Identification of a ubiquinone-binding site that affects autophosphorylation of the sensor kinase RegB.
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 approximately 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.
Swem
Lee R
LR
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Gong
Xing
X
Yu
Chang-An
CA
Bauer
Carl E
CE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D015152:Blotting, Northern; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry; D014451:Ubiquinone / Q000378:metabolism*
D001426:Bacterial Proteins; C091081:RegB protein, Rhodobacter; D014451:Ubiquinone; D011494:Protein Kinases