In vitro interactions between the PII proteins and the nitrogenase regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase-activating glycohydrolase (DraG) in Azospirillum brasilense.
The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the P(II) proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of P(II) uridylylation status and the P(II) effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when P(II) proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the P(II) ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity.
Huergo
Luciano F
LF
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, 81531-990 Curitiba-PR, Brazil. huergo@ufpr.br
Merrick
Mike
M
Monteiro
Rose A
RA
Chubatsu
Leda S
LS
Steffens
Maria B R
MB
Pedrosa
Fábio O
FO
Souza
Emanuel M
EM
D036002:ADP Ribose Transferases / Q000378:metabolism*; D015806:Azospirillum brasilense / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism
D001426:Bacterial Proteins; C100965:GlnZ protein, Azospirillum brasilense; D046912:Multiprotein Complexes; D051922:PII Nitrogen Regulatory Proteins; D000644:Quaternary Ammonium Compounds; C404143:PIID regulatory protein, Bacteria; D010088:Oxidoreductases; C029130:nitrogenase reductase; D036002:ADP Ribose Transferases; C057637:dinitrogenase reductase ADP-ribosyltransferase; D009699:N-Glycosyl Hydrolases; C036091:ADP-ribosyl-(dinitrogen reductase) hydrolase