title: The nitrate/nitrite ABC transporter of Phormidium laminosum: phosphorylation state of NrtA is not involved in its substrate binding activity.
abstract: Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr203, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.
authors.affiliation: Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, E-48080 Bilbao, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nagore
authors.forename: Begoña
authors.initials: B
authors.lastname: Sanz
authors.forename: Javier
authors.initials: J
authors.lastname: Soria
authors.forename: Marta
authors.initials: M
authors.lastname: Llarena
authors.forename: María J
authors.initials: MJ
authors.lastname: Llama
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Calvete
authors.forename: Juan L
authors.initials: JL
authors.lastname: Serra
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D000458:Cyanobacteria / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D000091285:Nitrate Transporters; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000737:chemistry
chemical_list: D018528:ATP-Binding Cassette Transporters; D027321:Anion Transport Proteins; D000091285:Nitrate Transporters; D009566:Nitrates; D009573:Nitrites; D020033:Protein Isoforms; D014443:Tyrosine