Developing a High-Throughput Assay for the Integral Membrane Glycerol 3-Phosphate Acyltransferase.

Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a potential target for the development of antibiotics. A convenient enzymatic assay should facilitate such drug discovery activities. Previously, we developed a continuous assay by monitoring phosphate, one of the enzymatic product, using a fluorescently labeled phosphate binding protein in a bilayer environment called lipid cubic phase (LCP). However, some intrinsic characteristics of LCP, such as high viscosity, make the assay incompatible with common high-throughput liquid-handling platforms. Here, we adapted the assay by hosting PlsY in detergent micelles, enabling us to conduct the assay using standard multi-channel pipets in a high-throughput manner. With optimal enzyme loading, the reaction velocity was linear up to 30 min. PlsY showed Michaelis-Menten kinetics behavior in micelles with a Vmax of 57.5 µmol min-1 mg-1, and Kmof 1.14 mM G3P and 6.2 µM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19 µM. The results principally demonstrated the feasibility of using the assay for high-throughput screening, and the protocol provided an encouraging starting point for further optimization and validation of the assay for automated platforms.

sn-Glycerol-3-phosphate acyltransferase from Escherichia coli.

This review attempts to capture the history of research involved in the understanding of lipid metabolism via investigation of the sn-glycerol-3-phosphate acyltransferase (glycerol-P acyltransferase), the first step in the synthesis of lipids in E. coli. We will review the original identification of this enzymatic activity and its subsequent characterization. The biochemical and genetic regulation of this enzyme and gene are discussed, as well as the unique structural characterization of this integral membrane protein. Future perspectives regarding the regulatory and structural aspects of this key enzyme are discussed.

Solubilisation, partial purification and properties of acyl-CoA: glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp.

Glycerol-3-phosphate acyltransferase (G3PAT) activity was studied using a microsomal membrane fraction from avocado (Persea americana) mesocarp. G3PAT was shown to be an integral membrane protein, having an active site that appeared to be accessible to the cytoplasmic face of the endoplasmic reticulum, in experiments using limited proteolytic digestion. CHAPS solubilisation (0.25%, w/v) of microsomal G3PAT activity was used as an initial step in purification of this enzyme. Both CHAPS-solubilised and microsomal G3PAT activities were characterised and compared. Affinity chromatography was used to purify microsomal G3PAT for the first time from a plant source. Glycerophosphorylethanolamine coupled to cyanogen bromide-activated Sepharose was used for this purpose. Specific elution of G3PAT by a solution of glycerol-3-phosphate resulted in about 150-fold purification. The significance of the results and the potential usefulness of the purification method for further studies of G3PAT in plants are discussed.

The triose-phosphate site of homogeneous reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

The sn-glycerol-3-phosphate (glycerol-phosphate) acyltransferase of Escherichia coli was purified to near homogeneity and its activity reconstituted with phospholipids (Green, P.R., Merrill, A.M., Jr. and Bell, R.M. (1981) J. Biol. Chem. 256, 11151-11159). The competency of glycerol-P analogues to serve as inhibitors and as substrates was investigated. Dihydroxyacetone-P, ethyleneglycol-P, 1,3-propanediol-P, 3,4-dihydroxybutylphosphonate and DL-glyceraldehyde-3-P were inhibitors of the reconstituted purified glycerol-phosphate acyltransferase. The kinetics of inhibition, while formally of the mixed type, most closely resembled that of a simple competitive inhibition with respect to glycerol-3-P. Inorganic phosphate was also found to be a competitive inhibitor. All of the glycerol-3-P analogues except DL-glyceraldehyde-3-P were substrates. Of these, dihydroxyacetone-P proved to be the best substrate. The secondary hydroxyl was not necessary for activity. Glycerol-phosphate acyltransferase catalyzed the hydrolysis of palmitoyl-CoA in the presence of DL-, but not D-glyceraldehyde-3-P. This suggests that the gem diol of L-glyceraldehyde-3-P may be a substrate, and that the acylated adduct may be unstable. The enzyme was inactivated by phenylglyoxal and butanedione, suggesting that arginine may be at or near the active site.

Characterization of reconstituted partially purified glycerophosphate acyltansferase from Escherichia coli.

A modification of the method of Snider and Kennedy (J. Bacteriol. (1977) 130, 1072-1083) was worked out to solubilize sn-glycero-3-phosphate acyltransferase from whole cells by Triton X-100. The solubilized preparation was used for a systematic study of the reconstitution of enzymatic activity as observed by addition of phospholipid vesicles. Although enzymatic activity was regained by addition of vesicles and not by addition of multilayered liposomes, subsequent Sepharose 4B chromatography revealed the enzyme to be incorporated in large lipid aggregates of undefined structures. Incorporation of glycerophosphate acyltransferase in single bilayer vesicles composed of phosphatidylcholine and phosphatidylglycerol (4:1) was obtained after removal to Triton X-100 from the enzyme solution, co-dispersion of enzyme and phospholipids with cholate and Sephadex G-50 gel filtration of this mixture to remove cholate. The optimal conditions for this reconstitution procedure with respect to phospholipid/protein and phosphatidylcholine/phosphatidylglycerol ratio were established. The active site of glycero-3-phosphate acyltransferase in the reconstituted system was localized for at least 90% at the outside surface of the vesicle, as revealed by proteolysis experiments under conditions of vesicle intactness as shown by C-NMR experiments. The reconstituted systems produced only lysophosphatidate from sn-[14C]glycero-3-phosphate and palmitoyl-CoA and showed identical apparent Km for sn-glycero-3-phosphate and identical pH- and temperature-dependencies as the enzyme in isolated Escherichia coli membranes.

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.

Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli.

Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.

Evidence for interactions of acyl carrier protein with glycerol-3-phosphate acyltransferase, an inner membrane protein of Escherichia coli.

We [(1989) FEBS Lett., in press] have previously shown that membrane vesicles from Escherichia coli contain protein-binding sites for the acyl carrier protein (ACP). We report now that membrane vesicles prepared from a strain amplified for glycerol-3-phosphate acyltransferase (GPAT) contain a higher number of ACP-binding sites than the membrane vesicles prepared from a wild type strain. In addition, we show that GPAT is retained specifically on an ACP-Sepharose affinity column and that [3H]ACP binds to the enzyme solubilized by detergent. We conclude that GPAT, an inner membrane protein which catalyses the transesterification of a fatty acyl group from acyl coenzyme A or acyl ACP to glycerol-3-phosphate, possesses a binding site for ACP.

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes.

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.

Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds.

The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis-Menten K(m) for the glycerol 3-phosphate was 354 muM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.

Localization and characterization of the sn-glycerol-3-phosphate acyltransferase in Rhodopseudomonas sphaeroides.

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds.

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.

Purification and characterization of thiol-reagent-sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus.

Glycerol-3-phosphate acyltransferase (GPAT), responsible for the first committed, rate-limiting, step of glycerolipid synthesis, was purified to homogeneity from the membrane fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the membrane fraction by pretreatment with 0.05% Triton X-100 and treatment of the resulting pellet with 0.3% Triton X-100. The enzyme was subsequently purified by column chromatography on heparin-Sepharose, Yellow 86 agarose, a second heparin-Sepharose column, Superdex-200 and hydroxylapatite Bio-Gel. Enzyme activity was finally enriched 1308-fold over that of the starting membrane fraction. SDS/PAGE of the purified fraction revealed a single band with a molecular mass of 45 kDa. Native PAGE showed a major band that corresponded to GPAT activity. Enzyme activity was inhibited by thiol reagents, suggesting that it originated from microsomes rather than mitochondria. Purified GPAT depended on exogenous oleoyl-CoA and sn-glycerol-3-phosphate, with the highest activity at approx. 50 and 250 microM, respectively, and preferred oleoyl-CoA 5.4-fold over palmitoyl-CoA as an acyl donor. Anionic phospholipids, such as phosphatidic acid and phosphatidylserine, were absolutely required for activity of the purified enzyme, and their ability to activate GPAT was influenced by the purity of the GPAT preparation. Bivalent cations, such as Mg(2+) and Ca(2+), inhibited purified GPAT activity, whereas 5 mM Mn(2+) elevated activity approx. 2-fold. These results provide new insights into the molecular characterization of microsomal GPAT, which has not been well characterized compared with mitochondrial and plastidic GPAT.

Comparison of 1-acylglycerophosphate and glycerophosphate acyltransferases from Euglena microsomes.

Both glycerophosphate and monoacylglycerophosphate acyltransferases from Euglena microsomes were inhibited by N-ethylmaleimide, but their responses to heat inactivation and sn-glyceraldehyde-3-phosphate differed. Glycerophosphate acyltransferase had a higher V with palmitoyl-CoA compared to oleoyl-CoA; the reverse was true for monoacylglycerophosphate acyltransferase. Km's (microM) for the glycerophosphate acyltransferase were: palmitoyl-CoA, 21; oleoyl-CoA, 14; and sn-glycerol-3-phosphate, 2900. Km's (microM) for monoacylglycerophosphate acyltransferase were: palmitoyl-CoA, 7; oleoyl-CoA, 4; and 1-palmitoyl-sn-glycerol-3-phosphate, 48.