The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae

Abstract

The presence of the acyl dihydroxyacetone phosphate (acyl DHIAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vm, of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHLAP (Km = 15 ,uM) by NADPH (Km = 20 ,uM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction.Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHIAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.Biosynthesis of phosphatidic acid, the precursor of glycerolipids in all organisms, proceeds via two different routes. In the glycerol phosphate pathway, phosphatidic acid is biosynthesized via the stepwise enzymatic acylation of snglycerol 3-phosphate (GP) by long-chain fatty acyl coenzyme A's (acyl-CoAs). In animals, phosphatidate is also biosynthesized by an alternate pathway, the acyl dihydroxyacetone phosphate (acyl DHAP) pathway. In this pathway, DHAP is acylated by a long-chain fatty acyl-CoA to form acyl DHAP. This compound is then reduced to 1-acyl GP, which is further acylated to yield phosphatidate (Fig. 1). The GP pathway is universal to all organisms, including yeasts (19,27). In contrast, the acyl DHAP pathway is absent in bacteria and plants (11, 41). In animals, the acyl DHAP pathway is obligatory for ether lipid biosynthesis; the ether bond is synthesized by the substitution of a long-chain alcohol for the acyl group of acyl DHAP, with the release of free fatty acid to form alkyl DHAP (13). The enzymatic conversion of alkyl DHAP to glycerol ether lipids proceeds via reduction of alkyl DHAP to 1-alkyl GP followed by acylation (13). The importance of the acyl DHAP pathway for the biosynthesis of non-ether glycerolipids is controversial (6,29,30,33,34,35). Except for some anaerobic bacteria which contain plasmalogens but not alkyl glycerol ethers, both aerobic bacteria and facultative anaerobe...

Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae

“…In the second pathway, dihydroxyacetone phosphate is first acylated to acyl dihydroxyacetone phosphate, which is subsequently reduced in an NADPH-dependent reaction to lysophosphatidic acid (1, 16) and further converted to phosphatidic acid in a second acylation step. At present it is not known which of these two pathways is followed in vivo and whether enzymes that catalyze the acylation of glycerol-3-phosphate also accept dihydroxyacetone phosphate as a substrate and vice versa (16,22,23).In mammalian cells, phosphatidic acid is synthesized from glycerol-3-phosphate by two acyltransferase reactions catalyzed sequentially by glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferase. Two isoforms of glycerol-3-phosphate acyltransferases are postulated to exist in mammalian cells-one in mitochondria and the other in the endoplasmic reticulum (2).…”

“…In the second pathway, dihydroxyacetone phosphate is first acylated to acyl dihydroxyacetone phosphate, which is subsequently reduced in an NADPH-dependent reaction to lysophosphatidic acid (1, 16) and further converted to phosphatidic acid in a second acylation step. At present it is not known which of these two pathways is followed in vivo and whether enzymes that catalyze the acylation of glycerol-3-phosphate also accept dihydroxyacetone phosphate as a substrate and vice versa (16,22,23).…”

Potential of aSaccharomyces cerevisiaerecombinant strain lacking ethanol and glycerol biosynthesis pathways in efficient anaerobic bioproduction

“…However, S. cerevisiae possess the bypass reactions for glycerol-3-phoshate biosynthesis; glycerol-3-phoshate is produced from 1-acyl-dihydroxyacetone phosphate which is produced by acylation of dihydroxyacetone phosphate. [22][23][24][25] Thus, the GPD1 and GPD2 knockout strain is viable without nutrient supplementation. Additionally, the PDC1 gene encoding pyruvate decarboxylase involved in acetaldehyde biosynthesis, which is necessary for cytosolic acetyl-CoA biosynthesis, is also disrupted in our recombinant strain.…”

1-Acyldihydroxyacetone-phosphate Reductase (Ayr1p) of the YeastSaccharomyces cerevisiae Encoded by the Open Reading Frame YIL124w Is a Major Component of Lipid Particles

“…1-Acyl-DHAP formed during PA synthesis through the DHAP pathway is reduced to LPA by 1-acyl-DHAP reductase (ADR) (13). In animal cells, ADR is a key enzyme for the formation of ether lipids and acylglycerolipids via the DHAP pathway (14).…”

“…Amino acid analysis of acyl/alkyl-DHAP reductase purified from guinea pig liver peroxisomes revealed that hydrophobic amino acids composed 27% of the molecule; the amino acid sequence of this protein, however, was not determined. In the yeast S. cerevisiae ADR has been detected by its enzymatic activity (13), but neither the gene nor the gene product was characterized at a molecular level. Yeast ADR activity is present in lipid particles and the ER (30,000 ϫ g microsomes), whereas mitochondria appear to be devoid of this enzyme (8).…”