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J. Biol. Chem., Vol. 283, Issue 30, 20888-20896, July 25, 2008

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Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803^*

Jakub Grzegorz Kopycki, Milton T. Stubbs, Wolfgang Brandt, Martin Hagemann^¶, Andrea Porzel, Jürgen Schmidt, Willibald Schliemann^||, Meinhart H. Zenk^**, and Thomas Vogt^||1

From the Department of Physical Biotechnology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale) 06099, Germany, the Departments of Bioorganic Chemistry and ^||Secondary Metabolism, Leibniz Institute of Plant Biochemistry, Halle (Saale) 06120, Germany, the ^¶Institute of Biological Sciences, University of Rostock, Rostock 18051, Germany, and the ^**Donald Danforth Plant Science Center, St. Louis, Missouri 63132

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-L-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys³ close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.

Received for publication, March 11, 2008 , and in revised form, May 22, 2008.

The atomic coordinates and structure factors (code 3CBG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* 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 Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 49-345-5582-1530; Fax: 49-345-5582-1509; E-mail: tvogt{at}ipb-halle.de.

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