibalism Affects Core Metabolic Processes in Helicoverpa armigera Larvae-A 2D NMR Metabolomics Study.

PubMed

Vergara, Fredd; Shino, Amiu; Kikuchi, Jun

2016-09-02

Cannibalism is known in many insect species, yet its impact on insect metabolism has not been investigated in detail. This study assessed the effects of cannibalism on the metabolism of fourth-instar larvae of the non-predatory insect Helicoverpa armigera (Lepidotera: Noctuidea). Two groups of larvae were analyzed: one group fed with fourth-instar larvae of H. armigera (cannibal), the other group fed with an artificial plant diet. Water-soluble small organic compounds present in the larvae were analyzed using two-dimensional nuclear magnetic resonance (NMR) and principal component analysis (PCA). Cannibalism negatively affected larval growth. PCA of NMR spectra showed that the metabolic profiles of cannibal and herbivore larvae were statistically different with monomeric sugars, fatty acid- and amino acid-related metabolites as the most variable compounds. Quantitation of ¹H-(13)C HSQC (Heteronuclear Single Quantum Coherence) signals revealed that the concentrations of glucose, glucono-1,5-lactone, glycerol phosphate, glutamine, glycine, leucine, isoleucine, lysine, ornithine, proline, threonine and valine were higher in the herbivore larvae.

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

PubMed Central

Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

2015-01-01

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

Quantum-mechanical predictions of electron-induced ionization cross sections of DNA components

NASA Astrophysics Data System (ADS)

Champion, Christophe

2013-05-01

Ionization of biomolecules remains still today rarely investigated on both the experimental and the theoretical sides. In this context, the present work appears as one of the first quantum mechanical approaches providing a multi-differential description of the electron-induced ionization process of the main DNA components for impact energies ranging from the target ionization threshold up to about 10 keV. The cross section calculations are here performed within the 1st Born approximation framework in which the ejected electron is described by a Coulomb wave whereas the incident and the scattered electrons are both described by a plane wave. The biological targets of interest, namely, the DNA nucleobases and the sugar-phosphate backbone, are here described by means of the GAUSSIAN 09 system using the restricted Hartree-Fock method with geometry optimization. The theoretical predictions also obtained have shown a reasonable agreement with the experimental total ionization cross sections while huge discrepancies have been pointed out with existing theoretical models, mainly developed within a semi-classical framework.

Hydration Changes upon DNA Folding Studied by Osmotic Stress Experiments

PubMed Central

Nakano, Shu-ichi; Yamaguchi, Daisuke; Tateishi-Karimata, Hisae; Miyoshi, Daisuke; Sugimoto, Naoki

2012-01-01

The thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems. PMID:22735531

Nutrient Sensing at the Plasma Membrane of Fungal Cells.

PubMed

Van Dijck, Patrick; Brown, Neil Andrew; Goldman, Gustavo H; Rutherford, Julian; Xue, Chaoyang; Van Zeebroeck, Griet

2017-03-01

To respond to the changing environment, cells must be able to sense external conditions. This is important for many processes including growth, mating, the expression of virulence factors, and several other regulatory effects. Nutrient sensing at the plasma membrane is mediated by different classes of membrane proteins that activate downstream signaling pathways: nontransporting receptors, transceptors, classical and nonclassical G-protein-coupled receptors, and the newly defined extracellular mucin receptors. Nontransporting receptors have the same structure as transport proteins, but have lost the capacity to transport while gaining a receptor function. Transceptors are transporters that also function as a receptor, because they can rapidly activate downstream signaling pathways. In this review, we focus on these four types of fungal membrane proteins. We mainly discuss the sensing mechanisms relating to sugars, ammonium, and amino acids. Mechanisms for other nutrients, such as phosphate and sulfate, are discussed briefly. Because the model yeast Saccharomyces cerevisiae has been the most studied, especially regarding these nutrient-sensing systems, each subsection will commence with what is known in this species.

Genetic Insights Into Pyralomicin Biosynthesis in Nonomuraea spiralis IMC A-0156

PubMed Central

Flatt, Patricia M.; Wu, Xiumei; Perry, Steven; Mahmud, Taifo

2013-01-01

The biosynthetic gene cluster for the pyralomicin antibiotics has been cloned and sequenced from Nonomuraea spiralis IMC A-0156. The 41-kb gene cluster contains 27 ORFs predicted to encode all of the functions for pyralomicin biosynthesis. This includes non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) required for the formation of the benzopyranopyrrole core unit, as well as a suite of tailoring enzymes (e.g., four halogenases, an O-methyltransferase, and an N-glycosyltransferase) necessary for further modifications of the core structure. The N-glycosyltransferase is predicted to transfer either glucose or a pseudosugar (cyclitol) to the aglycone. A gene cassette encoding C7-cyclitol biosynthetic enzymes was identified upstream of the benzopyranopyrrole-specific ORFs. Targeted disruption of the gene encoding the N-glycosyltransferase, prlH, abolished pyralomicin production and recombinant expression of PrlA confirms the activity of this enzyme as a sugar phosphate cyclase (SPC) involved in the formation of the C7-cyclitol moiety. PMID:23607523

Low source-sink ratio reduces reserve starch in grapevine woody canes and modulates sugar transport and metabolism at transcriptional and enzyme activity levels.

PubMed

Silva, Angélica; Noronha, Henrique; Dai, Zhanwu; Delrot, Serge; Gerós, Hernâni

2017-09-01

Severe leaf removal decreases storage starch and sucrose in grapevine cv. Cabernet Sauvignon fruiting cuttings and modulates the activity of key enzymes and the expression of sugar transporter genes. Leaf removal is an agricultural practice that has been shown to modify vineyard efficiency and grape and wine composition. In this study, we took advantage of the ability to precisely control the number of leaves to fruits in Cabernet Sauvignon fruiting cuttings to study the effect of source-sink ratios (2 (2L), 6 (6L) and 12 (12) leaves per cluster) on starch metabolism and accumulation. Starch concentration was significantly higher in canes from 6L (42.13 ± 1.44 mg g DW -1 ) and 12L (43.50 ± 2.85 mg g DW -1 ) than in 2L (22.72 ± 3.10 mg g DW -1 ) plants. Moreover, carbon limitation promoted a transcriptional adjustment of genes involved in starch metabolism in grapevine woody tissues, including a decrease in the expression of the plastidic glucose-6-phosphate translocator, VvGPT1. Contrarily, the transcript levels of the gene coding the catalytic subunit VvAGPB1 of the VvAGPase complex were higher in canes from 2L plants than in 6L and 12L, which positively correlated with the biochemical activity of this enzyme. Sucrose concentration increased in canes from 2L to 6L and 12L plants, and the amount of total phenolics followed the same trend. Expression studies showed that VvSusy transcripts decreased in canes from 2L to 6L and 12L plants, which correlated with the biochemical activity of insoluble invertase, while the expression of the sugar transporters VvSUC11 and VvSUC12, together with VvSPS1, which codes an enzyme involved in sucrose synthesis, increased. Thus, sucrose seems to control starch accumulation through the adjustment of the cane sink strength.

Surface coating-modulated toxic responses to silver nanoparticles in Wolffia globosa.

PubMed

Zou, Xiaoyan; Li, Penghui; Lou, Jie; Zhang, Hongwu

2017-08-01

With the omnipresence of silver nanoparticles (AgNPs) in our daily consumer products, their release has raised serious concerns. However, the biochemical mechanisms by which plants counteract the toxicity of nanoparticles are largely unknown. This study investigated the exposure of aquatic Wolffia globosa to ATP-nAg (AgNPs coated with adenosine triphosphate), cit-nAg (AgNPs coated with citrate), and Ag + . Hill reaction activity was basically lost in W. globosa treated with 10mg/L ATP-nAg and Ag + , while the activity was still maintained at 38.7%-38.9% of the respective controls at 10mg/L cit-nAg. The reduction of amounts of chlorophyll and soluble protein were shown in response to the Ag stresses. This was accompanied by the accumulation of sugar in W. globosa treated with cit-nAg. By contrast, the depletion of sugar was recorded after 10mg/L ATP-nAg and Ag + treatments. The superoxide dismutase and peroxidase activities were significantly increased after exposure to 10mg/L ATP-nAg and Ag + , which did not occurred in W. globosa treated with cit-nAg. The ratio between NADPH/NADP + was higher after cit-nAg and Ag + stresses than the respective controls. The accumulation of Ag was found to increase in a concentration-dependent manner. Ag + and ATP-nAg inhibited the uptake of P and K, and promoted the uptake of Fe and Cu. In contrast, cit-nAg only promoted the uptake of Cu. Our results implied that surface coating induced different physiological responses of W. globosa to AgNPs. Based on above results, we speculated that after exposure to cit-nAg, citrate possibly could serve as the substrate for the tricarboxylic acid cycle and accumulated sugar may promote pentose phosphate pathways. For ATP-nAg treatments, ATP would act as an exogenous energy source of plant metabolisms. Our findings demonstrate that surface coating regulates the physiological responses of plants to AgNPs through distinct mechanisms. Copyright © 2017. Published by Elsevier B.V.

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

PubMed

Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nectar minerals as regulators of flower visitation in stingless bees and nectar hoarding wasps.

PubMed

Afik, Ohad; Delaplane, Keith S; Shafir, Sharoni; Moo-Valle, Humberto; Quezada-Euán, J Javier G

2014-05-01

Various nectar components have a repellent effect on flower visitors, and their adaptive advantages for the plant are not well understood. Persea americana (avocado) is an example of a plant that secretes nectar with repellent components. It was demonstrated that the mineral constituents of this nectar, mainly potassium and phosphate, are concentrated enough to repel honey bees, Apis mellifera, a pollinator often used for commercial avocado pollination. Honey bees, however, are not the natural pollinator of P. americana, a plant native to Central America. In order to understand the role of nectar minerals in plant-pollinator relationships, it is important to focus on the plant's interactions with its natural pollinators. Two species of stingless bees and one species of social wasp, all native to the Yucatan Peninsula, Mexico, part of the natural range of P. americana, were tested for their sensitivity to sugar solutions enriched with potassium and phosphate, and compared with the sensitivity of honey bees. In choice tests between control and mineral-enriched solutions, all three native species were indifferent for mineral concentrations lower than those naturally occurring in P. americana nectar. Repellence was expressed at concentrations near or exceeding natural concentrations. The threshold point at which native pollinators showed repellence to increasing levels of minerals was higher than that detected for honey bees. The results do not support the hypothesis that high mineral content is attractive for native Hymenopteran pollinators; nevertheless, nectar mineral composition may still have a role in regulating flower visitors through different levels of repellency.

A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis.

PubMed

Miranda, José A; Avonce, Nelson; Suárez, Ramón; Thevelein, Johan M; Van Dijck, Patrick; Iturriaga, Gabriel

2007-11-01

Improving stress tolerance is a major goal for agriculture. Trehalose is a key molecule involved in drought tolerance in anhydrobiotic organisms. Here we describe the construction of a chimeric translational fusion of yeast trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. This construct was overexpressed in yeast cells displaying both TPS and TPP enzyme activities and trehalose biosynthesis capacity. In Arabidopsis thaliana, the gene fusion was overexpressed using either the 35S promoter or the stress-regulated rd29A promoter. Transgene insertion in the genome was checked by PCR and transcript expression by RT-PCR. Several independent homozygous lines were selected in the presence of kanamycin and further analyzed. Trehalose was accumulated in all these lines at low levels. No morphological or growth alterations were observed in lines overexpressing the TPS1-TPS2 construct, whereas plants overexpressing the TPS1 alone under the control of the 35S promoter had aberrant growth, color and shape. TPS1-TPS2 overexpressor lines were glucose insensitive, consistent with a suggested role of trehalose/T6P in modulating sugar sensing and carbohydrate metabolism. Moreover, TPS1-TPS2 lines displayed a significant increase in drought, freezing, salt and heat tolerance. This is the first time that trehalose accumulation in plants is shown to protect against freezing and heat stress. Therefore, these results demonstrate that engineering trehalose metabolism with a yeast TPS-TPP bifunctional enzyme confers multiple stress protection in plants, comprising a potential tool to improve stress-tolerance in crops.

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

NASA Astrophysics Data System (ADS)

Campos, Christiane Gonçalves; Veras, Henrique César Teixeira; de Aquino Ribeiro, José Antônio; Costa, Patrícia Pinto Kalil Gonçalves; Araújo, Katiúscia Pereira; Rodrigues, Clenilson Martins; de Almeida, João Ricardo Moreira; Abdelnur, Patrícia Verardi

2017-12-01

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. [Figure not available: see fulltext.

The Interplay between Carbon Availability and Growth in Different Zones of the Growing Maize Leaf1[OPEN

PubMed Central

Arrivault, Stéphanie; Lohse, Marc A.; Feil, Regina; Krohn, Nicole; Encke, Beatrice; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Stitt, Mark

2016-01-01

Plants assimilate carbon in their photosynthetic tissues in the light. However, carbon is required during the night and in nonphotosynthetic organs. It is therefore essential that plants manage their carbon resources spatially and temporally and coordinate growth with carbon availability. In growing maize (Zea mays) leaf blades, a defined developmental gradient facilitates analyses in the cell division, elongation, and mature zones. We investigated the responses of the metabolome and transcriptome and polysome loading, as a qualitative proxy for protein synthesis, at dusk, dawn, and 6, 14, and 24 h into an extended night, and tracked whole-leaf elongation over this time course. Starch and sugars are depleted by dawn in the mature zone, but only after an extension of the night in the elongation and division zones. Sucrose (Suc) recovers partially between 14 and 24 h into the extended night in the growth zones, but not the mature zone. The global metabolome and transcriptome track these zone-specific changes in Suc. Leaf elongation and polysome loading in the growth zones also remain high at dawn, decrease between 6 and 14 h into the extended night, and then partially recover, indicating that growth processes are determined by local carbon status. The level of Suc-signaling metabolite trehalose-6-phosphate, and the trehalose-6-phosphate:Suc ratio are much higher in growth than mature zones at dusk and dawn but fall in the extended night. Candidate genes were identified by searching for transcripts that show characteristic temporal response patterns or contrasting responses to carbon starvation in growth and mature zones. PMID:27582314

76 FR 50285 - Fiscal Year 2012 Tariff-Rate Quota Allocations for Raw Cane Sugar, Refined and Specialty Sugar...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-12

... for Raw Cane Sugar, Refined and Specialty Sugar and Sugar-Containing Products AGENCY: Office of the... quantity of the tariff-rate quotas for imported raw cane sugar, refined and specialty sugar and sugar...), the United States maintains tariff-rate quotas (TRQs) for imports of raw cane sugar and refined sugar...

21 CFR 173.320 - Chemicals for controlling microorganisms in cane-sugar and beet-sugar mills.

Code of Federal Regulations, 2014 CFR

2014-04-01

...-sugar and beet-sugar mills. 173.320 Section 173.320 Food and Drugs FOOD AND DRUG ADMINISTRATION...-sugar and beet-sugar mills. Agents for controlling microorganisms in cane-sugar and beet-sugar mills may... microorganisms in cane-sugar and/or beet-sugar mills as specified in paragraph (b) of this section. (b) They are...

Total and Free Sugar Content of Canadian Prepackaged Foods and Beverages.

PubMed

Bernstein, Jodi T; Schermel, Alyssa; Mills, Christine M; L'Abbé, Mary R

2016-09-21

A number of recommendations for policy and program interventions to limit excess free sugar consumption have emerged, however there are a lack of data describing the amounts and types of sugar in foods. This study presents an assessment of sugar in Canadian prepackaged foods including: (a) the first systematic calculation of free sugar contents; (b) a comprehensive assessment of total sugar and free sugar levels; and (c) sweetener and free sugar ingredient use, using the University of Toronto's Food Label Information Program (FLIP) database 2013 ( n = 15,342). Food groups with the highest proportion of foods containing free sugar ingredients also had the highest median total sugar and free sugar contents (per 100 g/mL): desserts (94%, 15 g, and 12 g), sugars and sweets (91%, 50 g, and 50 g), and bakery products (83%, 16 g, and 14 g, proportion with free sugar ingredients, median total sugar and free sugar content in Canadian foods, respectively). Free sugar accounted for 64% of total sugar content. Eight of 17 food groups had ≥75% of the total sugar derived from free sugar. Free sugar contributed 20% of calories overall in prepackaged foods and beverages, with the highest at 70% in beverages. These data can be used to inform interventions aimed at limiting free sugar consumption.

Total and Free Sugar Content of Canadian Prepackaged Foods and Beverages

PubMed Central

Bernstein, Jodi T.; Schermel, Alyssa; Mills, Christine M.; L’Abbé, Mary R.

2016-01-01

A number of recommendations for policy and program interventions to limit excess free sugar consumption have emerged, however there are a lack of data describing the amounts and types of sugar in foods. This study presents an assessment of sugar in Canadian prepackaged foods including: (a) the first systematic calculation of free sugar contents; (b) a comprehensive assessment of total sugar and free sugar levels; and (c) sweetener and free sugar ingredient use, using the University of Toronto’s Food Label Information Program (FLIP) database 2013 (n = 15,342). Food groups with the highest proportion of foods containing free sugar ingredients also had the highest median total sugar and free sugar contents (per 100 g/mL): desserts (94%, 15 g, and 12 g), sugars and sweets (91%, 50 g, and 50 g), and bakery products (83%, 16 g, and 14 g, proportion with free sugar ingredients, median total sugar and free sugar content in Canadian foods, respectively). Free sugar accounted for 64% of total sugar content. Eight of 17 food groups had ≥75% of the total sugar derived from free sugar. Free sugar contributed 20% of calories overall in prepackaged foods and beverages, with the highest at 70% in beverages. These data can be used to inform interventions aimed at limiting free sugar consumption. PMID:27657125

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development.

PubMed

Deluc, Laurent G; Grimplet, Jérôme; Wheatley, Matthew D; Tillett, Richard L; Quilici, David R; Osborne, Craig; Schooley, David A; Schlauch, Karen A; Cushman, John C; Cramer, Grant R

2007-11-22

Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (> or =2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development

PubMed Central

Deluc, Laurent G; Grimplet, Jérôme; Wheatley, Matthew D; Tillett, Richard L; Quilici, David R; Osborne, Craig; Schooley, David A; Schlauch, Karen A; Cushman, John C; Cramer, Grant R

2007-01-01

Background Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. Results Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. Conclusion These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing. PMID:18034876

75 FR 50796 - Fiscal Year 2011 Tariff-Rate Quota Allocations for Raw Cane Sugar, Refined and Specialty Sugar...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-17

... for Raw Cane Sugar, Refined and Specialty Sugar, and Sugar-Containing Products AGENCY: Office of the... quantity of the tariff-rate quotas for imported raw cane sugar, refined and specialty sugar, and sugar... imports of raw cane sugar and refined sugar. Pursuant to Additional U.S. Note 8 to Chapter 17 of the HTS...

29 CFR 780.815 - Basic conditions of exemption; second part, processing of sugar beets, sugar-beet molasses...

Code of Federal Regulations, 2010 CFR

2010-07-01

... sugar beets, sugar-beet molasses, sugarcane, or maple sap. 780.815 Section 780.815 Labor Regulations... Cotton and Processing of Sugar Beets, Sugar-Beet Molasses, Sugarcane, or Maple Sap into Sugar or Syrup... Quantities § 780.815 Basic conditions of exemption; second part, processing of sugar beets, sugar-beet...

29 CFR 780.815 - Basic conditions of exemption; second part, processing of sugar beets, sugar-beet molasses...

Code of Federal Regulations, 2011 CFR

2011-07-01

... sugar beets, sugar-beet molasses, sugarcane, or maple sap. 780.815 Section 780.815 Labor Regulations... Cotton and Processing of Sugar Beets, Sugar-Beet Molasses, Sugarcane, or Maple Sap into Sugar or Syrup... Quantities § 780.815 Basic conditions of exemption; second part, processing of sugar beets, sugar-beet...

29 CFR 780.815 - Basic conditions of exemption; second part, processing of sugar beets, sugar-beet molasses...

Code of Federal Regulations, 2014 CFR

2014-07-01

... sugar beets, sugar-beet molasses, sugarcane, or maple sap. 780.815 Section 780.815 Labor Regulations... Cotton and Processing of Sugar Beets, Sugar-Beet Molasses, Sugarcane, or Maple Sap into Sugar or Syrup... Quantities § 780.815 Basic conditions of exemption; second part, processing of sugar beets, sugar-beet...

29 CFR 780.815 - Basic conditions of exemption; second part, processing of sugar beets, sugar-beet molasses...

Code of Federal Regulations, 2013 CFR

2013-07-01

... sugar beets, sugar-beet molasses, sugarcane, or maple sap. 780.815 Section 780.815 Labor Regulations... Cotton and Processing of Sugar Beets, Sugar-Beet Molasses, Sugarcane, or Maple Sap into Sugar or Syrup... Quantities § 780.815 Basic conditions of exemption; second part, processing of sugar beets, sugar-beet...

29 CFR 780.815 - Basic conditions of exemption; second part, processing of sugar beets, sugar-beet molasses...

Code of Federal Regulations, 2012 CFR

2012-07-01

... sugar beets, sugar-beet molasses, sugarcane, or maple sap. 780.815 Section 780.815 Labor Regulations... Cotton and Processing of Sugar Beets, Sugar-Beet Molasses, Sugarcane, or Maple Sap into Sugar or Syrup... Quantities § 780.815 Basic conditions of exemption; second part, processing of sugar beets, sugar-beet...

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection.

PubMed

López-Agudelo, Víctor A; Baena, Andres; Ramirez-Malule, Howard; Ochoa, Silvia; Barrera, Luis F; Ríos-Estepa, Rigoberto

2017-11-21

Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb's vulnerability and metabolic adaptation have been carried out. A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis.

PubMed

Narasaki, Craig T; Mertens, Katja; Samuel, James E

2011-01-01

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.

Added sugars in kids' meals from chain restaurants.

PubMed

Scourboutakos, Mary J; Semnani-Azad, Zhila; L'Abbé, Mary R

2016-06-01

To analyze the added sugars in kids' meals from Canadian chain restaurants in relation to the World Health Organization's proposed sugar recommendation (less than 5% of total daily calories should come from added sugars) and current recommendation (less than 10% of total daily calories should come from added sugars). Total sugar levels were retrieved from the websites of 10 fast-food and 7 sit-down restaurants in 2010. The added sugar levels in 3178 kids' meals from Canadian chain restaurants were calculated in 2014 (in Toronto, Canada) by subtracting all naturally occurring sugars from the total sugar level. The average amount of added sugars in restaurant kids' meals (25 ± 0.36 g) exceeded the WHO's proposed daily recommendation for sugar intake. There was a wide range of added sugar levels in kids' meals ranging from 0 g to 114 g. 50% of meals exceeded the WHO's proposed daily sugar recommendation, and 19% exceeded the WHO's current daily sugar recommendation. There is a wide range of sugar levels in kids' meals from restaurants, and many contain more than a day's worth of sugar.

Total, Added, and Free Sugars: Are Restrictive Guidelines Science-Based or Achievable?

PubMed Central

Erickson, Jennifer; Slavin, Joanne

2015-01-01

Sugar consumption, especially added sugars, is under attack. Various government and health authorities have suggested new sugar recommendations and guidelines as low as 5% of total calories from free sugars. Definitions for total sugars, free sugars, and added sugars are not standardized, nor are there accepted nutrient databases for this information. Our objective was to measure total sugars and added sugars in sample meal plans created by the United States Department of Agriculture (USDA) and the Academy of Nutrition and Dietetics (AND). Utilizing the Nutrition Data System for Research (NDSR) nutritional database, results found that plans created by the USDA and AND averaged 5.1% and 3.1% calories from added sugar, 8.7% and 3.1% from free sugar, and 23.3% and 21.1% as total sugars respectively. Compliance with proposed added sugar recommendations would require strict dietary compliance and may not be sustainable for many Americans. Without an accepted definition and equation for calculating added sugar, added sugar recommendations are arbitrary and may reduce intakes of nutrient-rich, recommended foods, such as yogurt, whole grains, and tart fruits including cranberries, cherries, and grapefruit. Added sugars are one part of excess calorie intake; however, compliance with low added sugar recommendations may not be achievable for the general public. PMID:25884659

Total, added, and free sugars: are restrictive guidelines science-based or achievable?

PubMed

Erickson, Jennifer; Slavin, Joanne

2015-04-15

Sugar consumption, especially added sugars, is under attack. Various government and health authorities have suggested new sugar recommendations and guidelines as low as 5% of total calories from free sugars. Definitions for total sugars, free sugars, and added sugars are not standardized, nor are there accepted nutrient databases for this information. Our objective was to measure total sugars and added sugars in sample meal plans created by the United States Department of Agriculture (USDA) and the Academy of Nutrition and Dietetics (AND). Utilizing the Nutrition Data System for Research (NDSR) nutritional database, results found that plans created by the USDA and AND averaged 5.1% and 3.1% calories from added sugar, 8.7% and 3.1% from free sugar, and 23.3% and 21.1% as total sugars respectively. Compliance with proposed added sugar recommendations would require strict dietary compliance and may not be sustainable for many Americans. Without an accepted definition and equation for calculating added sugar, added sugar recommendations are arbitrary and may reduce intakes of nutrient-rich, recommended foods, such as yogurt, whole grains, and tart fruits including cranberries, cherries, and grapefruit. Added sugars are one part of excess calorie intake; however, compliance with low added sugar recommendations may not be achievable for the general public.