Identification of a Ubiquinone-binding Site That Affects Autophosphorylation of the Sensor Kinase RegB

• Published in: The Journal of biological chemistry Vol. 281; no. 10; pp. 6768 - 6775
• Main Authors: Swem, Lee R., Gong, Xing, Yu, Chang-An, Bauer, Carl E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.03.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Binding Sites; Blotting, Northern; DNA Mutational Analysis; Molecular Sequence Data; Oxidation-Reduction; Phosphorylation; Protein Kinases - genetics; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Rhodobacter capsulatus; Rhodobacter capsulatus - enzymology; Rhodobacter capsulatus - genetics; Rhodobacter capsulatus - metabolism; Spectrophotometry; Ubiquinone - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M509687200

Rhodobacter capsulatus regulates many metabolic processes in response to the level of environmental oxygen and the energy state of the cell. One of the key global redox regulators of the cell's metabolic physiology is the sensor kinase RegB that controls the synthesis of numerous energy generation and utilization processes. In this study, we have succeeded in purifying full-length RegB containing six transmembrane-spanning elements. Exogenous addition of excess oxidized coenzyme Q1 is capable of inhibiting RegB autophosphorylation ∼6-fold. However, the addition of reduced coenzyme Q1 exhibits no inhibitory effect on kinase activity. A ubiquinone-binding site, as defined by azidoquinone photo affinity cross-linking, was determined to lie within a periplasmic loop between transmembrane helices 3 and 4 that contains a fully conserved heptapeptide sequence of GGXXNPF. Mutation of the phenylalanine in this heptapeptide renders RegB constitutively active in vivo, indicating that this domain is responsible for sensing the redox state of the ubiquinone pool and subsequently controlling RegB autophosphorylation.