Cloning of an alkaline ceramidase from Saccharomyces cerevisiae -: An enzyme with reverse (CoA-independent) ceramide synthase activity
Cloning of an alkaline ceramidase from Saccharomyces cerevisiae -: An enzyme with reverse (CoA-independent) ceramide synthase activity
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DOI：
10.1074/jbc.275.10.6876
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发表时间：
2000-03-10
影响因子：
4.8
通讯作者：
Obeid, LM
中科院分区：
文献类型：
--
作者：
Mao, CG;Xu, RJ;Obeid, LM
神经酰胺不仅是鞘脂的核心中间体，而且是许多细胞事件的重要调节剂，包括细胞凋亡、细胞周期停滞、衰老、分化和应激反应。其营业额可能受到严格管制。然而，对其代谢的调节知之甚少，因为负责其合成和分解的大多数酶尚未被克隆。在这里，我们报告的酵母基因YPC 1（YBR 183 w）的克隆和表征筛选酿酒酵母基因的过度表达赋予伏马菌素B1的抗性。我们证明，酵母基因YPC 1编码的碱性神经酰胺酶活性负责分解的二氢神经酰胺和植物神经酰胺，但不饱和神经酰胺。通过使用大肠杆菌表达系统的体外研究证实了YPC 1神经酰胺酶活性。重要的是，YPC 1 p还具有反向活性，催化棕榈酸和植物鞘氨醇合成植物神经酰胺。这种神经酰胺合酶活性是CoA-独立的，并且对伏马菌素B1具有抗性，从而解释了为什么YPC 1被克隆为伏马菌素B1抗性基因。
Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.