Sphingolipids play varying roles within cells of eukaryotic organisms and are integral components in cell membranes, vesicles and as signalling molecules1. Within the de novo synthesis pathway, dihydroceramide desaturase (DES1) is a critical enzyme within the sphingolipid pathway, inserting a double bond into its substrate dihydroceramide to produce ceramide, which is a precursor for complex sphingolipids and their derivatives1. The irreversible Des1 desaturation reaction therefore, controls the saturation of the cellular sphingolipidome, as many downstream enzymes are capable of using both the saturated and desaturated forms of ceramide. Saturated (dihydro) and non-saturated sphingolipids have distinct cellular functions due to differences in their rigidity and packing structure in lipid membranes2. Research into the role of saturated sphingolipids is in its early stages, however their potentially pathogenic roles in human disease are becoming increasingly apparent, with implications in cancer, metabolic and cardiovascular diseases2. Hypomyelinating Leukodystrophy (HLD) is a rare neurogenerative disease caused by loss of myelination in white matter tissue with no current curative therapy. In recent years, three separate studies have linked HLD to loss-of-function mutations in dihydroceramide desaturase3-5. To investigate the mechanisms by which DES1 is regulated and by which activity is lost, a series of expression constructs containing non-pathogenic DES1 variants, point mutations linked to HLD and post-translational modification sites were made. DES1 variants were assessed using a High-Performance Liquid Chromatography (HPLC) assay and protein structural modelling. Pathogenic HLD variants were screened for their ability to bind co-factor protein Cytochrome B5 type A (CYB5A) by co-immunoprecipitation. DES1 phosphorylation null mutants were assessed for their impacts on enzyme activity and protein stability. Here we show the potential role of patient and phosphorylation mutants on DES1 enzyme activity, protein interactions and stabilisation. These studies are the first to investigate how phosphorylation may regulate DES1 function and uncover new mechanisms of de novo sphingolipid synthesis regulation. Uncovering new mechanisms of DES1 regulation presents novel targets for modulation of DES1 activity in disease.