title: Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling.
abstract: 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 Delta4,8-desaturated ceramide as substrates.
authors.affiliation: Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany. p.g.ternes@chem.uu.nl
authors.forename: Philipp
authors.initials: P
authors.lastname: Ternes
authors.forename: Petra
authors.initials: P
authors.lastname: Sperling
authors.forename: Sandra
authors.initials: S
authors.lastname: Albrecht
authors.forename: Stephan
authors.initials: S
authors.lastname: Franke
authors.forename: James M
authors.initials: JM
authors.lastname: Cregg
authors.forename: Dirk
authors.initials: D
authors.lastname: Warnecke
authors.forename: Ernst
authors.initials: E
authors.lastname: Heinz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019295:Computational Biology; D005656:Fungal Proteins / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D005963:Glucosylceramides / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005963:Glucosylceramides; D013107:Sphingolipids; D008780:Methyltransferases; C019964:cyclopropane synthetase