Removal of an adenine-like molecule during activation of dinitrogenase reductase from Rhodospirillum rubrum.

Abstract

During the activation of the inactive dinitro- Dinitrogenase reductase (Rr2) from Rhodospirillum rubrum is inactive as isolated (1) and has been shown to have two pentose, two phosphate, and two adenine-like molecules (Ad) attached per 60,000 molecular weight dimer (2). The role of these groups in activation is discussed in this paper.Other enzymes have been shown to have ribose, phosphate, and adenine covalently attached. The first of these was glutamine synthetase (GS) from Escherichia coli; it has up to 12 AMP molecules per dodecamer (3). The adenylylated form of the enzyme is much less active in the biosynthesis of glutamine than is the unadenylylated form. Cleavage of the bond between a tyrosine residue on the protein and the phosphate of AMP by a specific enzyme (ATase) isolated from E. coli or by snake venom diesterase (SVD) activates the enzyme. The E. coli enzyme can also adenylylate GS with ATP as an AMP donor.The RNA polymerase of E. coli has its template specificity altered by ADP-ribosylation (4). The ADP-ribose donor is NAD+, and linkage to the protein is through arginine-ribose. ADP-ribose is susceptible to hydrolysis either by SVD or by a glycohydrolase from chromatin material (5), but the two enzymes cleave the ADP-ribose in different places. The adenine/ribose/phosphate ratio is 1:2:2 for RNA polymerase and 1:1:1 for the adenylylated GS.The dinitrogenase reductases from other organisms do not appear to have the pentose-, phosphate-, and Ad-containing group (2), and they do not require activation. In this paper, evidence is presented to show that the Ad, but not the phosphate group, is removed upon activation. The role of ATP and metals in activation also is established.MATERIALS AND METHODS Enzymes. The Rrl and Rr2 of R. rubrum and the activating factor (AF) were purified as described (2, 6). Alkaline phosphatase from E. coli was obtained from Sigma. This preparation (type III-R) was essentially free from DNase and RNase activities and had a specific activity of 31 ,umol of p-nitrophenyl phosphate hydrolyzed per min per mg of protein. The stock solution of phosphatase was diluted 1:60 before use, and 50 MIl of the diluted solution was used per assay (0.07 unit per assay). SVD type II also was obtained from Sigma; 1.6 units of the enzyme was dissolved in 2 ml of buffer and 50 ,ul was used per assay (0.04 units per assay). Because of the lability of this preparation, its activity was checked before it was used on R. rubrum Rr2 by measuring its rate of hydrolysis of bis-p-nitrophenyl phosphate. The SVD used was low in 5'-nucleotidase activity, inorganic pyrophosphatase activity, and phosphatase activity but had considerable nucleotide pyrophosphatase activity. Spleen diesterase (SpD) from Sigma was dissolved in buffer so that 50 ,ul contained 0.3 unit; 50 ,ul was used in each assay. It had considerable phosphatase activity.Enzymatic Treatment of Rr2. Ten to 20 nmol of Rr2 was treated with enzymes (SVD, SpD, alkaline phosphatase) and AF in the presence of ATP, metal ions, and other compound...

Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium

“…Under activating assay conditions, which did not affect the activity significantly when cells had not been exposed to ammonium, the inhibition resulting from exposure of the culture to ammonium could be substantially eliminated in vitro by adding Mn2' and Mg2+ in concentrations known to be required for DRAG activity in an in vitro assay with purified proteins (41,42,51,59). These results are consistent with the in vivo ADP-ribosylation of wild-type component II in response to ammonium and the in vitro reactivation of this protein by DRAG.…”

“…This mixture (pH 7.8) consisted of 100 mM MOPS, 12.5 mM ATP, 80 mM creatine phosphate, 12.5 U of creatine phosphokinase, and 25 mM Na2S204 and contained different MgCl2 and MnCl2 concentrations: 10 mM MgCl2 for nonactivating conditions, in which DRAG is known to be inactive, and 25 mM MgCl2 and 0.5 mM MnCl2 for activating conditions, in which DRAG is capable of activating modified component II (41,42).…”

Regulation of N2 Fixation and Ammonia Assimilation in Rhodopseudomonas sphaeroides f. sp. denitrificans. Role of Glutamine

“…Ludden & Burris (1978) have reported that inactive nitrogenase from Rhodospirillum rubrum contains phosphate, ribose and adenine-like molecules attached to the Fe-protein of nitrogenase. It has also been found that a Mn2 +-dependent enzyme (Nordlund & Eriksson, 1979;Ludden & Burris, 1979), recently purified from Rhodospirillum rubrum (Gotto & Yoch, 1982), removes the adenine moiety from the Fe-protein of nitrogenase (Ludden & Burris, 1979), producing the 'switch on' effect.…”

Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue.

“…The inactive enzyme can be activated in vitro by the addition of an activating enzyme isolated from the chromatophore membrane fraction (8,9). This activation results from the removal of at least part of the modifying group (6, [8][9][10][11][12]. The modifying group may also be removed in vitro by mild heat treatment, resulting in activation of the enzyme (13).…”