Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl–enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery.

神经酰胺是调节细胞代谢至关重要的生物活性鞘脂。神经酰胺和二氢神经酰胺由六种神经酰胺合酶 （CerS） 酶合成，每种酶对不同的酰基辅酶 A 底物具有特异性。具有 16 碳酰基链的神经酰胺 （C16 神经酰胺） 与肥胖、胰岛素抵抗和肝病有关，而 C16 神经酰胺合成的 CerS6 被认为是肥胖相关疾病的有吸引力的药物靶点。尽管它们很重要，但 CerS 酶合成神经酰胺的分子机制仍然知之甚少。在这里，我们报告了人 CerS6 的冷冻电子显微镜结构，捕获共价中间体和产物结合状态。这些结构以及生化表征表明，CerS 催化通过涉及共价酰基-酶中间体的乒乓反应机制进行。值得注意的是，与霉菌毒素伏马菌素 B1 反应后获得产物结合结构，从而深入了解其对 CerS 的抑制作用。这些结果为理解 CerS 功能、选择性和抑制性提供了一个框架，并为未来的药物发现开辟了途径。