Study of The Divergent Acyl Chain Specificities of The Class I Ceramide Synthases in Plants

Abstract

Ceramide synthases catalyze the condensation of long-chain bases and fatty acyl-CoA to form the ceramide backbones of glycosphingolipids.The Class I and II ceramide synthases of plants have distinct substrate specificities that contribute to the divergent fatty acid chain-lengths and functions of plant sphingolipids. The Arabidopsis LOH2-encoded Class I ceramide synthase has been shown to have strong specificity for palmitoyl (16:0)-CoA that gives rise to ceramides with C16 fatty acids that are prevalent in dicot glucosylceramides. By contrast, glucosylceramides of many monocots and selected dicots are enriched in C18 and C20-ceramides. We hypothesized that these differences are due to functional and structural differences in LOH2 ceramide synthases. Consistent with this, we found that transgenic expression of a tomato LOH2 restored C16 fatty acid in glucosylceramides in an Arabidopsis loh2 mutant, whereas expression of a rice LOH2 resulted in C18- and C20-fatty acid-enriched glucosylceramides. To establish the structural basis for these different outcomes, we demonstrated that swapping a divergent 16 amino acid domain from the rice LOH2 to tomato LOH2 yielded C18- and C20-fatty acid-glucosylceramides when expressed in the loh2 mutant. By comparison of primary structures of LOH2 enzymes from diverse dicots and monocots, we identified a single amino acid that when mutated in the tomato LOH2 resulted in rice-type C18- and C20 fatty acid-glucosylceramides.We rationalize the altered LOH2 function in the context of the predicted LOH2 ceramide synthase secondary structure. Overall, our findings highlight the biochemical and genetic basis for divergent ceramide structures in dicots and monocots.