Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

Purification of ethanolaminephosphotransferase from bovine liver microsomes.

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.

CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase.

Ethanolaminephosphotransferase catalyzes the final step of the CDP-ethanolamine pathway for the de novo synthesis of phosphatidylethanolamine (PtdEtn) via transfer of a phosphoethanolamine moiety from CDP-ethanolamine to diacylglycerol for the formation of PtdEtn and CMP. Ethanolaminephosphotransferase is an integral membrane-bound enzyme whose intracellular location defines the site of PtdEtn synthesis by the CDP-ethanolamine pathway. Subcellular fractionation experiments have yet to resolve the precise subcellular location of ethanolaminephosphotransferase, although it is routinely associated with the microsomal fraction. Ethanolaminephosphotransferase has yet to be purified from any source and its cDNA has not been isolated from any mammalian source, thus preventing the generation of antibodies necessary to directly examine its intracellular location through immunofluorescence or electron microscopy approaches. An ethanolaminephosphotransferase gene has recently been isolated from the yeast Saccharomyces cerevisiae and structure/function analyses of the encoded enzyme identified several important characteristics including the catalytic site. The predicted amino acid sequence of the S. cerevisiae ethanolaminephosphotransferase gene should allow for the generation of antibodies required to directly define the site of PtdEtn synthesis in this organism, and it has provided the necessary information to pursue the isolation of a mammalian cDNA.

Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.

The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.

Hamster liver cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes.

CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) are microsomal enzymes that catalyze the final steps in the syntheses of phosphatidylcholine and phosphatidylethanolamine via the CDP-choline and CDP-ethanolamine pathways, respectively. Both enzyme activities were cosolubilized from hamster liver microsomes by Triton QS-15. Limited separation of these two activities was achieved by ion-exchange chromatography. The partially purified phosphotransferases displayed a higher sensitivity than microsomal phosphotransferases towards exogenous phospholipids and showed an absolute requirement for divalent cations. Upon purification, cholinephosphotransferase was more stable to heat treatment than ethanolaminephosphotransferase. The two enzymes exhibited distinct pH optima and responded differently to exogenous phospholipids. Our results clearly indicate that cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes.

An improved procedure for the purification of ethanolaminephosphotransferase. Reconstitution of the purified enzyme with lipids.

Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.

Partial purification of ethanolaminephosphotransferase from rat brain microsomes.

Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) was solubilized by treating rat brain microsomes with buffered solutions containing octyl glucoside or Triton X-100. The solubilized enzyme was stable both at 4 degrees C and at -18 degrees C. A partial purification was obtained using an ion-exchange chromatographic procedure. The partially purified enzyme showed four major bands in SDS-polyacrylamide gel electrophoresis; its specific activity was increased by a factor of 37 compared to that of the membrane-bound enzyme. Glycerol and diacylglycerol were effective as stabilizers. Phosphatidylcholine, lysophosphatidylcholine and phosphatidylserine increased both the specific activity and the stability of the partially purified enzyme.