Nagore Daniel, Sanz Begoña, Soria Javier, Llarena Marta, Llama María J, Calvete Juan J, Serra Juan L
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.
Biochim Biophys Acta. 2006 Feb;1760(2):172-81. doi: 10.1016/j.bbagen.2005.12.011. Epub 2006 Jan 6.
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.
大多数蓝细菌通过位于细胞质膜上的多亚基ABC转运蛋白（ATP结合盒）吸收硝酸盐或亚硝酸盐。培养基中铵的存在会立即阻断硝酸盐和亚硝酸盐的转运活性。先前的生化研究报道，在细长聚球藻PCC 6301的蓝细菌质膜中，存在与铵敏感激酶/磷酸酶活性相关的硝酸盐转运蛋白（NRT）的磷酸化/去磷酸化事件。在这项工作中，我们分析了嗜热非固氮蓝细菌层状席藻NRT的周质硝酸盐/亚硝酸盐结合亚基（NrtA）的生化特性。我们的结果表明，蓝细菌NrtA在体内被磷酸化。然而，体外底物结合活性不受蛋白质磷酸化状态的影响，排除了NrtA的磷酸化/去磷酸化参与NRT转运蛋白对硝酸盐/亚硝酸盐摄取调节的可能性。此外，NrtA以多种同工型存在，它们具有相同的分子量，但等电点不同，范围从pI 5到6。NrtA同工型的质谱表征表明，该蛋白在Tyr203残基处被磷酸化，并含有几个甲硫氨酸亚砜残基，这解释了观察到的同工型。磷酸化和非磷酸化形式的NrtA在体外均具有活性，对硝酸盐和亚硝酸盐表现出相当的结合亲和力。两种底物均表现为纯竞争性抑制剂，每个NrtA单体的阴离子结合化学计量为一分子。
