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J. Biol. Chem., Vol. 281, Issue 9, 5582-5592, March 3, 2006

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Identification of Fungal Sphingolipid C9-methyltransferases by Phylogenetic Profiling^*

Philipp Ternes¹, Petra Sperling, Sandra Albrecht, Stephan Franke, James M. Cregg^¶, Dirk Warnecke, and Ernst Heinz

From the Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany, the Organische Mikroanalytik, Institut für Organische Chemie, Universität Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany, and the ^¶Keck Graduate Institute, Claremont, California 91711

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24–71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and 4,8-desaturated ceramide as substrates.

Received for publication, December 1, 2005

^* This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB 470). 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY700777 [GenBank] , AY700778 [GenBank] , and DQ070247 [GenBank] .

The GenPept accession numbers of the deduced protein sequences are AAU10084 [GenBank] , AAU10085, and AAZ08581 [GenBank] .

The atomic coordinates and structure factors (code 2FAY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S4, Fig. S1, and Discussion.

¹ To whom correspondence should be addressed: Present address: Membrane Enzymology, Faculteit Scheikunde, Universiteit Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. Tel.: 31-30-253-7897; Fax: 31-30-252-2478; E-mail: p.g.ternes{at}chem.uu.nl.

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