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Paul W Ludden - One of the best experts on this subject based on the ideXlab platform.

  • genes required for rapid expression of nitrogenase activity in azotobacter vinelandii
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Leonardo Curatti, Paul W Ludden, Carolyn S Brown, Luis M Rubio
    Abstract:

    Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the Dinitrogenase Reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. Δrnf mutations cause two distinguishable effects on the nitrogenase system: (i), slower nifHDK gene expression and (ii), impairment of nitrogenase function. In these mutants, Dinitrogenase Reductase activity is lowered, whereas Dinitrogenase activity remains essentially unaltered. Further analysis indicates that Δrnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. Δrnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.

  • chapter 4 the gene products of the nif regulon
    Nitrogen Fixation at the Millennium, 2002
    Co-Authors: Luis M Rubio, Paul W Ludden
    Abstract:

    This chapter deals with the gene products required for molybdenum-dependent nitrogen fixation ( nif ) in the model organisms Azotobacter vinelandii and Klebsiella pneumoniae with references to a few other systems. The products of the structural genes of the Mo-containing nitrogenase are not initially competent for substrate reduction. Rather, the products of a number of other nitrogen-fixation ( nif )-specific genes are required for maturation of the nitrogenase components to their active forms, for electron transport to nitrogenase, and for activation or inhibition of nif -gene expression under the appropriate physiological conditions. The genes required for electron transport to nitrogenase in K. pneumoniae are nifF and nifJ , which encode a flavodoxin and a pyruvate:flavodoxin oxidoReductase, respectively. The pathways for electron transport to nitrogenase in A. vinelandii and other diazotrophs are not yet well defined. The nitrogenase enzyme is composed of two oxygen-labile metalloproteins: Dinitrogenase and Dinitrogenase Reductase. The genes encoding the Dinitrogenase component polypeptides, nifD and nifK , are not required for FeMo–co biosynthesis, suggesting that FeMo–co is assembled elsewhere in the cell and is then inserted into a semi-maturated apo-Dinitrogenase that already contains the P clusters.

  • functional characterization of three glnb homologs in the photosynthetic bacterium rhodospirillum rubrum roles in sensing ammonium and energy status
    Journal of Bacteriology, 2001
    Co-Authors: Yaoping Zhang, Paul W Ludden, Edward L Pohlmann, Gary P Roberts
    Abstract:

    The GlnB (PII) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other PII homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of Dinitrogenase Reductase catalyzed by the Dinitrogenase Reductase ADP-ribosyl transferase (DRAT)/Dinitrogenase Reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

  • effects of specific amino acid substitutions on activities of Dinitrogenase Reductase activating glycohydrolase from rhodospirillum rubrum
    Journal of Bacteriology, 2001
    Co-Authors: Babu S Antharavally, Yaoping Zhang, Gary P Roberts, Russell R Poyner, Paul W Ludden
    Abstract:

    Site-directed mutagenesis of the draG gene was used to generate altered forms of Dinitrogenase Reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

  • effect of p ii and its homolog glnk on reversible adp ribosylation of Dinitrogenase Reductase by heterologous expression of the rhodospirillum rubrum Dinitrogenase Reductase adp ribosyl transferase Dinitrogenase Reductase activating glycohydrolase regulatory system in klebsiella pneumoniae
    Journal of Bacteriology, 2001
    Co-Authors: Yaoping Zhang, Paul W Ludden, Edward L Pohlmann, Cale M Halbleib, Gary P Roberts
    Abstract:

    Reversible ADP-ribosylation of Dinitrogenase Reductase, catalyzed by the Dinitrogenase Reductase ADP-ribosyl transferase–Dinitrogenase Reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.

Gary P Roberts - One of the best experts on this subject based on the ideXlab platform.

  • identification of rhodospirillum rubrum glnb variants that are altered in their ability to interact with different targets in response to nitrogen status signals
    Journal of Bacteriology, 2006
    Co-Authors: Yu Zhu, Yaoping Zhang, Mary Conrad, Gary P Roberts
    Abstract:

    In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (Dinitrogenase Reductase ADP-ribosyl transferase)-DRAG (Dinitrogenase Reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

  • characterization of altered regulation variants of Dinitrogenase Reductase activating glycohydrolase from rhodospirillum rubrum
    FEBS Letters, 2004
    Co-Authors: Kitai Kim, Yaoping Zhang, Gary P Roberts
    Abstract:

    In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of Dinitrogenase Reductase by Dinitrogenase Reductase ADP-ribosyltransferase (DRAT) and Dinitrogenase Reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.

  • functional characterization of three glnb homologs in the photosynthetic bacterium rhodospirillum rubrum roles in sensing ammonium and energy status
    Journal of Bacteriology, 2001
    Co-Authors: Yaoping Zhang, Paul W Ludden, Edward L Pohlmann, Gary P Roberts
    Abstract:

    The GlnB (PII) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other PII homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of Dinitrogenase Reductase catalyzed by the Dinitrogenase Reductase ADP-ribosyl transferase (DRAT)/Dinitrogenase Reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

  • effects of specific amino acid substitutions on activities of Dinitrogenase Reductase activating glycohydrolase from rhodospirillum rubrum
    Journal of Bacteriology, 2001
    Co-Authors: Babu S Antharavally, Yaoping Zhang, Gary P Roberts, Russell R Poyner, Paul W Ludden
    Abstract:

    Site-directed mutagenesis of the draG gene was used to generate altered forms of Dinitrogenase Reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

  • effect of p ii and its homolog glnk on reversible adp ribosylation of Dinitrogenase Reductase by heterologous expression of the rhodospirillum rubrum Dinitrogenase Reductase adp ribosyl transferase Dinitrogenase Reductase activating glycohydrolase regulatory system in klebsiella pneumoniae
    Journal of Bacteriology, 2001
    Co-Authors: Yaoping Zhang, Paul W Ludden, Edward L Pohlmann, Cale M Halbleib, Gary P Roberts
    Abstract:

    Reversible ADP-ribosylation of Dinitrogenase Reductase, catalyzed by the Dinitrogenase Reductase ADP-ribosyl transferase–Dinitrogenase Reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.

Yaoping Zhang - One of the best experts on this subject based on the ideXlab platform.

  • © 1991 Kluwer Academic Publishers. Printed in the Netherlands. PLSO NL19 Control of nitrogenase in Azospirillum sp.
    2013
    Co-Authors: Robert H. Burris, Yaoping Zhang, Anton Hartmann, Key Words
    Abstract:

    Many N2-fixing organisms can turn off nitrogenase activity in the presence of NH] and turn it on again when the NH 4 is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of Dinitrogenase Reductase by Dinitrogenase Reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH~- is exhausted, by Dinitrogenase Reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genus Azospirillum possess the DRAT and DRAG systems (A. lip@rum and A. brasilense), whereas A. amazonense in the same genus lacks DRAT and DRAG. A. amazonense responds to NH 4 but does not exhibit modification of Dinitrogenase Reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them into A. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function in Acetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N 2 at a pH as low as 3.0. A. diazotrophicus does show a rather sluggish response to NH 4. A level of about 10/zM NH 4 is required to 'switch off ' the system. The response to NH 4 is influenced by the dissolved oxygen concentration (DOC) as has been reported for Azospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa 0 2 seems optimal for the response in A. diazotrophicus

  • identification of rhodospirillum rubrum glnb variants that are altered in their ability to interact with different targets in response to nitrogen status signals
    Journal of Bacteriology, 2006
    Co-Authors: Yu Zhu, Yaoping Zhang, Mary Conrad, Gary P Roberts
    Abstract:

    In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (Dinitrogenase Reductase ADP-ribosyl transferase)-DRAG (Dinitrogenase Reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

  • characterization of altered regulation variants of Dinitrogenase Reductase activating glycohydrolase from rhodospirillum rubrum
    FEBS Letters, 2004
    Co-Authors: Kitai Kim, Yaoping Zhang, Gary P Roberts
    Abstract:

    In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of Dinitrogenase Reductase by Dinitrogenase Reductase ADP-ribosyltransferase (DRAT) and Dinitrogenase Reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.

  • functional characterization of three glnb homologs in the photosynthetic bacterium rhodospirillum rubrum roles in sensing ammonium and energy status
    Journal of Bacteriology, 2001
    Co-Authors: Yaoping Zhang, Paul W Ludden, Edward L Pohlmann, Gary P Roberts
    Abstract:

    The GlnB (PII) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other PII homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of Dinitrogenase Reductase catalyzed by the Dinitrogenase Reductase ADP-ribosyl transferase (DRAT)/Dinitrogenase Reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

  • effects of specific amino acid substitutions on activities of Dinitrogenase Reductase activating glycohydrolase from rhodospirillum rubrum
    Journal of Bacteriology, 2001
    Co-Authors: Babu S Antharavally, Yaoping Zhang, Gary P Roberts, Russell R Poyner, Paul W Ludden
    Abstract:

    Site-directed mutagenesis of the draG gene was used to generate altered forms of Dinitrogenase Reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

Luciano F. Huergo - One of the best experts on this subject based on the ideXlab platform.

  • the nitrogenase regulatory enzyme Dinitrogenase Reductase adp ribosyltransferase drat is activated by direct interaction with the signal transduction protein glnb
    Journal of Bacteriology, 2013
    Co-Authors: Fábio O. Pedrosa, Vivian R. Moure, Karamatullah B Danyal, Shannon A Wendroth, Zhiyong Yang, Marcelo Mullersantos, Marcelo Scarduelli, Edileusa C M Gerhardt, Luciano F. Huergo
    Abstract:

    ABSTRACT Fe protein (Dinitrogenase Reductase) activity is reversibly inactivated by Dinitrogenase Reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the Dinitrogenase Reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling PII proteins. In A. brasilense, two PII proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with PII proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The PII effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated PII proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar Km value for NAD+. Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

  • crystal structure of Dinitrogenase Reductase activating glycohydrolase drag reveals conservation in the adp ribosylhydrolase fold and specific features in the adp ribose binding pocket
    Journal of Molecular Biology, 2009
    Co-Authors: Luciano F. Huergo, Fábio O. Pedrosa, Mike Merrick, Antonietta Gasperina, Fritz K Winkler
    Abstract:

    Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (Dinitrogenase Reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 A. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-alpha-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.

  • in vitro interactions between the pii proteins and the nitrogenase regulatory enzymes Dinitrogenase Reductase adp ribosyltransferase drat and Dinitrogenase Reductase activating glycohydrolase drag in azospirillum brasilense
    Journal of Biological Chemistry, 2009
    Co-Authors: Luciano F. Huergo, Maria B. R. Steffens, Fábio O. Pedrosa, Leda S. Chubatsu, Mike Merrick, Rose A Monteiro, Emanuel M. Souza
    Abstract:

    The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of PII uridylylation status and the PII effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when PII proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the PII ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity.

  • interactions between pii proteins and the nitrogenase regulatory enzymes drat and drag in azospirillum brasilense
    FEBS Letters, 2006
    Co-Authors: Luciano F. Huergo, Emanuel M. Souza, Maria B. R. Steffens, Fábio O. Pedrosa, Leda S. Chubatsu, Mike Merrick
    Abstract:

    In Azospirillum brasilense ADP-ribosylation of Dinitrogenase Reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by Dinitrogenase Reductase ADP-ribosyltransferase (DraT) and reversed by Dinitrogenase Reductase glycohydrolase (DraG). The PII proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation.

  • adp ribosylation of Dinitrogenase Reductase in azospirillum brasilense is regulated by amtb dependent membrane sequestration of drag
    Molecular Microbiology, 2006
    Co-Authors: Luciano F. Huergo, Emanuel M. Souza, Maria B. R. Steffens, Fábio O. Pedrosa, Leda S. Chubatsu, Mariana S Araujo, Mike Merrick
    Abstract:

    Summary Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of Dinitrogenase Reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This pro- cess is mediated by Dinitrogenase Reductase ADP- ribosyltransferase (DraT) and reversed by dinitroge- nase Reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P II proteins (GlnB and GlnZ), the ammonia channel pro- tein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP- ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane asso- ciation of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane seques- tration of a protein other than a P II family member. They also suggest a model for control of ADP-ribosy- lation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.

Peter Boger - One of the best experts on this subject based on the ideXlab platform.

  • proteolytic degradation of Dinitrogenase Reductase from anabaena variabilis atcc 29413 as a consequence of atp depletion and impact of oxygen
    Journal of Bacteriology, 1996
    Co-Authors: Jorg Durner, Ines Bohm, Oliver C Knorzer, Peter Boger
    Abstract:

    Both components of nitrogenase, Dinitrogenase and Dinitrogenase Reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of Dinitrogenase Reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of Dinitrogenase Reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin- and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of Dinitrogenase Reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of Dinitrogenase Reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation.

  • induction and modification of Dinitrogenase Reductase in the unicellular cyanobacterium synechocystis bo 8402
    Archives of Microbiology, 1992
    Co-Authors: Susanne Brass, Anneliese Ernst, Peter Boger
    Abstract:

    Oxygen is an important regulatory factor of nitrogenase induced in a unicellular cyanobacterium, Synechocystis BO 8402, during nitrogen starvation. Synthesis of the enzyme is limited by the efficiency of the cells to remove oxygen by respiration, supported by hydrogenases and, in the light, by inhibition of photosynthesis. With a polyclonal antibody against Dinitrogenase Reductase (the Fe protein of nitrogenase) a single polypeptide is detected, indicative of an active dimeric enzyme in dense cell suspensions. Inhibition of nitrogenase by addition of oxygen is accompanied by the appearance of a second polypeptide of the Fe protein having a 1.5 kDa higher molecular weight. This disappears upon removal of oxygen from the gas phase while nitrogenase activity is restored. No protein synthesis is required indicating that a fraction of the existing polypeptides is reversibly modified in response to oxygen. After induction of nitrogenase activity in dilute culture suspensions, both forms of the Fe-protein are found in variable amounts possibly due to oxygen contamination during the experiment.

  • in vitro activation of Dinitrogenase Reductase from the cyanobacterium anabaena variabilis atcc 29413
    Journal of Bacteriology, 1992
    Co-Authors: Ines Bohm, Anneliese Ernst, Andrea Halbherr, S Smaglinski, Peter Boger
    Abstract:

    Abstract Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Boger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) Dinitrogenase Reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.

  • modification and in vitro activation of Dinitrogenase Reductase from anabaena variabilis
    1991
    Co-Authors: Ines Bohm, Andrea Halbherr, Peter Boger
    Abstract:

    Cell-free preparations of inactivated nitrogenase from Anabaena variabilis were reactivated by a membrane fraction from A. variabilis and Rhodospirillum rubrum, known to contain the Dinitrogenase reduc-tase activating glycohydrolase (DRAG). These results imply that the modifying group at the cyanobac-terial Fe-protein (Dinitrogenase Reductase) is ADP-ribose as originally described for R. rubrum. The reactivating activity could only be found in membranes of heterocysts and when an active nitrogenase was present. A nitrogenase inactivating activity was observed in the supernatant from A. variabilis extracts, which was strongest in cells with highly regulated nitrogenase activity.