scholarly journals Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

2021 ◽  
Vol 17 (11) ◽  
pp. e1010052
Author(s):  
Chu Wang ◽  
Christian Nehls ◽  
Dirk Baabe ◽  
Olaf Burghaus ◽  
Robert Hurwitz ◽  
...  
Keyword(s):  
Transfer Reaction ◽  
Radical Mechanism ◽  
Zero Field ◽  
Paramagnetic Resonance ◽  
Methyl Transfer ◽  
Lysine Residues ◽  
Oxygen Sensitive ◽  
Flagellar Filament

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

ДИНИТРОЗИЛЬНЫЕ КОМПЛЕКСЫ ЖЕЛЕЗА C ТИОЛСОДЕРЖАЩИМИ ЛИГАНДАМИ ПРЕДСТАВЛЕНЫ В ЖИВЫХ ОРГАНИЗМАХ В ОСНОВНОМ ИХ БИЯДЕРНОЙ ФОРМОЙ

2020 ◽  
Vol 65 (6) ◽  
pp. 1142-1153
Author(s):  
В.Д. Микоян ◽  
◽  
Е.Н. Бургова ◽  
Р.Р. Бородулин ◽  
А.Ф. Ванин ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Sodium Nitrite ◽  
Iron Complexes ◽  
Paramagnetic Resonance ◽  
First Case ◽  
Dinitrosyl Iron ◽  
Electron Paramagnetic ◽  
Dinitrosyl Complexes

The number of mononitrosyl iron complexes with diethyldithiocarbamate, formed in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite or the vasodilating drug Isoket® was assessed by electron paramagnetic resonance (EPR). The number of the said complexes, in contrast to the complexes, formed after nitrite or Isoket administration, the level of which sharply increased after treatment of liver preparations with a strong reducing agent - dithionite, did not change in the presence of dithionite. It was concluded that, in the first case, EPR-detectable mononitrosyl iron complexes with diethyldithiocarbamate in the absence and presence of dithionite appeared as a result of the reaction of NO formed from nitrite with Fe2+-dieth- yldithiocarbamate and Fe3+-diethyldithiocarbamate complexes, respectively. In the second case, mononitrosyl iron complexes with diethyldithiocarbamate appeared as a result of the transition of iron-mononitosyl fragments from ready-made iron-dinitrosyl groups of binuclear dinitrosyl complexes, which is three to four times higher than the content of the mononuclear form of these complexes in the tissue...

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

scholarly journals Nitric oxide interactions with cobalamins: biochemical and functional consequences

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1857-1864 ◽  
Author(s):  
M Brouwer ◽  
W Chamulitrat ◽  
G Ferruzzi ◽  
DL Sauls ◽  
JB Weinberg
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
No Formation ◽  
Iron Nitrosyl ◽  
Wide Range ◽  
Functional Consequences

Abstract Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

scholarly journals The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

2020 ◽  
Vol 295 (51) ◽  
pp. 17770-17780
Author(s):  
Ikuko Hayashi
Keyword(s):  
Lipid Membranes ◽  
Binding Protein ◽  
Coiled Coil ◽  
Type Iii ◽  
Daughter Cells ◽  
Lysine Residues ◽  
Stable Maintenance ◽  
Biochemical Analyses

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

Hydrogen Evolution Catalyzed by Hydrogenase in Cultures of Cyanobacteria

1981 ◽  
Vol 36 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Patrick C. Hallenbeck ◽  
Leon V. Kochian ◽  
John R. Benemann
Keyword(s):  
Hydrogen Production ◽  
Hydrogen Evolution ◽  
High Frequency ◽  
Nitrogenase Activity ◽  
Inhibitory Effect ◽  
Hydrogenase Activity ◽  
Oxygen Inhibition ◽  
Oxygen Sensitive

Abstract Cultures of Anabaena cylindrica, grown on media containing 5 mᴍ NH4Cl (which represses heterocyst formation), evolved hydrogen after a period of dark incubation under an argon atmosphere. This hydrogen production was not due to nitrogenase activity, which was nearly undetectable, but was due to a hydrogenase. Cultures grown on media with tungsten substituted for molybdenum had a high frequency of heterocysts (15%) and inactive nitrogenase after nitrogen starvation. The hydrogenase activity of these cultures was three-fold greater than the activity of non-heterocystous cultures. The effects of oxygen inhibition on hydrogen evolution by hetero-cystous cultures suggest that two pools of hydrogenase activity exist - an oxygen sensitive hydrogen evolution in vegetative cells and a relatively oxygen-resistent hydrogen evolution in heterocysts. In either case, inhibition by oxygen was reversible. Light had an inhibitory effect on net hydrogen evolution. Hydrogen production in vitro was much higher than in vivo, indicating that in vivo hydrogenase activity is limited by endogenous reductant supply.

scholarly journals Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

2020 ◽  
Vol 295 (7) ◽  
pp. 1973-1984
Author(s):  
Detao Gao ◽  
Mohammad Z. Ashraf ◽  
Lifang Zhang ◽  
Niladri Kar ◽  
Tatiana V. Byzova ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cross Linking ◽  
Oxidized Phospholipids ◽  
Cross Link ◽  
In Vivo Detection ◽  
Lysine Residues ◽  
Cross Links ◽  
Human Atheroma

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2− system. We found that five histidine residues in helices 5–8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3–4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR−/− mice, including cross-link adducts of apoA-I His-165–apoA-I Lys-93, apoA-I His-154–apoA-I Lys-105, apoA-I His-154–apoA-IV Lys-149, and apoA-II Lys-30–apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

scholarly journals Regulation of Carbon and Electron Flow inClostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

2001 ◽  
Vol 183 (5) ◽  
pp. 1748-1754 ◽  
Author(s):  
Sylvie Saint-Amans ◽  
Laurence Girbal ◽  
Jose Andrade ◽  
Kerstin Ahrens ◽  
Philippe Soucaille
Keyword(s):  
Electron Flow ◽  
Sharp Decrease ◽  
Major Product ◽  
Glycerol Dehydrogenase ◽  
Hydrogenase Activity ◽  
Specific Rate ◽  
Oxygen Sensitive ◽  
High Level

ABSTRACT The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H+ ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

scholarly journals Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall

2008 ◽  
Vol 295 (2) ◽  
pp. H499-H508 ◽  
Author(s):  
Wael F. Alzawahra ◽  
M. A. Hassan Talukder ◽  
Xiaoping Liu ◽  
Alexandre Samouilov ◽  
Jay L. Zweier
Keyword(s):  
Nitric Oxide ◽  
Heme Proteins ◽  
Soluble Guanylyl Cyclase ◽  
Vascular Wall ◽  
Rat Aorta ◽  
No Production ◽  
Paramagnetic Resonance ◽  
Physiological Importance

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of 10 μM. Similarly, nitrite (10 μM-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO.

scholarly journals Widespread Distribution in Pathogenic Bacteria of Di-Iron Proteins That Repair Oxidative and Nitrosative Damage to Iron-Sulfur Centers

2008 ◽  
Vol 190 (6) ◽  
pp. 2004-2013 ◽  
Author(s):  
Tim W. Overton ◽  
Marta C. Justino ◽  
Ying Li ◽  
Joana M. Baptista ◽  
Ana M. P. Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Human Pathogens ◽  
Paramagnetic Resonance ◽  
Widespread Distribution ◽  
Iron Proteins ◽  
E Coli

ABSTRACT Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

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