Stabilization and partial characterization of the activating enzyme for dinitrogenase reductase (Fe protein) from rhodospirillum rubrum

Dinitrogenase reductase-activating glycohydrolase (DRAG) is responsible for removing the ADP-ribose moiety from post-translationally inactivated nitrogenase of Rhodospirillum rubrum. Using DRAG purified from an overexpressing strain (UR276), further properties of this enzyme were studied, including its u.v.-visible and fluorescence spectra and its stability in air. DRAG appears to require no covalently bound inorganic cofactors for its activity or regulation. Previously, purified DRAG was found to be rapidly inactivated in air. The air-catalysed lability originated with the presence of sodium dithionite and Mn2+ throughout the purification of the enzyme. This lability can be mimicked using H2O2, which is known to oxidatively inactivate proteins containing bivalent metals. Implications for the regulation of nitrogenase are discussed with respect to the lack of sensitivity to air of the regulatory enzyme, DRAG.

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Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen-fixing conditions

Biochemical Journal ◽

10.1042/bj2790155 ◽

1991 ◽

Vol 279 (1) ◽

pp. 155-158 ◽

Cited By ~ 37

Author(s):

E Brostedt ◽

S Nordlund

Keyword(s):

Klebsiella Pneumoniae ◽

Rhodospirillum Rubrum ◽

Nitrogenase Activity ◽

Photosynthetic Bacterium ◽

Molecular Properties ◽

Partial Characterization ◽

Nitrogen Fixing ◽

Pyruvate Oxidoreductase

A pyruvate oxidoreductase with the capacity to support pyruvate-dependent nitrogenase activity in vitro has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The enzyme requires CoA for activity and is irreversibly inactivated by oxygen. The molecular properties and Km values for the substrates have been studied. In supporting nitrogenase activity addition of ferredoxin is required. Overall the enzyme is similar to the nif-specific pyruvate: flavodoxin oxidoreductase purified from Klebsiella pneumoniae.

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Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes

Archives of Microbiology ◽

10.1007/s002030050739 ◽

1999 ◽

Vol 172 (1) ◽

pp. 51-58 ◽

Cited By ~ 9

Author(s):

Cale M. Halbleib ◽

P. W. Ludden

Keyword(s):

Rhodospirillum Rubrum ◽

Bacterial Membranes ◽

Dinitrogenase Reductase

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The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

Journal of Bacteriology ◽

10.1128/jb.01517-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 279-286 ◽

Cited By ~ 14

Author(s):

Vivian R. Moure ◽

Karamatullah Danyal ◽

Zhi-Yong Yang ◽

Shannon Wendroth ◽

Marcelo Müller-Santos ◽

...

Keyword(s):

Rhodospirillum Rubrum ◽

Metal Cluster ◽

Reductase Activity ◽

Direct Interaction ◽

Ammonium Concentration ◽

Content Type ◽

Fe Protein ◽

Dinitrogenase Reductase ◽

Adp Ribosyltransferase

ABSTRACTFe 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 inAzospirillum brasilense,Rhodospirillum rubrum, andRhodobacter 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 PIIproteins. InA. brasilense, two PIIproteins, GlnB and GlnZ, were identified. We used Fe protein fromAzotobacter vinelandiias the substrate to assess the activity ofA. brasilenseDraTin vitrocomplexed or not with PIIproteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The PIIeffector 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 PIIproteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that theA. brasilenseDraT-GlnB complex was at least 18-fold more efficient than DraT purified fromR. rubrum, but with a similarKmvalue 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.

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Mutagenesis and Functional Characterization of theglnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

Journal of Bacteriology ◽

10.1128/jb.182.4.983-992.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 983-992 ◽

Cited By ~ 49

Author(s):

Yaoping Zhang ◽

Edward L. Pohlmann ◽

Paul W. Ludden ◽

Gary P. Roberts

Keyword(s):

Nitrogen Fixation ◽

Rhodospirillum Rubrum ◽

Nitrogenase Activity ◽

Functional Characterization ◽

Photosynthetic Bacterium ◽

Wild Type ◽

Adp Ribosylation ◽

Dinitrogenase Reductase ◽

Adp Ribosyltransferase

ABSTRACT Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation ofnif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization ofglnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for PII) were constructed. While PII-Y51F showed a lower nitrogenase activity than that of wild type, a PIIdeletion mutant showed very little nif expression. This effect of PII on nif expression is apparently the result of a requirement of PII for NifA activation, whose activity is regulated by NH4 + in R. rubrum. The modification of glutamine synthetase (GS) in theseglnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of PII might exist inR. rubrum and regulate the modification of GS. PII also appears to be involved in the regulation of DRAT activity, since an altered response to NH4 + was found in a mutant expressing PII-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH4 + and darkness treatments.

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