神经酰胺合酶 (CerS) 在鞘脂代谢中发挥着至关重要的作用，并已成为代谢疾病、癌症和抗真菌治疗的有希望的药物靶标。然而，由于对小分子抑制机制的了解有限，CerS 的治疗靶向受到阻碍。伏马菌素 B1 (FB1) 作为真核细胞 CerS 的有效抑制剂已被广泛研究。在本研究中，我们表征了 FB1 对酵母 CerS (yCerS) 的抑制机制，并确定了 FB1 结合和 N-酰基-FB1 结合 yCerS 的结构。通过我们的结构分析和 yCerS 对 FB1 N-酰化的观察，我们提出了 yCerS 对 FB1 N-酰化的潜在乒乓催化机制。最后，我们证明与 C26-辅酶 A (CoA) 底物相比，FB1 对 yCerS 表现出较低的结合亲和力，这表明 FB1 对 yCerS 的有效抑制作用可能主要来自 yCerS 催化的 N-酰基-FB1，而不是通过直接绑定 FB1。版权所有 © 2024 Elsevier Inc. 保留所有权利。

Ceramide synthases (CerSs) play crucial roles in sphingolipid metabolism and have emerged as promising drug targets for metabolic diseases, cancers, and antifungal therapy. However, the therapeutic targeting of CerSs has been hindered by a limited understanding of their inhibition mechanisms by small molecules. Fumonisin B1 (FB1) has been extensively studied as a potent inhibitor of eukaryotic CerSs. In this study, we characterize the inhibition mechanism of FB1 on yeast CerS (yCerS) and determine the structures of both FB1-bound and N-acyl-FB1-bound yCerS. Through our structural analysis and the observation of N-acylation of FB1 by yCerS, we propose a potential ping-pong catalytic mechanism for FB1 N-acylation by yCerS. Lastly, we demonstrate that FB1 exhibits lower binding affinity for yCerS compared to the C26- coenzyme A (CoA) substrate, suggesting that the potent inhibitory effect of FB1 on yCerS may primarily result from the N-acyl-FB1 catalyzed by yCerS, rather than through direct binding of FB1.Copyright © 2024 Elsevier Inc. All rights reserved.