-
The Journal of Biological Chemistry Feb 1992Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the...
Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the endoplasmic reticulum and Golgi (sec 18, 20), within the Golgi (sec 7), and between the Golgi and plasma membrane (sec 1, 3, 4, 5, 6, 8, 9). The accumulation of vesicles was paralleled by the enrichment of a defined subset of proteins modified either via ester or amide linkages to myristic acid: Myristoylated proteins of 21, 32, 49, 56, 75, and 136 kDa were enriched between the endoplasmic reticulum and Golgi; proteins of 21, 32, 45, 56, 75, 136 kDa were enriched by blocks within the Golgi; and proteins of 18, 21, 32, 36, 49, 68, and 136 kDa were trapped in a myristoylated form by blocks between the Golgi and plasma membrane. This enrichment of myristoylated proteins was reversed upon returning the cells to the permissive temperature for secretion. The fatty acid was linked to the 21-kDa protein via a hydroxylamine-resistant amide linkage (N-myristoylation) and to the proteins of 24, 32, 49, 56, 68, 136 kDa via hydroxylamine-labile ester linkage (E-myristoylation). In addition, myristoylated proteins of 21, 56, and 136 kDa were glycosylated via amino linkages to asparagine. This suggests they are exposed to the lumen of the secretory pathway. Three proteins (24, 32, and 56) were E-myristoylated in the presence of protein synthesis inhibitors, indicating this modification can occur posttranslationally. After using cycloheximide to clear protein passengers from the secretory pathway the 21-, 32-, and 56-kDa proteins continued to accumulate in a myristoylated form when vesicular transport was blocked between the Golgi and plasma membrane. These data suggest that myristoylation occurs on a component of the secretory machinery rather than on a passenger protein.
Topics: Acylation; Autoradiography; Cerulenin; Chromatography, High Pressure Liquid; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fungal Proteins; Glycosylation; Golgi Apparatus; Hexosaminidases; Hydroxylamine; Hydroxylamines; Myristic Acid; Myristic Acids; Protein Processing, Post-Translational; Saccharomyces cerevisiae
PubMed: 1740440
DOI: No ID Found -
MSystems Mar 2024The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to...
The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB) can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase, lacks recognized nitrite reductase genes. In this study, was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.IMPORTANCENitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by , an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA). was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.
Topics: Nitrates; Ammonium Compounds; Nitrites; Ecosystem; Denitrification; Bacteria; Hydroxylamine; Nitrite Reductases; Nitrogen; Hydroxylamines; Sulfates
PubMed: 38323850
DOI: 10.1128/msystems.00967-23 -
Analytical Chemistry Jul 2016Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary...
Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.
Topics: Cell Line, Tumor; Chromatography, High Pressure Liquid; Diphosphoglyceric Acids; Glucose; Humans; Hydroxylamine; Isomerism; Metabolome; Tandem Mass Spectrometry
PubMed: 27314642
DOI: 10.1021/acs.analchem.6b02009 -
The Journal of Infectious Diseases Apr 2023Molnupiravir is an antiviral agent recently used for treating coronavirus disease 2019 (COVID-19). Here, we demonstrate that N4-hydroxycytidine (NHC), a molnupiravir...
Molnupiravir is an antiviral agent recently used for treating coronavirus disease 2019 (COVID-19). Here, we demonstrate that N4-hydroxycytidine (NHC), a molnupiravir metabolite, treated with cytidine deaminase (CDA) induced Cu(II)-mediated oxidative DNA damage in isolated DNA. A colorimetric assay revealed hydroxylamine generation from CDA-treated NHC. The site specificity of DNA damage also suggested involvement of hydroxylamine in the damage. Furthermore, Cu(I) and H2O2 play an important role in the DNA damage. We propose oxidative DNA damage via CDA-mediated metabolism as a possible mutagenic mechanism of NHC, highlighting the need for careful risk assessment of molnupiravir use in therapies for viral diseases, including COVID-19.
Topics: Humans; Antiviral Agents; SARS-CoV-2; Hydrogen Peroxide; COVID-19; Hydroxylamines; Oxidative Stress; DNA Damage
PubMed: 36461940
DOI: 10.1093/infdis/jiac477 -
Biochemical and Biophysical Research... May 2019O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan...
O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.
Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Glycomics; Glycoproteins; Glycosylation; Hydroxylamine; Mucins; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine
PubMed: 30952424
DOI: 10.1016/j.bbrc.2019.03.144 -
The Journal of Biological Chemistry May 1985The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward...
The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.
Topics: Animals; Argininosuccinate Lyase; Cattle; Diethyl Pyrocarbonate; Ethyldimethylaminopropyl Carbodiimide; Hydrogen-Ion Concentration; Hydroxylamine; Hydroxylamines; Kinetics; Liver; Lyases; Mathematics; Temperature
PubMed: 3988765
DOI: No ID Found -
Yakugaku Zasshi : Journal of the... 2021Several direct asymmetric Michael additions to α,β-unsaturated carboxylic acids with integrated catalysts comprising chiral bifunctional thiourea and arylboronic acid... (Review)
Review
Several direct asymmetric Michael additions to α,β-unsaturated carboxylic acids with integrated catalysts comprising chiral bifunctional thiourea and arylboronic acid were developed. First, the asymmetric aza-Michael addition of hydroxylamine derivatives efficiently afforded a variety of optically active β-amino acid derivatives. Furthermore, upon detailed investigation of the reaction, tetrahedral borate complexes, comprising two carboxylate molecules, were found to serve as reaction intermediates. Based on this observation, a drastic improvement in product enantioselectivity was achieved upon benzoic acid addition. Second, on merely changing the solvent, the asymmetric thia-Michael addition of arylthiols afforded both enantiomers of the adducts, which are important building blocks for biologically active compounds.
Topics: Amino Acids; Benzoic Acid; Boronic Acids; Carboxylic Acids; Catalysis; Hydroxylamines; Molecular Structure; Stereoisomerism; Thiourea
PubMed: 33642494
DOI: 10.1248/yakushi.20-00214 -
International Journal of Molecular... Mar 2015In the present study, a new magnetic powder based on magnetite can be used as a petroleum crude oil collector. Amidoximes based on rosin as a natural product can be...
In the present study, a new magnetic powder based on magnetite can be used as a petroleum crude oil collector. Amidoximes based on rosin as a natural product can be prepared from a reaction between hydroxylamine and rosin/acrylonitrile adducts. The produced rosin amidoximes were used as capping agents for magnetite nanoparticles to prepare hydrophobic coated magnetic powders. A new class of monodisperse hydrophobic magnetite nanoparticles was prepared by a simple and inexpensive co-precipitation method. Iron ions and iodine were prepared by the reaction between ferric chloride and potassium iodide. The structure and morphology of magnetite capped with rosin amidoxime were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), zeta potential, thermogravimetric analysis (TGA) and dynamic light scattering (DLS). The magnetic properties were determined from vibrating sample magnetometer (VSM) analyses. These prepared magnetite nanoparticles were tested as bioactive nanosystems and their antimicrobial effects were investigated. The prepared nanomaterials were examined as a crude oil collector using magnetic fields. The results show promising data for the separation of the petroleum crude oil from aqueous solution in environmental pollution cleanup.
Topics: Anti-Bacterial Agents; Escherichia coli; Hydroxylamines; Magnetite Nanoparticles; Microbial Sensitivity Tests; Molecular Structure; Particle Size; Petroleum Pollution; Resins, Plant; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus; X-Ray Diffraction
PubMed: 25822876
DOI: 10.3390/ijms16046911 -
Applied and Environmental Microbiology Dec 2023Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of...
Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of ammonium-based fertilizers in agriculture has disrupted this cycle, leading to groundwater pollution and greenhouse gas emissions. In this study, we have demonstrated the inhibitory effects of plant-derived juglone and related 1,4-naphthoquinones on the nitrification process in . Notably, the inhibition mechanism is elucidated in which 1,4-naphthoquinones interact with hydroxylamine oxidoreductase, disrupting the electron transfer to cytochrome , a physiological electron acceptor. These findings support the notion that phytochemicals can impede nitrification by interfering with the essential electron transfer process in ammonia oxidation. The findings presented in this article offer valuable insights for the development of strategies aimed at the management of nitrification, reduction of fertilizer utilization, and mitigation of greenhouse gas emissions.
Topics: Cytochromes c; Ammonia; Electrons; Greenhouse Gases; Naphthoquinones; Fertilizers; Oxidation-Reduction; Hydroxylamine; Nitrification
PubMed: 38009977
DOI: 10.1128/aem.01291-23 -
Ecotoxicology and Environmental Safety Nov 2023Hydroxylamine is a highly reactive inorganic nitrogen compound that not only has a toxic effect on microorganisms, but also makes wastewater treatment more difficult,...
Hydroxylamine is a highly reactive inorganic nitrogen compound that not only has a toxic effect on microorganisms, but also makes wastewater treatment more difficult, which in turn damages the environment and even endangers human health. This study reported a new method for converting of hydroxylamine by adding sodium carbonate or calcium bicarbonate to the hydroxylamine-polluted wastewater. The conversion efficiency of hydroxylamine was more than 99% in the presence of sodium carbonate or calcium bicarbonate under the reaction conditions of 25 °C, C/N ratio 15, and dissolved oxygen 7.4 mg/L. And its maximal conversion rate can reach 3.49 mg/L/h. This method overcomes various shortcomings of the reported hydroxylamine removal technologies that require a large material dosage and high cost. The technology in this report has many advantages: low cost, 'green' environmental protection, easy market promotion, and high economic benefits.
Topics: Humans; Wastewater; Hydroxylamine; Hydroxylamines; Dietary Supplements; Nitrogen; Calcium Carbonate
PubMed: 37856986
DOI: 10.1016/j.ecoenv.2023.115603