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J. Biol. Chem., Vol. 283, Issue 5, 2962-2972, February 1, 2008

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Functional Role for a Conserved Aspartate in the Spo0E Signature Motif Involved in the Dephosphorylation of the Bacillus subtilis Sporulation Regulator Spo0A^*

Alejandra R. Diaz, Sophie Stephenson, J. Michael Green, Vladimir M. Levdikov, Anthony J. Wilkinson, and Marta Perego¹

From the Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, La Jolla, California 92037 and the Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T³⁵IXXSQ ELDCLI⁴⁶ residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0AP was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr³⁵ and Cys⁴⁴ did not seem to have any critical functional or structural role. We propose that Asp⁴³ of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226[CrossRef][Medline] [Order article via Infotrieve] ).

Received for publication, November 2, 2007 , and in revised form, November 28, 2007.

^* This work was supported in part by NIGMS, National Institutes of Health Grant GM55594 (to M. P.), the Wellcome Trust, and European Union Grant SPINE2-Complexes C-T-2006-031220 (to A. J. W.). DNA sequencing reactions and oligonucleotide synthesis was supported in part by the Stein Beneficial Trust. This is manuscript number 18946 from The Scripps Research Institute. 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 Tables S1 and Figs. S1-S4.

¹ To whom correspondence should be addressed: 10550 N. Torrey Pines Rd., MEM-116, La Jolla, CA 92037. Tel.: 858-784-7912; Fax: 858-784-7966; E-mail: mperego{at}scripps.edu.

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