NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum

Grunwald, Sandra K.; Ludden, Paul W.

doi:10.1128/jb.179.10.3277-3283.1997

Cited by 14 publications

(24 citation statements)

References 40 publications

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“…The regulation of DRAT activity through its accessibility to NAD would be a very attractive hypothesis, based on the fact that NAD is required for the interaction between DRAT and dinitrogenase reductase in vitro (Grunwald & Ludden, 1997) and on reports showing that exogenous NAD can perturb the modification of dinitrogenase reductase (Nore! n et al, 1997 ;Soliman & Nordlund, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The purification of dinitrogenase reductases and DRAT has been reported previously (Grunwald & Ludden, 1997 ;Lowery & Ludden, 1988). On the Affi-gel column, DRAT-K103E showed substantially weaker binding than did DRAT-WT, eluting with 150 mM NaCl and 1 mM ADP.…”

Section: Methodsmentioning

confidence: 99%

“…NAD labelled with $#P was used as a donor of ADP-ribose for the in vitro DRAT activity assay, as described previously (Grunwald et al, 1995 ;Grunwald & Ludden, 1997 ;Lowery & Ludden, 1988). DRAT activity is expressed as nmol ADP-ribose transferred per min per mg protein.…”

Section: Methodsmentioning

confidence: 99%

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Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum

et al. 2001

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In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of NH M 4 or removal from light, resulting in a decrease in nitrogenase activity. DRAT is itself subject to post-translational regulation ; to investigate the mechanism for the regulation of DRAT activity, random PCR mutagenesis of draT (encoding DRAT) was performed and mutants with altered DRAT regulation were screened. Two mutants (with substitutions of K103E and N248D) were obtained in which DRAT showed activity under conditions where wild-type DRAT (DRAT-WT) did not. These mutants showed lower nitrogenase activity and a higher degree of ADP-ribosylation of dinitrogenase reductase under N 2 -fixing conditions than was seen in a wild-type control strain. DRAT-K103E was overexpressed and purified. DRAT-K103E displayed a much weaker affinity for an Affi-gel Blue matrix than did DRAT-WT, suggestive of a fairly striking biochemical change. However, there was no significant difference in kinetic constants, such as K m for NAD and V max , between DRAT-K103E and DRAT-WT. Like DRAT-WT, DRAT-K103E also modified reduced dinitrogenase reductase poorly. The biochemical properties of these variants are rationalized with respect to their behaviour in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum

et al. 2001

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“…The cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) can cross-link dinitrogenase reductase and DRAT in the presence of NAD. The crystal structure of A. vinelandii dinitrogenase reductase shows that the two arginine 100 side chains are located at the same surface as the metal cluster (15), which is the region speculated to interact with DRAT (17). The effects of substitutions at several other residues have been reported (18).…”

mentioning

confidence: 99%

“…A chemical cross-linking method has been used to study the interaction between these two proteins (17). The cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) can cross-link dinitrogenase reductase and DRAT in the presence of NAD.…”

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confidence: 99%

Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

Ludden

2001

J Bacteriol

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Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32 P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14 C]NAD individually upon UV irradiation, but most 14 C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14 C]NAD suggested that Arg 101 is not absolutely required for NAD binding.Various procaryotic microorganisms are capable of fixing nitrogen. The essential enzyme in this reaction is the nitrogenase complex, which catalyzes the reduction of nitrogen to ammonium (4). This complex is composed of two separable metalloenzymes designated dinitrogenase and dinitrogenase reductase. Dinitrogenase reductase transfers electrons from an electron donor to dinitrogenase, which then can catalyze the reduction of nitrogen to ammonium. Because nitrogen fixation is an energy-demanding process, it is tightly regulated in response to a number of environmental factors. In addition to transcriptional regulation, a posttranslational nitrogenase regulatory mechanism also exists in certain organisms, such as Rhodospirillum rubrum (33,35). R. rubrum is a free-living, purple, nonsulfur photosynthetic bacterium that fixes nitrogen (20). The activity of its nitrogenase is regulated by the reversible ADP-ribosylation of the arginine 101 of the dinitrogenase reductase (31, 42). Dinitrogenase reductase ADP-ribosyltransferase (DRAT) can transfer ADP-ribose from NAD to dinitrogenase reductase in response to darkness or the presence of ammonium. This ADP-ribosylation ...

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Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

Nordlund

Ludden

Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria

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The photosynthetic bacteria are remarkable for their range of metabolic capabilities and, as a result, the ability to survive under a wide range of environmental conditions, including nitrogen limitation. The photosynthetic bacteria are members of the alpha subdivision of the proteobacteria (Imhoff et al., 1984;Woese et al., 1984) and, with a single known exception, the wild-type strains of all purple sulfur bacteria and all purple non-sulfur bacteria are capable of N 2 fixation (Madigan et al., 1984). Most of the work that will be described in this chapter has been performed on a few species of purple, non-sulfur bacteria, including Rhodospirillum rubrum and Rhodobacter capsulatus. This chapter will focus on the biochemistry and physiology of N 2 fixation by photosynthetic bacteria and the regulation of nitrogenase activity by reversible ADP-ribosylation of the dinitrogenase reductase (Fe protein). ADP-ribosylation has also been found in Azospirillum and other genera and, where relevant, that work will be included. Other chapters in this series will cover the genes of the nif, anf and rnf regulons of the photosynthetic bacteria and the regulation of the expression of those genes. Although this chapter will discuss aspects of the nitrogenase enzyme, the mechanistic and structural details of nitrogenase will be covered in more detail in Volume 1 of this series.

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NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum

Cited by 14 publications

References 40 publications

Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum

Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum

Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

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