The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

Abstract

bFe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P II proteins. In A. brasilense, two P II proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P II proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P II effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P II proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K m value for NAD ؉ . Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

“…Likewise, GlnB can interact with and stimulate the activity of dinitrogenase reductase ADP‐ribosyltransferase (DraT), which mediates the ADP‐ribosylation and inactivation of NifH (Moure et al. ). GlnB also associates with and regulates activity of the adenyltransferase (ATase) that covalently modifies and inactivates glutamine synthetase (Mangum et al.…”

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense

“…For these assays, either GlnZ or a mixture of GlnZ and AmtB were incubated with DraG in the presence of ADP (to stimulate protein complex formation) and purified ADP‐ribosylated Fe‐protein (to act as substrate for DraG). After 20 min, purified MoFe‐protein was added to the reaction to assess nitrogenase activity by the H 2 evolution method . Addition of GlnZ at a molar ratio of 1 : 40 (DraG : GlnZ) did not reduce DraG activity (Fig.…”

“…DraG activity was also determined indirectly by measuring the Fe‐protein activity based on H 2 evolution under anaerobic conditions, as reported previously . Firstly, Azotobacter vinelandii Fe‐protein ADP‐ribosylated in vitro or unmodified Fe‐protein was incubated with DraG and then Fe‐protein activity was determined by adding MoFe‐protein.…”

Endogenous ADP-Ribosylation

“…1a). ADP-ribosylation of dinitrogenase reductase R101 prevents its association with the dinitrogenase, inhibiting the electron transfer cycles and thereby inactivating nitrogenase (Moure et al 2013;Murrell et al 1988) (Fig. 1b).…”

“…The formation of the DraT-GlnB complex results in DraT conformational changes activating the ADP-ribosyltransferase and therefore promoting dinitrogenase reductase ADP-ribosylation and nitrogenase inactivation (Moure et al 2013) (Fig. 3).…”