Mechanism of ceramide synthase inhibition by fumonisin B₁

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.