PMID:15781452
| Citation |
Kneuper, H, Janausch, IG, Vijayan, V, Zweckstetter, M, Bock, V, Griesinger, C and Unden, G (2005) The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli. J. Biol. Chem. 280:20596-603 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M502015200 |
| Keywords |
Amino Acid Sequence; Binding Sites; Citric Acid/metabolism; Citric Acid/pharmacology; Dicarboxylic Acid Transporters/genetics; Dicarboxylic Acids/chemistry; Dicarboxylic Acids/pharmacology; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fumarates/metabolism; Fumarates/pharmacology; Gene Expression Regulation, Bacterial/drug effects; Lac Operon/genetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitro Compounds; Peptide Fragments/chemistry; Peptide Fragments/metabolism; Phosphorylation; Propionates/pharmacology; Protein Kinases/chemistry; Protein Kinases/genetics; Protein Kinases/metabolism; Recombinant Fusion Proteins/genetics; Sequence Alignment; Structure-Activity Relationship; Tartrates/pharmacology |
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