The biosynthesis of dihydrosphingosine in cell-free preparations of Hansenula ciferri.

Studies of the biosynthesis of sphingosine and dihydrosphingosine in whole animals'-4 and in brain tissue homogenates5-8 indicate that the first two carbon atoms of these compounds are derived from serine while the remainder of the molecule is derived from palmitic acid or palmitaldehyde. Brady, Formica, and Koval6 reported that pyridoxal phosphate (PLP), Mn++, nicotinamide, cytidine 5'-diphosphate (CDP)-choline, and several other cofactors stimulated the synthesis of sphingosine from serine in cell-free particulate preparations of rat brain. In subsequent experiments by these and other investigators, PLP and Mn++ were routinely added to the reaction mixtures.9 Even though several PLP-dependent mechanisms for the condensation of serine with a 16-carbon derivative have been suggested,4a requirement for this coenzyme in this specific reaction has never been clearly demonstrated. The very low rate of sphingolipid base synthesis in brain extracts has directed attention to the yeast Hansenula ciferri as a potential source of an enzyme system capable of synthesizing sphingolipids at a higher rate. 10 This yeast is able to produce large quantities of acetylated phytosphingosine and, to a lesser extent, acetylated dihydrosphingosine."1-"4 In experiments with intact cells and radioactive precursors, Green, Kaneshiro, and Law10 showed that phytosphingosine and dihydrosphingosine are derived from serine and palmitic acid, presumably by a pathway resembling that found in brain tissue. These investigators, however, were unable to demonstrate the formation of these lipids in vitro. More recently, Haskell and Snell" showed that the vitamin B6-deficient yeast, Hanseniaspora valbyensis, contained lowered amounts of phytosphingosine, thus providing nutritional evidence for a role of this vitamin in sphingolipid biosynthesis. The postulated role for pyridoxal phosphate in the synthesis of sphingolipids is unusual for PLP-enzymes in that the serine carboxyl is replaced by a carbon chain derived from palmitate rather than by a hydrogen atom. We have therefore investigated the enzymatic aspects of this reaction, and present herein experiments that demonstrate synthesis of dihydrosphingosine from palmityl-CoA and serine by a cell-free particulate fraction of H. ciferri, and that PLP is required in the condensation reaction. Experimental Procedures.-Materials: Substrates and cofactors not specified below were commercially available products. Palmityl-CoA was synthesized according to the procedure of Seubert.'6 Chromatographic samples of authentic sphingolipid bases were gifts from Dr. H. E. Carter. Growth of organisms and preparation of enzyme: Hansenula ciferri (NRRL Y-1031, mating type F-60-10), provided through the courtesy of Dr. L. J. Wickerham, was grown in the New Brunswick fermentor on yeast maintenance medium", 14 containing 20 gm of glucose per liter. An aeration rate of 5 liters per minute and a temperature of 250 provided an optimal enzyme yield when the cells were harvested at 36 hr. The fresh cells were washed twice with 10 volumes of cold, distilled water, then suspended in 2 volumes of 0.05 M potassium phosphate buffer (pH 7.0) containing dithjothrejto (U milV) and disrupted in an Aminco pressure cell at a pressure of 20,000 psi. Whole