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 b5 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, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent K(m) and V(max) of Des2 for the substrate N-octanoylsphinganine were 35 microM and 40 nmol x h(-1) x mg of protein(-1) respectively. The K(m) of the hydroxylase for mb5 was 0.8 microM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, 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 b5R (NADH-cytochrome b5 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 mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.
Enomoto
Ayako
A
Sphingolipid Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan.
Omae
Fumio
F
Miyazaki
Masao
M
Kozutsumi
Yasunori
Y
Yubisui
Toshitsugu
T
Suzuki
Akemi
A
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002518:Ceramides / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004583:Electrons; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D007313:Insecta; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry*; D017830:Octoxynol / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry
D002518:Ceramides; D003902:Detergents; D009097:Multienzyme Complexes; C109343:dihydroceramide; D017830:Octoxynol; D015786:Cytochromes b5; C489141:Degs2 protein, mouse; D010088:Oxidoreductases; D013110:Sphingosine; C005682:safingol