Acyl coenzyme a preference of diacylglycerol acyltransferase from the maturing seeds of cuphea, maize, rapeseed, and canola

In their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations from maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of triacylglycerols from the acyl groups at 24, 32, and 40°C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24°C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40°C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology.     

Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed

The acyl coenzyme A (CoA) preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm (Butia capitata Becc.), maize (Zea mays L.), and rapeseed (Brassica napus L.) was tested. Each microsomal preparation was incubated with [¹⁴C-U]-glycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the ¹⁴C-lipid products were separated and quantitated. In the presence of oleoyl CoA, the microsomes from each of the three species produced lysophosphatidic acid, phosphatidic acid, diacylglycerol, and triacylglycerol with kinetics consistent with the operation of the glycerol phosphate pathway. In the presence of erucoyl CoA, the microsomes from all the three species did not produce di- or tri-acyl lipids. In the presence of lauroyl CoA, only the microsomes from palm, but not those from maize or rapeseed, synthesized di- and tri-acyl lipids. This lack of reactivity of lauroyl CoA was also observed in the microsomes from maturing castor bean, peanut, and soybean. In maize seed and rapeseed, but not palm seed, the kinetics of labeling suggest that lauroyl and erucoyl moieties of the acyl CoAs were incorporated into lysophosphatidic acid but failed to enter into phosphatidic acid and thus the subsequent lipid products. We propose that the high degree of acyl specificity of lysophosphatidyl acyltransferase is the blocking step in the synthesis of triacylglycerols using lauroyl CoA or erucoyl CoA. The significance of the findings in seed oil biotechnology is discussed.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Metabolism of hepatic haem and `green pigments'' in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover

The acyl specificities of several acyltransferases located in the microsomal fraction of lactating rat mammary gland have been investigated using palmitate and oleate as substrates along with CoA, ATP and Mg²⁺, bovine serum albumin and NaF. With either sn-glycerol 3-phosphate or dihydroxyacetone phosphate (plus NADPH) as acyl acceptor, phosphatidic acid containing palmitate preferentially esterified at position-2 and oleate at position-1 was the major product. Dihydroxyacetone phosphate and sn-glycerol 3-phosphate competitively inhibited each other's acylations, suggesting that a single enzyme might be responsible for both esterifications and oleate was the preferred substrate for the formation of acyldihydroxyacetone phosphate. The specificities of the acyl-CoA–1-monoacyl-sn-glycerol 3-phosphate and the acyl-CoA–2-monoacyl-sn-glycerol 3-phosphate acyltransferases were also studied. The specificities observed combined with the relative velocities of these reactions suggest that phosphatidic acid is formed in the mammary gland with the first acylation occurring at position-1 favouring oleate followed by the second acylation at position-2 favouring palmitate. This is consistent with the unusual structure found in the triacylglycerols of rat milk. When a mouse liver microsomal fraction was used the opposite specificities were observed consistent with the structure of the triacylglycerols of mouse liver. The microsomal acylation of the monoacyl-sn-glycerol 3-phosphocholines was also investigated. Although no marked acyl specificity could be detected when the 2-monoacyl-sn-glycerol 3-phosphocholine was used as the acyl acceptor, both oleate and linoleate were esterified in preference to palmitate to the 1-monoacyl-sn-glycerol 3-phosphocholine.     

Diacylglycerol acyltransferase in maturing oil seeds of maize and other species

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts.     

Hepatic lipid metabolism: effect of spermine, albumin, and Z protein on microsomal lipid formation.

Effects of spermine, bovine serum albumin, and Z protein on microsomal lipid formation from sn-glycerol 3-phosphate and [¹⁴C]palmitoyl CoA were investigated. In the presence of these agents, microsomal lipid formation was stimulated. This was attributed to the activation of sn-glycerol 3-phosphate acyltransferase and to the inhibition of palmitoyl CoA hydrolase. In addition to palmitoyl CoA, spermine also reacted with microsomal membranes in causing their aggregation, and ATP reversed the effect of spermine. Further studies indicated that the interaction of spermine with palmitoyl CoA, rather than with microsomal membranes, was responsible for the activation of glycerolipid formation or to the inhibition of palmitoyl CoA reductase. Examination of the intravesicular distribution of sn-glycerol 3-phosphate acyltransferase and palmitoyl CoA hydrolase and the effects of structural integrity of microsomal vesicles on these two membrane-bound enzymes indicated that the activation of glycerolipid formation and the inhibition of palmitoyl CoA hydrolase by spermine, bovine serum albumin, or Z protein may be closely linked with the structural integrity of microsomal vesicles.     

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B₁₂-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.     

A NOVEL ASSAY OF ACYL‐COA:DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY UTILIZING FLUORESCENT SUBSTRATE1

Common methods for assaying acyl‐CoA:diacylglycerol acyltransferase (DGAT) enzymatic activity rely upon radiolabeled substrates or product assay. We developed a novel assay that directly quantifies endogenous DGAT activity through the use of a fluorescently labeled substrate. We performed this assay with microsomal protein, 2‐(6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)hexanoyl‐1‐hexadecanoyl‐sn‐glycero‐3‐diacylglycerol (NBD‐DAG), and oleoyl‐CoA substrates. DGAT activity was analyzed in three species of algae as well as rat liver. The protocol proved to be sensitive and reliable. This assay may be used to facilitate research in the areas of biodiesel, oilseed crops, and triacylglycerol‐related human pathologies.     

Update and nomenclature proposal for mammalian lysophospholipid acyltransferases,which create membrane phospholipid diversity

The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A_1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.     

Phosphatidate phosphatase from avocado (Persea americana) – purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signalling in plants

Phosphatidate phosphatase (PAP; EC 3.1.3.4) is a pivotal enzyme in plant lipid metabolism positioned at a major branchpoint between the biosyntheses of phospholipids and triacylglycerols. The enzyme has been purified 7000-fold from the microsomes of maturing Avocado fruit. The enzyme has a subunit molecular mass, as determined on SDS-PAGE, of 49 kDa, and gel filtration studies revealed that it is monomeric. The enzyme was examined for the ability to hydrolyse sn-1,2-dioleoylglycerol-3-phosphate (PA), sn-1-oleoylglycerol-3-phosphate (LPA), sn-2-oleoylglycerol-3-phosphate (sn-2-LPA), and ceramide-1-phosphate. All substrates were used, but the apparent V_max values for ceramide-1-phosphate and sn-2-LPA were considerably lower than for PA or LPA. A kinetic analysis of the purified enzyme was conducted using PA and LPA in Triton X-100 mixed micelles according to the surface dilution kinetic model. The V_max and interfacial Michaelis constant (Km^B) were calculated for PA as 47 μmol min^–1 mg^–1 and 1.49 mol%, respectively. LPA was found to be a better substrate with a V_max and Km^B of 100 μmol min^–1 mg^–1 and 1.06 mol%, respectively. A detailed kinetic analysis of the effect of LPA on PA hydrolysis revealed LPA to be a potent competitive inhibitor of the reaction, with a K_i of 1.4 mol%. This possibly indicates that strict metabolic channelling is in operation in the Kennedy Pathway, with very low steady state concentrations of lyso-phosphatidate with respect to phosphatidate in vivo.     

Evolution of Acyl-Substrate Recognition by a Family of Acyl-Homoserine Lactone Synthases

Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family.     

Metabolic interrelations of hydroxy-substituted ether-linked glycerolipids in the pink portion of the rabbit harderian gland.

The pink portion of the rabbit harderian gland is known to contain a preponderance of ether-linked glycerolipids consisting primarily of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols and smaller amounts of 1-alkyl-2,3-diacyl-sn-glycerols. In the present study, we have used a combination of chemical, enzymatic, and chromatographic techniques to identify two minor lipid components in the gland as 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols. The long-chain acyl groups occurring in the 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols are almost exclusively hexadecanoic acid, whereas the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols have a ratio of hexadecanoic acid to octadecanoic acid of $\text{2}\text{1}$. The 1-(O-acyl) hydroxyalkyl-2,3-diacyl-sn-glycerols and the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols also contain a short-chain acyl moiety identified as 3-methylbutanoic acid (isovaleric acid). This acid was found to occupy the 3-position of the glycerol backbone in these lipid classes.Metabolic experiments demonstrate that 3-methylbutanoic acid in the lipids of the gland is derived from the catabolism of l-leucine. Pulse-chase data show a precursor-product relation between the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols and 1-(O-acyl-hydroxyalkyl-2,3-diacyl-sn-glycerols and rule out direct hydroxylation of 1-alkyl-2,3-diacyl-sn-glycerols as a possible biosynthetic route to the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols.Characterization of the alkyl and acyl groups and the positional distributions of the acyl moieties in combination with the metabolic information indicated the acylation sequence involved in the formation of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerol is 1-hydroxyalkyl-2-acyl-sn-glycerols → 1-hydroxyalkyl-2,3-diacyl-sn-glycerols → 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols. The data also suggest that hydroxylation of the alkyl side-chain occurs before or at the alkylacylglycerol stage.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.