In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein.
Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30-40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.
Araujo
Mariana S
MS
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP19046, Curitiba, PR 81531-990, Brazil.
Baura
Valter A
VA
Souza
Emanuel M
EM
Benelli
Elaine M
EM
Rigo
Liu U
LU
Steffens
M Berenice R
MB
Pedrosa
Fabio O
FO
Chubatsu
Leda S
LS
D000255:Adenosine Triphosphate / Q000378:metabolism; D015806:Azospirillum brasilense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005973:Glutamine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction
D001426:Bacterial Proteins; C100965:GlnZ protein, Azospirillum brasilense; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C022515:regulatory protein uridylyltransferase