REVERSIBLE ADP-RIBOSYLATION OF NITROGENASE

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DESCRIPTION (Adapted from the applicant's abstract): 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.