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Journal of Bacteriology, August 2008, p. 5663-5671, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00346-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Compatible-Solute-Binding Protein OpuAC from Bacillus subtilis: Ligand Binding, Site-Directed Mutagenesis, and Crystallographic Studies

Sander H. J. Smits,^1, Marina Höing,^2, Justin Lecher,¹ Mohamed Jebbar,^3, Lutz Schmitt,^1* and Erhard Bremer²

Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany,¹ Laboratory for Microbiology, Department of Biology, Philipps University Marburg, Karl-von-Frisch Str., 35032 Marburg, Germany,² Departement Osmoregulation chez les Bacteries, Universite de Rennes 1, UMR-CNRS 6026, Rennes, France³

Received 10 March 2008/ Accepted 9 June 2008

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-Å resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.

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* Corresponding author. Mailing address: Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. Phone: 49(0)211-81-10773. Fax: 49(0)211-81-15310. E-mail: lutz.schmitt{at}uni-duesseldorf.de

Published ahead of print on 20 June 2008.

Both authors contributed equally to this work.

Present address: Laboratory of Extreme Environments, Microbiology, University of West Brittany (Brest), European Institute of Marine studies, Technopôle Brest-Iroise, F-29280 Plouzané, France.

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Journal of Bacteriology, August 2008, p. 5663-5671, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00346-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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