Regulatory Properties of Glutamine Synthetase from the Nitrogen-Fixing Phototrophic Bacterium Rhodopseudomonas palustris

1981 ◽  
Vol 36 (9-10) ◽  
pp. 784-789 ◽  
Author(s):  
Kassem Alef ◽  
Walter G. Zumft
Keyword(s):  
Catalytic Activity ◽  
Glutamine Synthetase ◽  
Kinetic Parameters ◽  
Rhodopseudomonas Palustris ◽  
Nitrogen Fixing ◽  
Substrate Interaction ◽  
Phototrophic Bacterium ◽  
Enzyme Substrate ◽  
Regulatory Properties

Abstract Rhodopseudomonas palustris, Glutamine Synthetase Regulation, Adenylylation Glutamine synthetase from Rhodopseudomonas palustris is regulated via an adenylylation/deadenylylation mechanism. The enzyme purified from ammonia-grown cells, released AMP upon treatment with phosphodiesterase, along with drastic changes in its pH and metal dependency. Kinetic parameters for enzyme-substrate interaction were also dependent on the adenylylation state of the enzyme, as was the influence of several nitrogenous feedback inhibitors on the catalytic activity. The adenylylation state of the enzyme was modified in vivo by the availability of ammonia.

Molecular Characterization of Glutamine Synthetase from the Nitrogen-Fixing Phototrophic Bacterium Rhodopseudomonas palustris1

1981 ◽  
Vol 36 (3-4) ◽  
pp. 246-254 ◽  
Author(s):  
Kassem Alef ◽  
Hans-Joachim Burkardt ◽  
Hans-Joachim Horstmann ◽  
Walter G. Zumft
Keyword(s):  
Electron Microscopy ◽  
Glutamine Synthetase ◽  
Rhodopseudomonas Palustris ◽  
Glutamate Synthase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molecular Entity ◽  
Phototrophic Bacterium ◽  
Electrophoretic Techniques

The phototrophic bacterium Rhodopseudomonas palustris assimilated ammonium via glutamine synthetase and glutamate synthase. Diazotrophic and ammonium-grown cells had high levels of both enzymes, whereas enzymes of alternative assimilatory pathways were absent or had only low activities. Glutamine synthetase was purified to electrophoretic homogeneity within three steps by dye-ligand and ion exchange chromatography. Electron microscopy revealed a dodecameric molecular entity which was in accordance with parameters derived from electrophoretic techniques. The molecular weight of the enzyme monomer was 55 800; that of the dodecamer 670 000. The amino acid composition of R. palustris glutamine synthetase was determined and compared by a statistical method with other known enzyme compositions from prokaryotic and eukaryotic origins

In Vivo Control of Glutamine Synthetase in the Facultative Phototrophic Bacterium Rhodopseudomonas sphaeroides

1981 ◽  
Vol 36 (5-6) ◽  
pp. 407-410 ◽  
Author(s):  
Harald Engelhardt ◽  
Jobst-Heinrich Klemme
Keyword(s):  
Light Intensity ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Catalytic Properties ◽  
Rhodopseudomonas Sphaeroides ◽  
Phototrophic Bacterium

Abstract In vivo control of glutamine synthetase (GS) in the facultative phototrophic bacterium Rhodopseudomonas sphaeroides was studied. The enzyme was partially purified from cells grown photosynthetically in ammonium-malate-medium. Its catalytic properties were characteristically changed by incubation with phosphodiesterase indicating an in vivo regulation by adenylylation/deadenylylation. Adenylylation states of GS were measured as a function of variations in the nitrogen source and the light intensity during photosynthetic growth.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds

1991 ◽  
Vol 279 (2) ◽  
pp. 495-501 ◽  
Author(s):  
F E Podestá ◽  
W C Plaxton
Keyword(s):  
Pyruvate Kinase ◽  
Castor Oil ◽  
Mixed Type ◽  
Competitive Inhibition ◽  
Ricinus Communis ◽  
Cytosolic Ph ◽  
Oil Seeds ◽  
Saturation Kinetics ◽  
Regulatory Properties

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

scholarly journals Protection Elicited by Two Glutamine Auxotrophs of Mycobacterium tuberculosis and In Vivo Growth Phenotypes of the Four Unique Glutamine Synthetase Mutants in a Murine Model

2006 ◽  
Vol 74 (11) ◽  
pp. 6491-6495 ◽  
Author(s):  
Sunhee Lee ◽  
Bo-Young Jeon ◽  
Svetoslav Bardarov ◽  
Mei Chen ◽  
Sheldon L. Morris ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Mycobacterium Bovis ◽  
Murine Model ◽  
Triple Mutant ◽  
Auxotrophic Mutants ◽  
Bcg Vaccination ◽  
In Vivo Growth ◽  
Subcutaneous Immunization

ABSTRACT We generated four individual glutamine synthetase (GS) mutants (ΔglnA1, ΔglnA2, ΔglnA3, and ΔglnA4) and one triple mutant (ΔglnA1EA2) of Mycobacterium tuberculosis to investigate the roles of GS enzymes. Subcutaneous immunization with the ΔglnA1EA2 and ΔglnA1 glutamine auxotrophic mutants conferred protection on C57BL/6 mice against an aerosol challenge with virulent M. tuberculosis, which was comparable to that provided by Mycobacterium bovis BCG vaccination.

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