ADP-ribosylation is an important mechanism of regulation of enzyme activity in eucaryotes and procaryotes. This protein modification plays an important role in a number of human diseases. The dinitrogenase reductase protein of the nitrogenase enzyme complex from the photosynthetic bacterium Rhodospirillum rubrum is regulated by reversible ADP-ribosylation of arg-101. ADP-ribosylation occurs in vivo in response to darkness or ammonium and is catalyzed by the dinitrogenase reductase ADP-ribosyltransferase (DRAT). The Mn-dependent removal of ADP-ribose is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG). Both DRAT and DRAG activities are regulated in vivo and the goal of this proposal is to determine the mechanism(s) of in vivo regulation of these two enzymes. Purified DRAT and DRAG are fully active, and thus the regulation is thought to occur by inhibition of their activities. A model that involves regulation of DRAT and DRAG activities by formation of protein:protein complexes that contain DRAT:DRAG or DRAT:dinitrogenase reductase has been developed.
The specific aims of this proposal are to characterize the complexes of DRAT with dinitrogenase reductase and of DRAT with DRAG. Effectors that control the formation of complexes will be sought and identified. Evidence for the DRAT:dinitrogenase reductase complex has been obtained using chemical crosslinking agent EDC. Formation of the DRAT:dinitrogenase reductase complex requires the presence of NAD and is stimulated by ADP. Preliminary evidence also suggests the existence of the DRAT:DRAG complex. The model calls for small molecule effectors to be involved in these complexes and a goal of the proposal is to isolate effectors, identify them and demonstrate changes in concentration of the effectors in concert with in vivo ADP-ribosylation of dinitrogenase reductase. Physiological experiments have implicated the product of an orf downstream of the dratTG genes in the regulation of DRAT and DRAG and the purification of the orf product from an overexpressing strain is a goal of this project. Particular attention will be paid to the ability of the orf product to participate in any protein complex. The Mn-binding site of DRAG will be characterized and the role of Mn in stimulating/inhibiting any complex of DRAG with other proteins will be investigated. The hypothesis that Mn constitutes a binuclear cluster in DRAG will be tested.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054910-03
Application #
2734820
Study Section
Special Emphasis Panel (ZRG5-BM-1 (01))
Project Start
1996-07-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
3
Fiscal Year
1998
Total Cost
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
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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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