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Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from Escherichia coli K-12

Chris E. Noriega,¹ Radomir Schmidt,^1, Michael J. Gray,^2, Li-Ling Chen,¹ and Valley Stewart^1,2*

Department of Microbiology,¹ Food Science Graduate Group, University of California, Davis, California 95616-8665²

Received 17 January 2008/ Accepted 17 March 2008

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K_m) for ADP were similar for both proteins, about 2 µM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 µM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 x 10^–4 s^–1) was lower than that observed for MBP-NarQ (about 2.2 x 10^–4 s^–1). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s^–1 for MBP-NarX and about 0.01 s^–1 for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 µM (KinA) to about 40 µM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.

Published ahead of print on 28 March 2008.

Present address: Department of Land, Air and Water Resources, University of California, Davis, CA 95616.

Present address: Microbiology Doctoral Training Program, University of Wisconsin, Madison, WI 53706.