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J. Biol. Chem., Vol. 280, Issue 21, 20596-20603, May 27, 2005

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The Nature of the Stimulus and of the Fumarate Binding Site of the Fumarate Sensor DcuS of Escherichia coli^*

Holger Kneuper, Ingo G. Janausch, Vinesh Vijayan, Markus Zweckstetter¶, Verena Bock, Christian Griesinger, and Gottfried Unden||

From the Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany and Max Planck Institut für Biophysikalische Chemie, 37077 Göttingen, Germany

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C₄ -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C₄-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 Å apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2–3 mM for the C₄-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 (Arg¹⁰⁷-X₂-His¹¹⁰-X₉-Phe¹²⁰-X₂₆-Arg¹⁴⁷-X-Phe¹⁴⁹) 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 C₄-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 C₄-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

Received for publication, February 22, 2005 , and in revised form, March 21, 2005.

^* This work was supported by grants of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to G. U. and C. G.) and the Max-Planck-Gesellschaft (to C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact..

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

^¶ Supported by Emmy Noether Grant ZW 71/1-4 of the Deutsche Forschungsgemeinschaft.

^|| To whom correspondence should be addressed: Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany. Tel.: 49-6131-3923550; Fax: 49-6131-3922695; E-Mail: unden{at}uni-mainz.de.

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