title: NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii.
abstract: Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by ADP-ribosylation of a specific arginine residue of dinitrogenase reductase based on the cellular nitrogen or energy status. In this paper, we have investigated the ability of nicotinamide adenine dinucleotide, NAD (the physiological ADP-ribose donor), and its analogs to support covalent modification of dinitrogenase reductase in vitro. R. rubrum dinitrogenase reductase can be modified by DRAT in the presence of 2 mM NAD, but not with 2 mM nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide phosphate (NADP). We also found that the apo- and the all-ferrous forms of R. rubrum dinitrogenase reductase are not substrates for covalent modification. In contrast, Azotobacter vinelandii dinitrogenase reductase can be modified by the dinitrogenase reductase ADP-ribosyl transferase (DRAT) in vitro in the presence of either 2 mM NAD, NMN or NADP as nucleotide donors. We found that: (1) a simple ribose sugar in the modification site of the A. vinelandii dinitrogenase reductase is sufficient to inactivate the enzyme, (2) phosphoADP-ribose is the modifying unit in the NADP-modified enzyme, and (3) the NMN-modified enzyme carries two ribose-phosphate units in one modification site. This is the first report of NADP- or NMN-dependent modification of a target protein by an ADP-ribosyl transferase.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
authors.forename: Rajdevi K
authors.initials: RK
authors.lastname: Ponnuraj
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
authors.forename: Sandra K
authors.initials: SK
authors.lastname: Grunwald
authors.forename: Paul W
authors.initials: PW
authors.lastname: Ludden
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D016948:Azotobacter vinelandii / Q000201:enzymology*; D017037:Dinitrogenase Reductase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000494:pharmacology; D009249:NADP / Q000737:chemistry / Q000494:pharmacology; D009537:Nicotinamide Mononucleotide / Q000737:chemistry / Q000494:pharmacology; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012265:Ribonucleotides / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D017037:Dinitrogenase Reductase; D012265:Ribonucleotides; D009243:NAD; D009537:Nicotinamide Mononucleotide; D000246:Adenosine Diphosphate Ribose; D009249:NADP