Biosynthesis of sphingosine and dihydrosphingosine by cell-free systems from Hansenula ciferri

Cells of Hanseniaspora valbyensis, a yeast auxotrophic for vitamin B 6, show a marked decrease in its palmitoleic acid and sphingolipid content when grown under conditions of vitamin B 6 deficiency. In studying the metabolic basis of the latter observation, we found that a particulate preparation from another yeast, Hansenula ciferri, synthesized both dihydrosphingosine and sphingosine (but not phytosphingosine) from palmityl CoA and serine. Synthesis of dihydrosphingosine proceeds in two steps: (a) condensation of palmityl CoA with serine to yield CO 2 and a previously unrecognized biosynthetic intermediate, 3-ketodihydrosphingosine; and (b) TPNH-dependent reduction of this ketonic intermediate to erythro-dihydrosphingosine. The synthesis and properties of labeled 3-ketodihydrosphingosine are described. Action of the particulate enzyme on this compound yields only dihydrosphingosine, whereas the same enzyme preparation forms both sphingosine and dihydrosphingosine from palmityl CoA and serine. The desaturation reaction involved in sphingosine biosynthesis thus appears to occur at the fatty acyl CoA level, although the possibility that it occurs at the ketonic level remains open. Synthesis of sphingosine also proceeds via a new ketonic intermediate, 3-ketosphingosine, by reactions analogous to (a) and (b). Palmityl CoA is the preferred substrate of the condensing enzyme, stearyl CoA and possibly myristyl CoA are reasonably good substrates, whereas lauryl CoA is not utilized. Trans-2-hexadecenoyl CoA is a good substrate for the particulate enzyme preparation, but like palmityl CoA, leads to mixtures of sphingosine and dihydrosphingosine, α-Hydroxypalmityl CoA is not a substrate, and appears, therefore, not to be a precursor of phytosphingosine, at least in enzyme preparations of this nature.