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J. Biol. Chem., Vol. 282, Issue 43, 31147-31155, October 26, 2007

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Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase^*

From the Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, D-35032 Marburg, Germany

By using natural abundance ¹³C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

Received for publication, May 16, 2007 , and in revised form, July 18, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 395, the Graduiertenkolleg "Proteinfunktion auf atomarer Ebene," the Bundesministerium für Bildung und Forschung through the "Genom Netzwerk Göttingen," European Union Contract IAC-CT-2000-30041, the Fonds der Chemischen Industrie, and the Max Planck Institute for Terrestrial Microbiology (Marburg). 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 "Experimental Procedures" and additional references.

¹ To whom correspondence should be addressed: Laboratory for Microbiology, Dept. of Biology, Philipps-University Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Germany. Tel.: 49-6421-2821529; Fax: 49-6421-2828979; E-mail: bremer{at}staff.uni-marburg.de.

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