hide
Free keywords:
-
Abstract:
Staphylococci are able to use nitrate as an alternative electron acceptor during anaerobic respiration. The regulation of energy metabolism is dependent on the presence of oxygen and nitrate. Under anaerobic conditions, staphylococci employ the nitrate regulatory element (Nre) for transcriptional activation of genes involved in reduction and transport of nitrate and nitrite. Of the three proteins that constitute the Nre system, NreB has been characterized as an oxygen sensor kinase and NreC has been characterized as its cognate response regulator. Here, we present structural and functional data that establish NreA as a new type of nitrate receptor. The structure of NreA with bound nitrate was solved at 2.35Å resolution, revealing a GAF domain fold. Isothermal titration calorimetry experiments showed that NreA binds nitrate with low micromolar affinity (KD=22μM). Two crystal forms for NreA were obtained, with either bound nitrate or iodide. While the binding site is hydrophobic, two helix dipoles and polar interactions contribute to specific binding of the ions. The expression of nitrate reductase (NarGHI) was examined using a narG-lip (lipase) reporter gene assay in vivo. Expression was regulated by the presence of NreA and nitrate. Structure-guided mutations of NreA reduced its nitrate binding affinity and also affected the gene expression, thus providing support for the function of NreA as a nitrate receptor.
