title: The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB.
abstract: Fe 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.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA.
authors.forename: Vivian R
authors.initials: VR
authors.lastname: Moure
authors.forename: Karamatullah
authors.initials: K
authors.lastname: Danyal
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Yang
authors.forename: Shannon
authors.initials: S
authors.lastname: Wendroth
authors.forename: Marcelo
authors.initials: M
authors.lastname: Müller-Santos
authors.forename: Fabio O
authors.initials: FO
authors.lastname: Pedrosa
authors.forename: Marcelo
authors.initials: M
authors.lastname: Scarduelli
authors.forename: Edileusa C M
authors.initials: EC
authors.lastname: Gerhardt
authors.forename: Luciano F
authors.initials: LF
authors.lastname: Huergo
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D036002:ADP Ribose Transferases / Q000302:isolation & purification / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D015806:Azospirillum brasilense / Q000201:enzymology*; D016948:Azotobacter vinelandii / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism*; D003067:Coenzymes; D004791:Enzyme Inhibitors / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D004791:Enzyme Inhibitors; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D009243:NAD; C404143:PIID regulatory protein, Bacteria; D000244:Adenosine Diphosphate; D010088:Oxidoreductases; C029130:nitrogenase reductase; D036002:ADP Ribose Transferases; C057637:dinitrogenase reductase ADP-ribosyltransferase; D008274:Magnesium