The goal of this project is to understand the regulation of dinitrogenase reductase by reversible ADP-ribosylation. The dinitrogenase reductase protein of the nitrogenase enzyme in Rhodospirillum rubrum is inactivated by ADP{-ribosylation of arg101 in response to ammonium or darkness. DRAT is the ADP-ribosyltransferase responsible for inactivation and DRAG is the glycohydrolase that activates dinitrogenase reductase by removing ADP-ribose. Both DRAT and DRAG are independently regulated in the cell, and the overall goal of this project is to understand the signal transduction pathways that regulate DRAT and DRAG. Structural information for both DRAT and DRAG will be obtained by crystallography, sequence modeling and site-specific chemical modification of the enzymes. This information will be used to direct mutagenesis of genes encoding DRAT and DRAG, and the mutant forms of DRAT, DRAG, and dinitrogenase reductase will be tested for their activities and regulation. NAD serves as the ADP-ribose donor, and the site of NAD binding to DRAT and dinitrogenase reductase will be established. The hypothesis that occupancy of the NAD binding sites regulates DRAT activity will be tested in vitro and in vivo; UV photoaffinity techniques will be developed for the in vivo tests. Binding of NAD to DRAT and to dinitrogenase reductase will be quantified. Effectors of DRAT and DRAG activities will be sought using in vitro assays. Mutant forms of DRAT and DRAG that are unregulated """"""""always active"""""""" will serve as controls in these assays. Effectors will be isolated and identified. ADP-ribosylation is increasingly recognized as an important mechanism of regulation in eucaryotes and procaryotes. A number of pathogenic bacteria produce toxins that ADP-ribosylate crucial protein targets in their eucaryotic hosts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054910-07
Application #
6519779
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Ikeda, Richard A
Project Start
1996-07-01
Project End
2002-12-31
Budget Start
2002-07-01
Budget End
2002-12-31
Support Year
7
Fiscal Year
2002
Total Cost
$41,007
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Ponnuraj, Rajdevi K; Rubio, Luis M; Grunwald, Sandra K et al. (2005) NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Lett 579:5751-8
Ma, Y; Ludden, P W (2001) Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage. J Bacteriol 183:250-6
Zhang, Y; Pohlmann, E L; Ludden, P W et al. (2001) Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J Bacteriol 183:6159-68
Zhang, Y; Pohlmann, E L; Ludden, P W et al. (2000) Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 182:983-92
Halbleib, C M; Ludden, P W (2000) Regulation of biological nitrogen fixation. J Nutr 130:1081-4
Halbleib, C M; Zhang, Y; Roberts, G P et al. (2000) Effects of perturbations of the nitrogenase electron transfer chain on reversible ADP-ribosylation of nitrogenase Fe protein in Klebsiella pneumoniae strains bearing the Rhodospirillum rubrum dra operon. J Bacteriol 182:3681-7
Halbleib, C M; Zhang, Y; Ludden, P W (2000) Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein. J Biol Chem 275:3493-500
Grunwald, S K; Ryle, M J; Lanzilotta, W N et al. (2000) ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase. J Bacteriol 182:2597-603
Halbleib, C M; Ludden, P W (1999) Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes. Arch Microbiol 172:51-8
Grunwald, S K; Ludden, P W (1997) NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. J Bacteriol 179:3277-83

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