Ternes Philipp, Sperling Petra, Albrecht Sandra, Franke Stephan, Cregg James M, Warnecke Dirk, Heinz Ernst
Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany.
J Biol Chem. 2006 Mar 3;281(9):5582-92. doi: 10.1074/jbc.M512864200. Epub 2005 Dec 8.
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.
真菌葡糖神经酰胺在植物与病原体的相互作用中发挥着重要作用，使植物能够识别真菌攻击并启动特定的防御反应。真菌葡糖神经酰胺与植物和动物的葡糖神经酰胺的一个主要结构特征区别在于鞘氨醇碱基的C9位上有一个甲基，其生物合成从未被研究过。利用不同真菌物种中C9甲基化葡糖神经酰胺存在与否的信息，我们开发了一种生物信息学策略来鉴定负责该C9甲基生物合成的基因。这种系统发育分析使得从每个含有C9甲基化葡糖神经酰胺的真菌物种中存在的24 - 71个甲基转移酶序列中选择出一个单一的候选基因成为可能。构建了一个缺乏候选鞘脂C9甲基转移酶的毕赤酵母敲除菌株，实际上，该菌株只含有未甲基化的葡糖神经酰胺。在一种互补方法中，对酿酒酵母菌株进行改造，使其产生适合作为C9甲基化底物的葡糖神经酰胺。在表达来自毕赤酵母的候选基因的该菌株中检测到了C9甲基化鞘脂，证明了其作为鞘脂C9甲基转移酶的功能。该酶属于S - 腺苷甲硫氨酸（SAM）依赖性甲基转移酶超家族，与植物和细菌的环丙烷脂肪酸合酶具有最高的序列相似性。体外试验表明，鞘脂C9甲基化是膜结合的，需要SAM和Δ4,8 - 去饱和神经酰胺作为底物。
