Functional Analysis of the Escherichia coli Molybdopterin Cofactor Biosynthesis Protein MoeA by Site-Directed Mutagenesis

2002 ◽  
Vol 383 (2) ◽  
pp. 319-323 ◽  
Author(s):  
C. Sandu ◽  
R. Brandsch
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Sitedirected Mutagenesis ◽  
Cofactor Biosynthesis

AbstractFive moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Momolybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

Changes in Leaf Nitrate Reductase Activity In Vivo and In Vitro During Light-Dark Transitions

1999 ◽  
Vol 8 (1) ◽  
pp. 37-40 ◽  
Author(s):  
B. K. Salalkar ◽  
R. S. Shaikh ◽  
R. M. Naik ◽  
S. V. Munjal ◽  
B. B. Desai ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Leaf Nitrate

scholarly journals Effect of lead on nitrate reductase activity and nitrate assimilation in pea leaves

2014 ◽  
Vol 57 (4) ◽  
pp. 457-463 ◽  
Author(s):  
S. K. Sinha ◽  
H. S. Srivastava ◽  
S. N. Mishra
Keyword(s):  
Enzyme Activity ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Organic Nitrogen ◽  
Reductase Activity ◽  
Total Organic Nitrogen ◽  
Intact Leaf ◽  
Leaf Tissues

The effect of Pb on nitrate reductase activity, protein, total organic nitrogen and on the chlorophyll content in excised and intact leaf tissues of <em>Pisum sativum</em> was examine. Enzyme activity assayed in vitro or in vivo in the excised leaves showed marked increase at lower concentrations of Pb while being inhibited at higher concentrations. In intact leaf tissues, the enzyme activity (in vivo or in vitro) was unaffected at lower concentrations but was inhibited at higher concentrations of Pb. Chlorophyll, carotenoids (non-nitrogenous pigments), soluble protein and organic nitrogen contents remained almost unaffected at all concentrations of Pb tested. It seems that nitrate reductase has a different response towards Pb pollution in this species, which is more tolerant to heavy metal pollution, especially Pb.

scholarly journals Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

2002 ◽  
Vol 184 (5) ◽  
pp. 1314-1323 ◽  
Author(s):  
Valley Stewart ◽  
Yiran Lu ◽  
Andrew J. Darwin
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Null Alleles ◽  
E Coli ◽  
Periplasmic Nitrate Reductase ◽  
Nitrate Concentrations ◽  
K 12

ABSTRACT Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

Effects of Glufosinate on Anion Uptake in Lemna gibba G 1

1989 ◽  
Vol 44 (1-2) ◽  
pp. 33-38 ◽  
Author(s):  
Gudrun D. Trogisch ◽  
Helmut Köcher ◽  
Wolfram R. Ullrich
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Nitrate Uptake ◽  
Dark Respiration ◽  
Reductase Activity ◽  
Lemna Gibba ◽  
Proton Cotransport ◽  
Chlorophyll Formation

Abstract The duckweed Lemna gibba G 1 was used as a model to study inhibitory sites with the herbicide and glutamate analogue glufosinate (PPT). Growth and chlorophyll formation were partly inhibited by 25 n-M, completely suppressed by 250 (im PPT. Photosynthesis showed partial inhibition within few hours, dark respiration ( 0 2 consumption) increased already within one hour. In the presence of 1 mM PPT in the light, the ammonium pool of Lemna increased to 600% within few hours, later to 1000%. The overall amino acid pool exhibited a slower increase to 300%, the nitrate pool only a slight increase, while total phosphate remained almost unchanged. In the dark all these effects were less pronounced than in the light. Nitrate, nitrite and phosphate uptake were partially inhibited by PPT, especially after 19 h PPT pretreatment. Nitrate reductase activity in vitro, after PPT treatment in vivo, showed an inhibition similar to that of nitrate uptake. Ammonium was not taken up but released under the same conditions. The data are explained by a combined effect of PPT, by inhibition of glutamine synthetase leading to accumulation of ammonium from photorespiration and proteolysis, by membrane depolarization and inhibition of anion/proton cotransport, by secondary uncoupling of phosphorylation, and by secondary inhibition of nitrate reductase activity.

scholarly journals Effect of vanadium on nitrate reductase activity in tomato leaves

2015 ◽  
Vol 42 (2) ◽  
pp. 223-232 ◽  
Author(s):  
J. Buczek
Keyword(s):  
Nitrate Reductase ◽  
Enzyme Inhibition ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Tomato Seedlings ◽  
Low Concentrations ◽  
Edta Solution ◽  
Vanadium Ions

The activity of nitrate reductase in cell-free extracts from tomato leaves is completely inhibited by 100 μM NaVO<sub>3</sub> or VOCl<sub>2</sub>. In experiments in vivo vanadium ions inhibit the activity of the enzyme in 50 to 60 per cent. Addition of l mM vanadium to the medium on which tomato seedlings are grown causes after 24 h almost complete inhibition of nitrate reductase activity in cell-free extracts of the enzyme. Inhibition with vanadium may be abolished in experiments <i>in vitro</i> if the extract is treated with a rather concentrated EDTA solution or Sephadex G-25. The data obtained in this study indicate that vanadium ions in relatively low concentrations inhibit the activity of nitrate reductase in tomato seedlings both <i>in vivo</i> and <i>in nitro</i>.

Sign in / Sign up

Export Citation Format

Share Document