Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum

Nordlund, Stefan; Eriksson, Urban; Baltscheffsky, Herrick

doi:10.1016/0005-2728(77)90201-8

Cited by 97 publications

(54 citation statements)

References 14 publications

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“…In R. rubrum DraG has been shown to reversibly associate to the membrane as part of a regulatory mechanism (15)(16)(17). DraG has long served as the model enzyme for the de-ADP-ribosylation process in numerous biochemical and functional studies (14,(18)(19)(20)(21), but structural and mechanistic data have been lacking.…”

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confidence: 99%

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Berthold

Wang²,

Nordlund³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.binuclear dinuclear ͉ bioinorganic chemistry ͉ covalent modification ͉ nitrogen fixation ͉ posttranslational modifications P osttranslational modification of proteins by the attachment of chemical groups is used by cells from all kingdoms of life and occurs in several forms. Through the reversible covalent modification of specific amino acid side chains, enzyme activity, or protein function can be switched on and off as a rapid response to environmental stimuli. ADP-ribosylation is a posttranslational modification existing in two distinct forms, mono-and poly-ADPribosylation, resulting from the attachment of a single ADP-ribose moiety or a polymeric ADP-ribose chain structure, respectively, to the protein (1, 2).Mono-ADP-ribosylation was first discovered as a process used in the action of several bacterial toxins, including cholera-, diphtheria-, and pertussis toxin (3, 4). The pathogenesis of these diseases involves ADP-ribosylation of specific human proteins, catalyzed by the toxins, thereby affecting the activity of the modified proteins. In human physiology, mono-ADP-ribosylation is found in the regulation of cell-cell and cell-matrix interactions, as well as part of immune function (5, 6). Poly-ADP-ribosylation is involved in many fundamental processes such as DNA repair, transcription and cell differentiation, and is also implicated in carcinogenesis (6-8). Poly-ADP-ribosylation inhibitors are currently in phase II clinical trials for treatment of breast and ovarian cancer (9).Mono-ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and is specifically targeted to amino acids residues such as Arg, Asn, Glu, Asp, Cys, diphthamide, or phosphoserine. The reaction is stereospecific, using ␤-NAD ϩ as a substrate, forming the ␣-anomer of the ADP-ribosylated amino acid and releasing nicotinamide (10) (Fig. 1). PolyADP-ribosylation i...

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confidence: 99%

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Berthold

Wang²,

Nordlund³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The molecular basis for the inhibition is the ADP-ribosylation of dinitrogenase reductase (the iron protein) at a specific arginyl residue (26). Enzymes that attach (14) and remove (15,23,29) the ADP-ribosyl residue have been isolated from R. rubrum and have been found under a variety of culturing conditions (14,33).Factors that lead to the loss of activity in vivo include ammonia (10, 22, 31), glutamine (22), darkness (12, 40), and phenazine methosulfate (PMS) (12,25). Cells grown on N2 or glutamate as the N source are competent to switch-off nitrogenase activity (switch-off is the term given by Zumft and Castillo [44] to the reversible, in vivo loss of nitrogenase activity), whereas cells grown on limiting-ammonia (i.e., N-starved) medium are incapable of switch-off (38).…”

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confidence: 99%

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Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity

Kanemoto¹,

Ludden²

1987

J Bacteriol

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The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions. Nitrogenase activity in the phototrophic bacterium Rhodospirillum rubrum is regulated at the level of expression and by posttranslational modification (8,10,12,28,33, and references therein). Oxygen and fixed sources of N such as glutamine and ammonia lead to the repression of nitrogenase; when glutamate is provided as the N source, nitrogenase is expressed.The inhibition of nitrogenase activity in vivo by ammonia was first noted by Gest et al. (8). The molecular basis for the inhibition is the ADP-ribosylation of dinitrogenase reductase (the iron protein) at a specific arginyl residue (26). Enzymes that attach (14) and remove (15,23,29) the ADP-ribosyl residue have been isolated from R. rubrum and have been found under a variety of culturing conditions (14,33).Factors that lead to the loss of activity in vivo include ammonia (10, 22, 31), glutamine (22), darkness (12, 40), and phenazine methosulfate (PMS) (12,25). Cells grown on N2 or glutamate as the N source are competent to switch-off nitrogenase activity (switch-off is the term given by Zumft and Castillo [44] to the reversible, in vivo loss of nitrogenase activity), whereas cells grown on limiting-ammonia (i.e., N-starved) medium are incapable of switch-off (38). Nitrogen assimilation in R. rubrum under nif-derepressed conditions is via glutamine synthetase (GS) and glutamate synthase (GOGAT) (3,21,32,36), and ...

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“…The supernatants of R rubrum were partially purified by passage through a DEAE-cellulose column (1.5 by 11 cm) as described by Nordlund et al (18).…”

Section: Methodsmentioning

confidence: 99%

“…This reaction requires Mg-ATP and Me2 . The enzyme was found to be membrane associated (13) and extremely oxygen labile (14,18 and partially purified dinitrogenase reductase from both organisms, thereby yielding active nitrogenase (measured as acetylene formation). Furthermore, the activities of membrane fractions of different cell types and from filaments of different physiological states fromA.…”

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In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413)

et al. 1992

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Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Böger, 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.

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Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum

Cited by 97 publications

References 14 publications

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity

In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413)

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