Identification of Fungal Sphingolipid C9-methyltransferases by Phylogenetic Profiling

• Published in: The Journal of biological chemistry Vol. 281; no. 9; pp. 5582 - 5592
• Main Authors: Ternes, Philipp, Sperling, Petra, Albrecht, Sandra, Franke, Stephan, Cregg, James M., Warnecke, Dirk, Heinz, Ernst
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.03.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Computational Biology; Fungal Proteins - classification; Fungal Proteins - genetics; Fungal Proteins - metabolism; Glucosylceramides - chemistry; Glucosylceramides - metabolism; Methyltransferases - classification; Methyltransferases - genetics; Methyltransferases - metabolism; Molecular Sequence Data; Molecular Structure; Phylogeny; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Saccharomyces cerevisiae; Sequence Alignment; Sphingolipids - chemistry; Sphingolipids - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M512864200

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 Δ4,8-desaturated ceramide as substrates.