Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Abstract

Soluble acyl-CoAsn-glycerol 3-phosphate acyltransferases (EC 23.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oke.aca and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.The enriched enzyme fractions possessed a specificity for glycerol 3-phosphate as acyl acceptor and did not use dihydroxyacetone phosphate. Besides acyl-CoA, acyl-acyl carrier protein also can function as acyl donor. With acyl-CoA as acyl donor, the enzyme shows a high positional specificity, since the predominant product is 1-acylglycerol 3-phosphate.Different acyl-CoAs, when offered separately, were all accepted as substrates, whereas incubations with mixtures of palmitoyl-, stearoyl-, and oleoyl-CoA demonstrated a preference for olec acid. The acyltransferase from spinach displays higher selectivity than does the enzyme from pea and, therefore, may be responsible for the preferred esterification of oleic acid at the C-1 position in chloroplast lipids and the exclusion of palmitic acid from this position as observed during in vivo labeling experiments.When membrane lipids contain different fatty acids, they are usually distributed asymmetrically between positions C-1 and C-2 of the glycerol backbone. In leaf lipids, palmitic acid and unsaturated C18-fatty acids such as linoleic and linolenic acid predominate. In so-called 16:3-plants, in addition to linolenic acid, the lower homologue hexadecatrienoic acid is present and accumulates specifically in monogalactosyl diacylglycerol (13). Positional analyses have shown that this acid is confined to C-2 in this glycolipid, whereas linolenic acid is found in both positions (12). When these analyses were extended to labeled lipids from leaves or chloroplasts of 16:3-plants, common patterns were observed in all chloroplast lipids. After short labeling times, they carried mainly oleate at C-I and palmitate at C-2 (16,22,26,27), which suggests a common diacylglycerol precursor. The only exception in chloroplasts is phosphatidyl choline, which carries palmitate esterified at the C-1 position.The discovery that, apart from other organelles also chloroplasts (3, 15) contain acyl-CoA:glycerol 3-P acyltransferase activity suggested that this enzyme may be involved in the control of fatty acid distribution in chloroplast lipids, since it catalyzes the first acylation step in the de novo synthesis ofcomplex lipids. Therefore, we purified this chloroplast enzyme to investigate its general 'This work was supported by the Deutsche Forschungsgemeinschaft.properties as well as its positional and fatty acid specificity. The results show tha...

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus

“…We studied the response of the HaPLSB enzyme to pH using palmitoyl-ACP as an acyl donor, observing maximum HaPLSB activity at pH 6.9 that decreased significantly up to pH 8.0. This pH curve ( Figure 6C) is similar to that reported for the pea chloroplast GPAT (Bertrams and Heinz, 1981), for which the optimal pH value is 7.4. A recent study reported that the activity of the plastidial GPAT from eight different plant species at pH 7.4 is lower than at pH 8.0 (Zhu et al, 2009), showing that plastidial GPAT activity responds to pH changes.…”

Phosphatidylglycerol Synthesis in Spinach Chloroplasts: Characterization of the Newly Synthesized Molecule

“…Under the conditions described previously (20), total acetate incorporation into lipid was linear up to I h after which the incorporation tapered off and by 3 …”

A distinct type of glycerol-3-phosphate acyltransferase with sn -2 preference and phosphatase activity producing 2-monoacylglycerol

“…Immediately after GPAT assay, 0.1 M borate buffer (pH 7.5, 1 mL) and 0.1 M Tris·HCl (pH 7.5, 1 mL) were added. Dephosphorylation (17) was started by adding 1 μL of Escherichia coli alkaline phosphatase (0.25 unit) and continued for 30 min at room temperature. Products were extracted by diethyl ether, and MAGs were separated by borate-TLC.…”