Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b(5) and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG-Des2, mb(5) (the membrane form of cytochrome b(5)) and bovine erythrocyte membrane. The apparent K-m and V-max of Des2 for the substrate N-octanoylsphinganine were 35 mu M and 40 nmol (.) h(-1) (.) mg of protein(-1) respectively. The K-m of the hydroxylase for mb(5) was 0.8 mu M. Interestingly, mb(5) was not replaced with the soluble form of cytochrome b(5), which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b(5)R (NADH-cytochrome b(5) reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains; The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb(5) via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb(5) by b(5)R.