Characterization of the signaling domain of the NO-responsive regulator NorR from Ralstonia eutropha H16 by site-directed mutagenesis.
In Ralstonia eutropha H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.
Klink
Andrea
A
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany.
Elsner
Bettina
B
Strube
Katja
K
Cramm
Rainer
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020118:Cupriavidus necator / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction; D015398:Signal Transduction; D013053:Spectrophotometry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D015534:Trans-Activators; D010088:Oxidoreductases; C034747:nitric-oxide reductase