Structure-activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus

► We have examined how substrate structure affects sphingomyelinase activity. ► We have computationally docked the substrate into the active site. ► 2N-methylation of sphingomyelin interferes with induced fit stabilization. ► Head-group methyls are important for proper active site alignment. ► Phytosphingomyelin is a good substrate despite additional hydroxyl.