Purification and properties of NrtC and NrtD, the ATP-binding subunits of the ABC nitrate/nitrite transporter of Phormidium laminosum

• Published in: Biochimica et biophysica acta 2006-12, Vol.1760 (12), p.1819-1826
• Main Authors: Llarena, Marta, Llama, María J., Serra, Juan L.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; ATP hydrolysis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Base Sequence; Cloning, Molecular; Cyanobacteria; Cyanobacteria - metabolism; DNA Primers; Electrophoresis, Polyacrylamide Gel; Molecular Weight; Nitrate/nitrite transport; NrtC; NrtD; Phormidium laminosum; Protein Binding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2006.08.006

A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759–766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His 6NrtC and His 6NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45–50 °C and pH 8.0, and similar K m (0.49 and 0.43 mM) and V max (0.085 and 0.114 U mg − 1 protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg 2+) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His 6NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed.