The negative transcriptional regulator NmrA discriminates between oxidized and reduced dinucleotides

Abstract

NmrA, a transcription repressor involved in the regulation of nitrogen metabolism in Aspergillus nidulans, is a member of the short-chain dehydrogenase reductase superfamily. Isothermal titration calorimetry and differential scanning calorimetry have been used to show NmrA binds NAD + and NADP+ with similar affinity (average KD 65 μM) but has a greatly reduced affinity for NADH and NADPH (average K D 6.0 mM). The structure of NmrA in a complex with NADP+ reveals how repositioning a His-37 side chain allows the different conformations of NAD+ and NADP+ to be accommodated. Modeling NAD(P)H into NmrA indicated that steric clashes, attenuation of electrostatic interactions, and loss of aromatic ring stacking can explain the differing affinities of NAD(P)+/NAD(P)H. The ability of NmrA to discriminate between the oxidized and reduced forms of the dinucleotides may be linked to a possible role in redox sensing. Isothermal titration calorimetry demonstrated that NmrA and a C-terminal fragment of the GATA transcription factor AreA interacted with a 1:1 stoichiometry and an apparent KD of 0.26 μM. NmrA was unable to bind the nitrogen metabolite repression signaling molecules ammonium or glutamine.