Ceramides are essential lipids involved in forming complex sphingolipids and acting as signaling molecules. They result from the N-acylation of a sphingoid base and a CoA-activated fatty acid, a reaction catalyzed by the ceramide synthase (CerS) family of enzymes. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here we used cryo-electron microscopy single-particle analysis to unravel the structure of the yeast CerS complex in both an active and a fumonisin B1-inhibited state. Our results reveal the complex’s architecture as a dimer of Lip1 subunits bound to the catalytic subunits Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice connecting the cytosolic site with the intermembrane space. The active site, located centrally in the tunnel, was resolved in a substrate preloaded state, representing one intermediate in ceramide synthesis. Our data provide evidence for competitive binding of fumonisin B1 to the acyl-CoA-binding tunnel.

神经酰胺是参与形成复杂鞘脂并充当信号分子的必需脂质。它们由鞘氨醇碱基和 CoA 激活的脂肪酸的 N-酰化引起，这是一种由神经酰胺合酶 （CerS） 酶家族催化的反应。然而，CerSs 的精确结构细节和催化机制仍然难以捉摸。在这里，我们使用冷冻电子显微镜单颗粒分析来揭示酵母 CerS 复合物在活性和伏马菌素 B1 抑制状态下的结构。我们的结果揭示了该复合物的结构是与催化亚基 Lag1 和 Lac1 结合的 Lip1 亚基的二聚体。每个催化亚基形成一个疏水裂缝，将胞质位点与膜间空间连接起来。活性位点位于隧道中央，在底物预加载状态下分离，代表神经酰胺合成中的一种中间体。我们的数据为伏马菌素 B1 与酰基辅酶 A 结合隧道的竞争性结合提供了证据。