The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli.
DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.
Kneuper
Holger
H
Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany.
Janausch
Ingo G
IG
Vijayan
Vinesh
V
Zweckstetter
Markus
M
Bock
Verena
V
Griesinger
Christian
C
Unden
Gottfried
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D019343:Citric Acid / Q000378:metabolism / Q000494:pharmacology; D029382:Dicarboxylic Acid Transporters / Q000235:genetics; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D000071677:Histidine Kinase; D007763:Lac Operon / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009574:Nitro Compounds; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011422:Propionates / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013644:Tartrates / Q000494:pharmacology
D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D029968:Escherichia coli Proteins; D005650:Fumarates; D009574:Nitro Compounds; D010446:Peptide Fragments; D011422:Propionates; D011993:Recombinant Fusion Proteins; D013644:Tartrates; C086256:dcuB protein, E coli; D019343:Citric Acid; C032005:fumaric acid; D011494:Protein Kinases; C469359:DcuS protein, E coli; D000071677:Histidine Kinase; C015392:3-nitropropionic acid; C029768:tartaric acid