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Imke Schroder - One of the best experts on this subject based on the ideXlab platform.

  • adaptation to a high tungsten environment pyrobaculum aerophilum contains an active tungsten Nitrate Reductase
    Biochemistry, 2010
    Co-Authors: Simon De Vries, Milica Momcilovic, Marc J F Strampraad, Julian P Whitelegge, Ashkan Baghai, Imke Schroder
    Abstract:

    Nitrate Reductases (Nars) belong to the DMSO Reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits Nitrate Reductase (Nar) activity even at...

  • properties of a thermostable Nitrate Reductase from the hyperthermophilic archaeon pyrobaculum aerophilum
    Journal of Bacteriology, 2001
    Co-Authors: Sepideh Afshar, Eric E. Johnson, Simon De Vries, Imke Schroder
    Abstract:

    The Nitrate Reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49mol/mol complex) as cofactors. The P. aerophilum Nitrate Reductase distinguishes itself from Nitrate Reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (Vmax with Nitrate, 1,162 s 21 (326 U/mg); Vmax with chlorate, 1,348 s 21 (378 U/mg) [assayed at 75°C]). The Km values for Nitrate and chlorate were 58 and 140 mM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the Nitrate Reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified Nitrate Reductase had a half-life of 1.5 h. This study constitutes the first description of a Nitrate Reductase from a hyperthermophilic archaeon. Nitrate serves as electron acceptor to many prokaryotic microbes that thrive under anaerobic conditions. Nitrate respiration occurs via two independent pathways, the denitrification pathway and the ammonification pathway (3). Nitrate is reduced sequentially to dinitrogen gas in the denitrification pathway, while ammonium is the product of the two-step ammonification pathway. The first reaction, in which Nitrate is reduced to nitrite via the membrane-bound Nitrate Reductase, is identical in both pathways (3, 21). In general, the dissimilatory Nitrate Reductase is conserved among bacteria and archaea that have been investigated thus far. The enzyme has been extensively studied in mesophilic Nitrate reducing bacteria such as the ammonifier Escherichia coli and the denitrifiers Paracoccus denitrificans, Pseudomonas stuzeri, Pseudomonas denitrificans, and others (5, 6, 11, 12, 16). The E. coli NarGHI enzyme is one of the best-characterized enzymes (3). The enzyme complex consists of three subunits (11, 16). The a subunit (NarG) has an Mr of 145,000 and contains a molybdopterin cofactor at its active site, where Nitrate is reduced to nitrite. The b subunit (NarH) has an Mr of 58,000 and is the location of one [3Fe-4S] center and three [4Fe-4S] centers. Both the a and b subunits are attached to the cytoplasmic membrane by the 25,000-Da g subunit (NarI). This polypeptide contains cytochrome b and functions to oxidize the menaquinol or ubiquinol of the quinone pool. Electrons are transferred from the quinol pool via the b subunit to the a subunit active site (3). Recently, Nitrate Reductases from several archaeal species have been described. While Haloferax volcanii contains a heterotrimeric enzyme complex similar to the bacterial dissimilatory Nitrate Reductases, the Nitrate Reductase from Haloferax denitrificans was purified as a heterodimeric enzyme possibly lacking the

Tsuneo Hino - One of the best experts on this subject based on the ideXlab platform.

  • molecular characterization and transcriptional regulation of Nitrate Reductase in a ruminal bacterium selenomonas ruminantium
    Journal of General and Applied Microbiology, 2004
    Co-Authors: Narito Asanuma, Miwa Iwamoto, Takahiro Yoshii, Tsuneo Hino
    Abstract:

    Nitrate Reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced. The 6.4 kbp nar gene consisted of narG, H, J, and I in this order. The deduced amino acid sequences of these subunits resembled those of membrane-bound Nitrate Reductase-A reported for Escherichia coli. It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon). The level of intracellular nar-mRNA was higher when S. ruminantium was grown with Nitrate than when grown without Nitrate, suggesting that nar transcription is enhanced by Nitrate. The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply. Therefore, NaR synthesis in S. ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons. S. ruminantium reduced Nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription. Addition of fumarate stimulated Nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction.

  • effects of energy substrates on Nitrate reduction and Nitrate Reductase activity in a ruminal bacterium selenomonas ruminantium
    Anaerobe, 2001
    Co-Authors: Miwa Iwamoto, Narito Asanuma, Tsuneo Hino
    Abstract:

    Abstract The factors affecting the rate of Nitrate reduction and the Nitrate Reductase content in Selenomonas ruminantium were examined. The rate of Nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The Nitrate reduction rate was parallel to the Nitrate Reductase content in cells, suggesting that the amount of Nitrate Reductase limits the rate of Nitrate reduction. The amount of Nitrate Reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD + was related to the rate of Nitrate reduction and to the amount of Nitrate Reductase. From these results, it is conceivable that the synthesis of Nitrate Reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of Nitrate Reductase was increased by the presence of Nitrate, suggesting that the synthesis of Nitrate Reductase is enhanced by Nitrate. In addition, Nitrate reduction altered the fermentation pattern as a result of electron consumption.

Manuel Losada - One of the best experts on this subject based on the ideXlab platform.

  • purification and properties of assimilatory Nitrate Reductase nad p h from ankistrodesmus braunii
    FEBS Journal, 2005
    Co-Authors: Miguel A. Rosa, Jesús Diez, Jose M Vega, Manuel Losada
    Abstract:

    Assimilatory Nitrate Reductase [NAD(P)H] (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by a simple procedure that utilizes as the main step affinity chromatography on Blue-Sepharose. The best enzyme preparation has a specific activity of 61.25 units/mg protein. The enzyme has a sedimentation coefficient of 10.9 S by sucrose-density-gradient centrifugation, and a Stokes radius of 9.8 nm was estimated by gel filtration techniques. Its molecular weight is 460000, but only one single band of 58000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of eight subunits. The Nitrate Reductase absorption spectrum shows wavelength maxima at 280 and 416 nm and a broad shoulder at 450 nm. Reduced enzyme shows maxima at 424 (Soret), 527 (β) and 557 (α) nm, and a bleaching at 450 nm. The reduced extracted heme chromophore, in pyridine and KOH, shows absorption bands at 414, 522 and 552 nm. These properties indicate the presence of a b-type cytochrome and flavin as prosthetic groups of A. braunii Nitrate Reductase. A minimum of four molecules of heme has been calculated per molecule of the enzyme complex. Redox titration of the enzyme shows a midpoint potential for the heme of –73 mV at pH 7.0. In the presence of p-hydroxymercuribenzoate, which inhibits the NAD(P)H-dependent activities of the complex, the enzyme-bound heme can be reduced with dithionite, but not with NAD(P)H.

Miwa Iwamoto - One of the best experts on this subject based on the ideXlab platform.

  • molecular characterization and transcriptional regulation of Nitrate Reductase in a ruminal bacterium selenomonas ruminantium
    Journal of General and Applied Microbiology, 2004
    Co-Authors: Narito Asanuma, Miwa Iwamoto, Takahiro Yoshii, Tsuneo Hino
    Abstract:

    Nitrate Reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced. The 6.4 kbp nar gene consisted of narG, H, J, and I in this order. The deduced amino acid sequences of these subunits resembled those of membrane-bound Nitrate Reductase-A reported for Escherichia coli. It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon). The level of intracellular nar-mRNA was higher when S. ruminantium was grown with Nitrate than when grown without Nitrate, suggesting that nar transcription is enhanced by Nitrate. The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply. Therefore, NaR synthesis in S. ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons. S. ruminantium reduced Nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription. Addition of fumarate stimulated Nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction.

  • effects of energy substrates on Nitrate reduction and Nitrate Reductase activity in a ruminal bacterium selenomonas ruminantium
    Anaerobe, 2001
    Co-Authors: Miwa Iwamoto, Narito Asanuma, Tsuneo Hino
    Abstract:

    Abstract The factors affecting the rate of Nitrate reduction and the Nitrate Reductase content in Selenomonas ruminantium were examined. The rate of Nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The Nitrate reduction rate was parallel to the Nitrate Reductase content in cells, suggesting that the amount of Nitrate Reductase limits the rate of Nitrate reduction. The amount of Nitrate Reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD + was related to the rate of Nitrate reduction and to the amount of Nitrate Reductase. From these results, it is conceivable that the synthesis of Nitrate Reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of Nitrate Reductase was increased by the presence of Nitrate, suggesting that the synthesis of Nitrate Reductase is enhanced by Nitrate. In addition, Nitrate reduction altered the fermentation pattern as a result of electron consumption.

Simon De Vries - One of the best experts on this subject based on the ideXlab platform.

  • adaptation to a high tungsten environment pyrobaculum aerophilum contains an active tungsten Nitrate Reductase
    Biochemistry, 2010
    Co-Authors: Simon De Vries, Milica Momcilovic, Marc J F Strampraad, Julian P Whitelegge, Ashkan Baghai, Imke Schroder
    Abstract:

    Nitrate Reductases (Nars) belong to the DMSO Reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits Nitrate Reductase (Nar) activity even at...

  • properties of a thermostable Nitrate Reductase from the hyperthermophilic archaeon pyrobaculum aerophilum
    Journal of Bacteriology, 2001
    Co-Authors: Sepideh Afshar, Eric E. Johnson, Simon De Vries, Imke Schroder
    Abstract:

    The Nitrate Reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49mol/mol complex) as cofactors. The P. aerophilum Nitrate Reductase distinguishes itself from Nitrate Reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (Vmax with Nitrate, 1,162 s 21 (326 U/mg); Vmax with chlorate, 1,348 s 21 (378 U/mg) [assayed at 75°C]). The Km values for Nitrate and chlorate were 58 and 140 mM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the Nitrate Reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified Nitrate Reductase had a half-life of 1.5 h. This study constitutes the first description of a Nitrate Reductase from a hyperthermophilic archaeon. Nitrate serves as electron acceptor to many prokaryotic microbes that thrive under anaerobic conditions. Nitrate respiration occurs via two independent pathways, the denitrification pathway and the ammonification pathway (3). Nitrate is reduced sequentially to dinitrogen gas in the denitrification pathway, while ammonium is the product of the two-step ammonification pathway. The first reaction, in which Nitrate is reduced to nitrite via the membrane-bound Nitrate Reductase, is identical in both pathways (3, 21). In general, the dissimilatory Nitrate Reductase is conserved among bacteria and archaea that have been investigated thus far. The enzyme has been extensively studied in mesophilic Nitrate reducing bacteria such as the ammonifier Escherichia coli and the denitrifiers Paracoccus denitrificans, Pseudomonas stuzeri, Pseudomonas denitrificans, and others (5, 6, 11, 12, 16). The E. coli NarGHI enzyme is one of the best-characterized enzymes (3). The enzyme complex consists of three subunits (11, 16). The a subunit (NarG) has an Mr of 145,000 and contains a molybdopterin cofactor at its active site, where Nitrate is reduced to nitrite. The b subunit (NarH) has an Mr of 58,000 and is the location of one [3Fe-4S] center and three [4Fe-4S] centers. Both the a and b subunits are attached to the cytoplasmic membrane by the 25,000-Da g subunit (NarI). This polypeptide contains cytochrome b and functions to oxidize the menaquinol or ubiquinol of the quinone pool. Electrons are transferred from the quinol pool via the b subunit to the a subunit active site (3). Recently, Nitrate Reductases from several archaeal species have been described. While Haloferax volcanii contains a heterotrimeric enzyme complex similar to the bacterial dissimilatory Nitrate Reductases, the Nitrate Reductase from Haloferax denitrificans was purified as a heterodimeric enzyme possibly lacking the