Biosynthesis of Sphingolipid Bases: II. Keto Intermediates in Synthesis of Sphingosine and Dihydrosphingosine by Cell-Free Extracts of Hansenula Ciferri

Abstract

Abstract In the presence of TPNH, particulate cell-free preparations from Hansenula ciferri convert palmityl coenzyme A and serine-14C to labeled dihydrosphingosine and sphingosine. Both the free bases and their dinitrophenyl derivatives migrated identically with authentic standards on thin layer chromatograms. The identity of sphingosine, not previously reported as a biosynthetic product in yeast, was further confirmed by its catalytic reduction to dihydrosphingosine and by recrystallization of its triacetyl derivative to constant specific radioactivity in the presence of unlabeled authentic carrier. Both the biosynthetic sphingosine and dihydrosphingosine have the erythro configuration. In the absence of added TPNH, no sphingosine or dihydrosphingosine is formed by this system. Instead, two less stable, labeled compounds are formed, (a) 2-amino-1-hydroxyoctadecan-3-one (3-ketodihydrosphingosine), and (b) a compound which may be the 3-keto analogue of sphingosine. These intermediates are converted either enzymatically in the presence of TPNH or nonenzymatically with sodium borohydride to dihydrosphingosine and sphingosine. The most prominent labeled component of the mixture on acetylation chromatographs identically with authentic N-acetyl-3-ketodihydrosphingosine. Like the pyridoxal-P-dependent over-all reaction leading to dihydrosphingosine (1), formation of the labile intermediate, but not its reduction to dihydrosphingosine, is inhibited by cysteine. On the basis of these experiments, synthesis of dihydrosphingosine appears to proceed by a pyridoxal-P-dependent condensation of palmityl-CoA with serine to yield 3-ketodihydrosphingosine. TPNH-dependent reduction of this 3-keto intermediate then leads to dihydrosphingosine. The origin of other sphingolipid bases is discussed. 2-Hexadecenoate, but not α-hydroxypalmitate, replaces palmitate as a substrate in this crude cell-free system, but does not change the distribution of products. Phytosphingosine, the major sphingolipid base excreted by the growing yeast, is not formed in the cell-free system.