Eleven residues determine the acyl chain specificity of ceramide synthases

Lipids display large structural complexity, with approximate to 40,000 different lipids identified to date, approximate to 4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of approximate to 150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5((299-309CerS2)), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4((291-301CerS2)), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.