Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro.
The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate. DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane. The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer. His(6)-DcuS was reconstituted into liposomes made from E. coli phospholipids. Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by [gamma-(33)P]ATP with an approximate K(D) of 0.16 mm for ATP. Up to 7% of the reconstituted DcuS was phosphorylated. Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates. The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.
Janausch
Ingo G
IG
Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany.
Garcia-Moreno
Inma
I
Unden
Gottfried
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D005650:Fumarates / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008954:Models, Biological; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000502:physiology*; D011510:Proteolipids / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000494:pharmacology
D004268:DNA-Binding Proteins; C115005:DcuR protein, E coli; D003902:Detergents; D029968:Escherichia coli Proteins; D005650:Fumarates; D008081:Liposomes; D011510:Proteolipids; D014157:Transcription Factors; C020485:proteoliposomes; C032005:fumaric acid; D000255:Adenosine Triphosphate; D004247:DNA; D011494:Protein Kinases; C469359:DcuS protein, E coli; D000071677:Histidine Kinase