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Science Advances Sep 2018Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the...
Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH/NH) by the distinct members of this family is still a matter of controversy. Using cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH/NH conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in N relative to N when compared to the external ammonium source. We observed N depletion over a wide range of external pH, indicating its independence of NH formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K. We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor N over N, owing to the dissociation of NH into NH + H in the protein, leading to N depletion in the cell and allowing NH passage or NH/H cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH/K ratio, allowing organisms to specifically scavenge NH. We show that N isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.
Topics: Ammonia; Ammonium Compounds; Arabidopsis Proteins; Biochemistry; Cation Transport Proteins; Hydrogen-Ion Concentration; Ion Transport; Microorganisms, Genetically-Modified; Nitrogen Isotopes; Plant Proteins; Potassium; Potassium Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30214933
DOI: 10.1126/sciadv.aar3599 -
Journal of Bacteriology Apr 1964Genghof, Dorothy S. (Yeshiva University, New York, N.Y.), and Olga Van Damme. Biosynthesis of ergothioneine and hercynine by mycobacteria. J. Bacteriol. 87:852-862....
Genghof, Dorothy S. (Yeshiva University, New York, N.Y.), and Olga Van Damme. Biosynthesis of ergothioneine and hercynine by mycobacteria. J. Bacteriol. 87:852-862. 1964.-Ergothioneine and hercynine were found to be synthesized by a wide variety of mycobacteria grown in chemically defined media free from these compounds. The cultures examined included 53 recently isolated and laboratory strains of Mycobacterium tuberculosis, 26 "unclassified" mycobacteria (Runyon groups I to IV), and representatives of most other species in the genus. Purification and separation of the betaines was achieved by means of chromatography on two successive alumina columns. Photometric measurement of the diazotized effluents from the second column permitted amounts of each compound to be determined. Measurement of hercynine by this method was made possible for the first time by the development of a standard curve. The pathway of ergothioneine biosynthesis in mycobacteria, as judged by the use S(35)-sulfate and l-histidine-2-C(14) as tracers, appears similar to that found in Neurospora crassa and Claviceps purpurea, that is, from histidine to ergothioneine via hercynine. None of a small group of bacteria other than mycobacteria was found to produce ergothioneine. Two strains of group A streptococci and one of Escherichia coli produced hercyninelike material, as yet unidentified.
Topics: Ammonium Compounds; Bacteriological Techniques; Betaine; Carbon Isotopes; Chromatography; Ergothioneine; Histidine; Metabolism; Mycobacterium; Mycobacterium bovis; Mycobacterium leprae; Mycobacterium tuberculosis; Quaternary Ammonium Compounds; Research; Sulfur Isotopes
PubMed: 14137624
DOI: 10.1128/jb.87.4.852-862.1964 -
Synergistic Flame Retardancy of Phosphatized Sesbania Gum/Ammonium Polyphosphate on Polylactic Acid.Molecules (Basel, Switzerland) Jul 2022Phosphating sesbania gum (DESG) was obtained by modifying sesbania gum (SG) with 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and endic anhydride (EA). The...
Phosphating sesbania gum (DESG) was obtained by modifying sesbania gum (SG) with 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and endic anhydride (EA). The structure of DESG was determined using Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (H-NMR). Flame-retardant polylactic acid (PLA) composites were prepared by melt-blending PLA with DESG, which acted as a carbon source, and ammonium polyphosphate (APP), which acted as an acid source and a gas source. The flame retardancy of the PLA composite was investigated using vertical combustion (UL-94), the limiting oxygen index (LOI) and the cone calorimeter (CONE) test. Thermal properties and morphology were characterized via thermogravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM), respectively. Experimental results indicated that when the mass ratio of DESG/APP was equal to 12/8 the LOI value was 32.2%; a vertical burning test (UL-94) V-0 rating was achieved. Meanwhile, the sample showed a lowest total heat release (THR) value of 52.7 MJ/m, which is a 32.5% reduction compared to that of neat PLA. Using FESEM, the uniform distribution of DESG and APP in the PLA matrix was observed. The synergistic effect of DESG and APP effectively enhanced the flame retardancy of PLA. Additionally, the synergistic mechanism of DESG and APP in PLA was proposed.
Topics: Ammonium Compounds; Polyesters; Polyphosphates; Sesbania
PubMed: 35897921
DOI: 10.3390/molecules27154748 -
Plant Physiology Jan 2023A metabolite of ammonium assimilation was previously theorized to be involved in the coordination of the overall nitrate response in plants. Here we show that...
A metabolite of ammonium assimilation was previously theorized to be involved in the coordination of the overall nitrate response in plants. Here we show that 2-hydroxy-5-oxoproline, made by transamination of glutamine, the first product of ammonium assimilation, may be involved in signaling a plant's ammonium assimilation status. In leaves, 2-hydroxy-5-oxoproline met four foundational requirements to be such a signal. First, when it was applied to foliage, enzyme activities of nitrate reduction and ammonium assimilation increased; the activities of key tricarboxylic acid cycle-associated enzymes that help to supply carbon skeletons for amino acid synthesis also increased. Second, its leaf pools increased as nitrate availability increased. Third, the pool size of its precursor, Gln, reflected ammonium assimilation rather than photorespiration. Fourth, it was widely conserved among monocots, dicots, legumes, and nonlegumes and in plants with C3 or C4 metabolism. Made directly from the first product of ammonium assimilation, 2-hydroxy-5-oxoproline acted as a nitrate uptake stimulant. When 2-hydroxy-5-oxoproline was provided to roots, the plant's nitrate uptake rate approximately doubled. Plants exogenously provided with 2-hydroxy-5-oxoproline to either roots or leaves accumulated greater biomass. A model was constructed that included the proposed roles of 2-hydroxy-5-oxoproline as a signal molecule of ammonium assimilation status in leaves, as a stimulator of nitrate uptake by roots and nitrate downloading from the xylem. In summary, a glutamine metabolite made in the ω-amidase pathway stimulated nitrate uptake by roots and was likely to be a signal of ammonium assimilation status in leaves. A chemical synthesis method for 2-hydroxy-5-oxoproline was also developed.
Topics: Nitrates; Ammonium Compounds; Glutamine; Pyrrolidonecarboxylic Acid; Plants
PubMed: 36303326
DOI: 10.1093/plphys/kiac501 -
Microbial Cell Factories Apr 2022The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has...
BACKGROUND
The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has an inherent flaw, which is difficult to overcome. Organisms also prioritize using different nitrogen sources, which is called nitrogen catabolite repression. To date, few gene regulatory systems based on nitrogen catabolite repression have been reported.
RESULTS
In this study, we constructed a nitrogen switching auto-inducible expression system (NSAES) based on nitrogen catabolite regulation and nitrogen utilization in Aspergillus nidulans. The P promoter that is highly induced by nitrate and inhibition by ammonia was used as the promoter. Glucuronidase was the reporter protein. Glucuronidase expression occurred after ammonium was consumed in an ammonium and nitrate compounding medium, achieving stage auto-switching for cell growth and gene expression. This system maintained a balance between cell growth and protein production to maximize stress products. Expressions of glycosylated and secretory proteins were successfully achieved using this auto-inducible system.
CONCLUSIONS
We described an efficient auto-inducible protein expression system based on nitrogen catabolite regulation. The system could be useful for protein production in the laboratory and industrial applications. Simultaneously, NSAES provides a new auto-inducible expression regulation mode for other filamentous fungi.
Topics: Ammonium Compounds; Catabolite Repression; Glucuronidase; Nitrates; Nitrogen
PubMed: 35484589
DOI: 10.1186/s12934-022-01794-5 -
Clinical Journal of the American... Apr 2018
Topics: Acid-Base Equilibrium; Adult; Ammonium Compounds; Female; Humans; Kidney Transplantation; Male; Middle Aged; Osmolar Concentration; Randomized Controlled Trials as Topic; Urine
PubMed: 29519951
DOI: 10.2215/CJN.13311117 -
Scientific Reports Sep 2019N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of N labeled amino acids in metabolite studies have...
N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of N labeled amino acids in metabolite studies have not been pursued extensively, presumably due to line broadening present under standard experimental conditions. In this work, we show that lowering the temperature to -5 °C allows facile characterization of N-labeled amino acids. Further, we show that this technique can be exploited to measure NH produced in an enzyme catalyzed reaction and the transport and metabolism of individual amino acids in mammalian cell culture. With respect to C-labeled amino acids, N-labeled amino acids are less costly and enable direct characterization of nitrogen metabolism in complex biological systems by NMR. In summary, the present work significantly expands the metabolite pools and their reactions for study by NMR.
Topics: Amino Acids; Ammonium Compounds; Cell Line, Tumor; Humans; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Temperature
PubMed: 31488858
DOI: 10.1038/s41598-019-49208-8 -
The Biochemical Journal 1945
Topics: Ammonia; Ammonium Compounds; Quaternary Ammonium Compounds; Saccharomyces cerevisiae; Yeasts
PubMed: 16747922
DOI: 10.1042/bj0390368 -
PloS One 2019River restoration projects focused on altering flow regimes through use of in-channel structures can facilitate ecosystem services, such as promoting nitrogen (N)...
River restoration projects focused on altering flow regimes through use of in-channel structures can facilitate ecosystem services, such as promoting nitrogen (N) storage to reduce eutrophication. In this study we use small flux chambers to examine ammonium (NH4+) and nitrate (NO3-) cycling across the sediment-water interface. Paired restored and unrestored study sites in 5 urban tributaries of the River Thames in Greater London were used to examine N dynamics following physical disturbances (0-3 min exposures) and subsequent biogeochemical activity (3-10 min exposures). Average ambient NH4+ concentrations were significantly different amongst all sites and ranged from 28.0 to 731.7 μg L-1, with the highest concentrations measured at restored sites. Average NO3- concentrations ranged from 9.6 to 26.4 mg L-1, but did not significantly differ between restored and unrestored sites. Average NH4+ fluxes at restored sites ranged from -8.9 to 5.0 μg N m-2 sec-1, however restoration did not significantly influence NH4+ uptake or regeneration (i.e., a measure of release to surface water) between 0-3 minutes and 3-10 minutes. Further, average NO3- fluxes amongst sites responded significantly between 0-3 minutes ranging from -33.6 to 97.7 μg N m-2 sec-1. Neither NH4+ nor NO3- fluxes correlated to sediment chlorophyll-a, total organic matter, or grain size. We attributed variations in overall N fluxes to N-specific sediment storage capacity, biogeochemical transformations, potential legacy effects associated with urban pollution, and variations in river-specific restoration actions.
Topics: Ammonium Compounds; Ecosystem; Eutrophication; Geologic Sediments; Nitrates; Nitrogen; Rivers; Water; Water Pollutants, Chemical
PubMed: 30865649
DOI: 10.1371/journal.pone.0212690 -
Proceedings of the National Academy of... Jul 2014Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea,...
Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.
Topics: Ammonium Compounds; Archaeoglobus fulgidus; Carrier Proteins; Hydrogen-Ion Concentration; Ion Transport
PubMed: 24958855
DOI: 10.1073/pnas.1406409111