Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities

Abstract

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.

Phospholipid‐synthesizing enzymes in Golgi membranes of the yeast, Saccharomyces cerevisiae

“…CDP [methyl-~4C]choline (final concentration 0.3 mM) was incubated with 125 mg protein in the presence of 50 mM MOPS/NaOH, pH 7.5, 20 mM MgC12 in a total volume of 0.2 ml. Glycerol-3-phosphate acyltransferase was assayed by the method of Schlossmann and Bell [21]. Phosphatidylethanolamine N-methyltransferase and phospholipid N-methyltransferase were assayed essentially as reported by Kodaki and Yamashita [22] with the modifications described by Gaigg et al [23].…”

Phosphatidic acid, a key intermediate in lipid metabolism

“…Whereas the Gro3 P pathway is the ‘universal’ route of PtdOH formation in prokaryotes, plants, yeast and mammalian cells, enzymes needed for the first two steps of PtdOH formation in the Grn P pathway, namely Grn P AT and 1‐acyl‐Grn P reductase, are only present in yeast and mammals [4–8]. The rate limiting reaction in PtdOH formation is the first step of acylation catalyzed either by Gro3 P AT or Grn P AT [9].…”

“…In yeast, PtdOH can be synthesized either by using Gro3 P or Grn P as a precursor [7,8,28]. Yeast acyltransferases involved in the first step of acylation of Gro3 P or Grn P have not yet been identified at a molecular level.…”

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

“…Preparation of substrates. [2-3H]glycero-3-P was synthesized by the glycerol kinase-dependent phosphorylation of [2-3H]glycerol by the procedure of Chang and Kennedy (6) with the modifications of Schlossman and Bell (26). CDP-diacylglycerol synthesized from soybean lechithin-derived 1,2-diacyl-snglycerol-3-P (16) was prepared by the method of Agranoff and Suomi (1) with the modifications of Raetz and Kennedy (25).…”