EPR Spectral Evidence for a Binuclear Mn(II) Center in Dinitrogenase Reductase-Activating Glycohydrolase from<i>Rhodospirillum rubrum</i>

Antharavally, Babu S.; Poyner, and Russell R.; Ludden, Paul W.

doi:10.1021/ja9818912

Cited by 20 publications

(25 citation statements)

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“…The active site of the monomeric holoenzyme contains a dinuclear manganese site (21). One of the metal positions (designated Mn A ) appears fully occupied while the other (designated Mn B ) was refined at half occupancy.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

“…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.…”

mentioning

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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“…It is known that calcium is not required for the DRAG regulation in vivo or in vitro [30], although an e¥ux of calcium has been reported to coincide with the ADPribosylation of dinitrogenase reductase and therefore with the inactivation of DRAG [31]. However, some divalent cations clearly play an important role in the activity of DRAG, since a binuclear Mn 2þ center has been demonstrated in the puri¢ed DRAG [1,32] and Fe 2þ is also able to support DRAG activity, although Mg 2þ does not. DRAG and DRAG-N100K were both similarly inhibited by the presence of 0.5 mM CaCl 2 in terms of their ability to remove the ADP-ribose group from dinitrogenase reductase in the presence of 25 mM MgCl 2 and 0.5 mM of MnCl 2 (data not shown).…”

Section: +mentioning

confidence: 99%

Characterization of altered regulation variants of dinitrogenase reductase‐activating glycohydrolase from Rhodospirillum rubrum

2004

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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 di¡erent mutants were found and the DRAG variants displayed di¡erent biochemical properties including an altered a⁄nity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.

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“…In R. rubrum the mutation of draB caused a decrease in DRAG activity and protein level, and the total DRAG activity in draB mutant is approximately 35% of that of the wild type (30). Recently, DRAG has been shown to have a binuclear manganese center when it is treated with Mn 2ϩ (1). Based on its sequence similarity to other metal processing proteins, therefore, DRAB might be involved in the processing of a metal center in DRAG.…”

Section: Vol 183 2001mentioning

confidence: 99%

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

et al. 2001

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Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADPribosyl 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 NH 4 ؉ 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 NH 4 ؉ 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.Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonium, is catalyzed by the nitrogenase complex, which consists of two proteins: dinitrogenase (or MoFe protein) and dinitrogenase reductase (or Fe protein) (7). It is a very energy-demanding process and is thus tightly regulated at both transcriptional and posttranslational levels.Transcriptional regulation of the nif genes has been found in all studied nitrogen-fixing bacteria and is best characterized in Klebsiella pneumoniae, a free-living nitrogen-fixing bacterium, where it involves the general nitrogen regulation (ntr) system (36). Analysis of the ntr regulatory system in K. pneumoniae and Escherichia coli (36, 39) has shown that it controls the transcription of many genes involved in nitrogen fixation and assimilation, such as glnA (encoding glutamine synthetase [GS]) and nifA (encoding the transcriptional activator for the other nif genes). The ntr system involves a number of gene products, including those of glnD, ntrA, ntrB, ntrC, and glnB, glnD encodes a bifunctional, uridylyltransferase-uridylyl-removing enzyme (UTase-UR) that is believed to be the sensor of the intracellular concentration of glutamine in the cell. UTase-UR reversibly controls the activity of the P II protein (the gene product of glnB) by uridylylation or deuridylylation. P II is responsible for sensing ␣-ketoglutarate (␣-KG) in E. coli (24), and it controls NtrB (NRII) activity. NtrB and NtrC (the gene products of ntrB and ntrC) belong to the family of twocomponent regulators. NtrB is a histidine kinase that phosphorylates NtrC (NRI) under nitrogen-limiting conditions and also can act as a phosphatase to dephosphorylate NtrC unde...

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EPR Spectral Evidence for a Binuclear Mn(II) Center in Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

Cited by 20 publications

References 22 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

Characterization of altered regulation variants of dinitrogenase reductase‐activating glycohydrolase from Rhodospirillum rubrum

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

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EPR Spectral Evidence for a Binuclear Mn(II) Center in Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

Cited by 20 publications

References 22 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

Characterization of altered regulation variants of dinitrogenase reductase‐activating glycohydrolase from Rhodospirillum rubrum

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

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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