Posttranslational modification of nitrogenase

Abstract

In the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulatus posttranslational regulation of nitrogenase is due to ADP-ribosylation of the Fe-protein, the dinitrogenase reductase [Burris, R. H, (1991) J. Biol. Chem. 266,. This mechanism has been assumed to be responsible for nitrogenase modification in a variety of organisms. In the present study, we examined whether ADP-ribosylation holds true for the filamentous cyanobacterium Anabaena vuriabilis. Genes coding for the nitrogenase-modifying enzymes dinitrogenase reductase-activating glycohydrolase (DRAG) and dinitrogenase reductase ADP-ribosyl transferase (DRAT) from R. rubrum have been subcloned and overexpressed in Escherichia coli. After isolation under anaerobic conditions, both proteins were functional as determined by in-vitro assays using nitrogenase from R. rubrum as substrate. In contrast to the R. rubrum enzyme, nitrogenase from A. variabilis was not affected by DRAG or DRAT. Neither could inactive nitrogenase be restored by DRAG, nor nitrogenase activity suppressed by DRAT. Using specific antibodies against arginine-bound ADP-ribose The reduction of dinitrogen to ammonia during nitrogen fixation is catalyzed by a multi-enzyme complex consisting of dinitrogenase (MoFe-protein) and dinitrogenase reductase (usually referred to as Fe-protein; see [ l ] for a review). In the cyanobacterium Anabaena variabilis this enzyme complex is located in the heterocysts [2], cells with a thick, laminated envelope that functions as a diffusion barrier for gases. High respiration rates and lack of photosynthetic oxygen evolution enables these cells to fix nitrogen aerobically. Heterocysts do not catalyze CO, fixation and are dependent on adjacent photosynthetic vegetative cells for supply of fixed carbon. Since nitrogen fixation is an energy-demanding process (20-30 molecules ATP are required for the fixation of one molecule N2), regulation of nitrogenase requires both transcriptional and posttranslational events [ 11. Expression of nitrogenase in cyanobacteria depends on combined nitrogen, oxygen and cell differentiation [3, 41. In addition to transcriptional control, enzyme activity is influenced by external factors which limit the C supply of heterocysts 151 and by excess ammonium chloride at alkaline pH 161. These treatments cause inactivation of the enzyme ('ammonia switch-off'). In a number of diazotrophs, nitrogenase activity is regulated posttranslationally by reversible covalent ADP-ribosylation of the Fe-protein, as was most thoroughly investigated with the photosynthetic bacterium Rhodospirillum rubrum 17, 81. The site of ADP-ribosylation is a specific arginine residue (ArglOl or ArglOO in case of Azotobacter vinelandii) in one of the two identical subunits of the Fe-protein, thereby blocking an effective complex formation between the Fe-protein and . The process of addition and cleavage of the modifying group is catalyzed by specific enzymes. Modification by ADP-ribosylation (inactivation) is performed by dinitrogenase reductase AD...

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase

“…(A) The whole-cell nitrogenase activity was measured in duplicate at the indicated times. The culture was exposed to two cycles of 40 min of darkness (Dk) and 20 min of reillumination (Lt), followed by addition of NH 4 Cl to a final concentration of 2 mM. (B) Immunoblot analysis of dinitrogenase reductase from samples withdrawn from the culture at the indicated times and culture conditions and processed as described in Materials and Methods.…”

“…Growth of overexpression strains and NH 4 Cl or darkness treatment. Growth conditions were similar to those used by Kanemoto and Ludden (15).…”

“…The light source was then removed, and the nitrogenase activity of culture samples was monitored. At the end of several light-dark cycles, a degassed solution of NH 4 Cl was added to the culture so that the final concentration of NH 4 ϩ was 2 mM. After the final activity measurement and sample processing, the remaining culture cell paste was collected and stored in liquid nitrogen for DRAG-DRAT activity measurements and nitrogenase protein quantitation.…”

“…The R. rubrum strain containing this plasmid construct overexpressed DRAG under nif-derepressing conditions but failed to overexpress DRAT, presumably because of interference with translation of the draT gene by translation of the nifH coding region placed upstream of draT. Recently, Durner et al reported on the overexpression of functional DRAT and DRAG in Escherichia coli (4).…”

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

“…The best characterized mechanism of switch-off involves the reversible ADP-ribosylation of an arginine residue in NifH by the dinitrogenase reductase ADP ribosyltransferase͞dinitrogenase reductase-activating glycohydrolase (DraT͞DraG) system (3). This mode of regulation occurs in various Proteobacteria, however other diazotrophs apparently do not effect switch-off by this process (4)(5)(6)(7).…”