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Journal of Bacteriology, January 2009, p. 588-599, Vol. 191, No. 2
0021-9193/09/$08.00+0     doi:10.1128/JB.01104-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes{triangledown}

Anke Schilhabel ,1,{dagger},{ddagger} Sandra Studenik,1,{ddagger} Martin Vödisch,2 Sandra Kreher,1 Bernhard Schlott,3 Antonio Y. Pierik,4 and Gabriele Diekert1*

Institut für Mikrobiologie, Friedrich-Schiller-Universität Jena, Lehrstuhl für Angewandte und Ökologische Mikrobiologie, Philosophenweg 12, 07743 Jena, Germany,1 Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie, Abteilung Molekulare und Angewandte Mikrobiologie, Beutenbergstrasse 11a, 07745 Jena, Germany,2 Leibniz-Institut für Altersforschung-Fritz-Lipmann-Institut, Beutenbergstrasse 11, 07745 Jena, Germany,3 Institut für Zytobiologie und Zytopathologie, Phillipps-Universität-Marburg, Robert-Koch-Strasse 6, 35037 Marburg, Germany4

Received 7 August 2008/ Accepted 3 November 2008

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.

* Corresponding author. Mailing address: Institut für Mikrobiologie, Friedrich-Schiller-Universität Jena, Lehrstuhl für Angewandte und Ökologische Mikrobiologie, Philosophenweg 12, 07743 Jena, Germany. Phone: 49 3641 949300. Fax: 49 3641 949302. E-mail: gabriele.diekert{at}uni-jena.de

{triangledown} Published ahead of print on 14 November 2008.

{dagger} Present address: Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.

{ddagger} A. Schilhabel and S. Studenik contributed equally to this work.

Journal of Bacteriology, January 2009, p. 588-599, Vol. 191, No. 2
0021-9193/09/$08.00+0     doi:10.1128/JB.01104-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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