title: Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria.
abstract: Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba-PR, Brazil.
authors.forename: Luciano F
authors.initials: LF
authors.lastname: Huergo
authors.forename: Mike
authors.initials: M
authors.lastname: Merrick
authors.forename: Fábio O
authors.initials: FO
authors.lastname: Pedrosa
authors.forename: Leda S
authors.initials: LS
authors.lastname: Chubatsu
authors.forename: Luíza M
authors.initials: LM
authors.lastname: Araujo
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D015806:Azospirillum brasilense / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C100965:GlnZ protein, Azospirillum brasilense; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D009591:Nitrogenase; D006026:Glycoside Hydrolases; D009584:Nitrogen