Long-chain Acyl Moieties Research Articles

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of thelatter binding sites and to identify substrates for which channeling is crucial.

Up to Date of Cyanobacterial Natural Products

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.

Alteration to the Skin Ceramide Profile Following Broad-Spectrum UV Exposure.

The epidermal stratum corneum (SC) lipid matrix, principally consisting of an equimolar ratio of ceramides, free fatty acids, and cholesterol, plays a crucial role in maintaining proper skin barrier function. Conditions which impair barrier integrity, such as in atopic dermatitis, correlate with the alternation of key ceramide subclasses and reduced chain length of acyl moieties. However, there is limited knowledge about the impact of unprotected repeat sun exposure on the skin lipid composition, especially ceramide profiles.This study investigated the effects of ultraviolet (UV) radiation on the ceramide profile using both an ex vivo skin and a clinical model. Lipidomic analysis of UV-exposed skin showed shifts to the composition of ceramide subclasses essential in repairing and strengthening the SC barrier (including CER1[EOS], CER3[NP], and CER6[AP]) and reduced very long-chain acyl moieties. Gene expression analysis and immunohistochemical staining of key enzymes (aSMase, DES1, CerS5, CerS3) suggested that lipid alterations can be attributed to changes within the ceramide biosynthesis process. Topical application of ceramide-containing suncare products help maintain SC-essential ceramide subclasses and proper ceramide chain length, demonstrating the importance of proper photoprotection to maintain healthy skin barrier and ceramide quality during daily sun exposure. J Drugs Dermatol. 2022;21(1):77-85. doi:10.36849/JDD.6331.

Alteration to the Skin Ceramide Profile Following Broad-Spectrum UV Exposure

The epidermal stratum corneum (SC) lipid matrix, principally consisting of an equimolar ratio of ceramides, free fatty acids, and cholesterol, plays a crucial role in maintaining proper skin barrier function. Conditions which impair barrier integrity, such as in atopic dermatitis, correlate with the alternation of key ceramide subclasses and reduced chain length of acyl moieties. However, there is limited knowledge about the impact of unprotected repeat sun exposure on the skin lipid composition, especially ceramide profiles.This study investigated the effects of ultraviolet (UV) radiation on the ceramide profile using both an ex vivo skin and a clinical model. Lipidomic analysis of UV-exposed skin showed shifts to the composition of ceramide subclasses essential in repairing and strengthening the SC barrier (including CER1[EOS], CER3[NP], and CER6[AP]) and reduced very long-chain acyl moieties. Gene expression analysis and immunohistochemical staining of key enzymes (aSMase, DES1, CerS5, CerS3) suggested that lipid alterations can be attributed to changes within the ceramide biosynthesis process. Topical application of ceramide-containing suncare products help maintain SC-essential ceramide subclasses and proper ceramide chain length, demonstrating the importance of proper photoprotection to maintain healthy skin barrier and ceramide quality during daily sun exposure. J Drugs Dermatol. 2022;21(1):77-85. doi:10.36849/JDD.6331Broad-Spectrum UV Exposure

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeoleiVT8.

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.

High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes

BackgroundBiotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.ResultsFor optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of wax esters, containing 27–34 mol% oleyl oleate.ConclusionsThe overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.

Validated method for the quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples.

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil.

Seed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1∆c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1∆c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.

Application of lipid biomarker analysis to evaluate the function of ‘slab-lined pits’ in Arctic Norway

Gas chromatography–mass spectrometry (GC–MS) and bulk carbon isotope determinations have been performed on samples (‘cemented organic residues’, charcoal, sediment and fire-cracked rock) excavated from 12 slab-lined pits from various locations in Arctic Norway to test the premise that these archaeological features were used for the extraction of oil from the blubber of marine mammals, such as seal, whale and walrus. A wide range of lipid compound classes were detected especially in the cemented organic residues and in the charcoal samples. The presence of long-chain unsaturated and isoprenoid fatty acids together with oxidation and thermal alteration products of unsaturated acids such as dicarboxylic acids, dihydroxyfatty acids and ω-(o-alkylphenyl)alkanoic acids suggests that these features were used for marine oil extraction at elevated temperatures. Notably the location of the hydroxyl groups in the dihydroxyfatty acids provides a record of the positional isomer of the precursor fatty acid and allows confirmation that 11-docosenoic (cetoleic) acid, the most abundant C 22:1 isomer in marine oil, was a major component of the original lipid. Further information was provided by the presence of long-chain fatty acyl moieties in surviving triacylglycerols and the presence of cholesterol. A fungal metabolite, mycose (trehalose), was found in all samples apart from a fire-cracked rock and points to microbiological activity in the pits. Bulk isotope analysis conducted on the ‘cemented organic residues’ is consistent with modern reference samples of blubber and oil from seal and whale. These data provide clear analytical evidence of the function of slab-lined pits in the archaeological record and suggest widespread exploitation of marine mammals for producing oil for heating, lighting and myriad other uses in the past.

Molecular species-specific differences in composition of triacylglycerols of mouse adipose tissue and diet

It has been demonstrated that the composition of molecular species of triacylglycerol (TG) in adipose tissue broadly reflects dietary TG molecular species composition. Medium-chain fatty acids (FAs) are not accumulated easily relative to long-chain FAs in body fat. The objective of the present study was to examine whether medium-chain FAs were incorporated into body fats. To this end, the composition of TG molecular species stored in the epididymal adipose tissues of mice fed a coconut oil diet was analyzed by gas chromatography. Most TG molecular species detected in mice adipose tissues contained long-chain fatty acyl moieties, even though dietary coconut oil is rich in TG molecular species composed of medium-chain fatty acyl moieties. The major medium-chain FAs in the dietary oil, decanoic, and lauric acids were seen in TG molecular species having 1 or 2 long-chain acyl moieties, such as palmitoyl and oleoyl. These results suggest that molecular species composition of TG in mice adipose tissue is affected by coconut oil. In summary, our findings demonstrate that the metabolic processes for TG molecular species with long- and/or medium-chain FAs in TG synthesis and storage differ in the intestinal lumen and adipose tissue.

Effect of Modification of the Length and Flexibility of the Acyl Carrier Protein−Thioesterase Interdomain Linker on Functionality of the Animal Fatty Acid Synthase

A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain acyl moiety in the thioesterase active site.

Fatty Acid Synthase Dimers Containing Catalytically Active β-Ketoacyl Synthase or Malonyl/Acetyltransferase Domains in Only One Subunit Can Support Fatty Acid Synthesis at the Acyl Carrier Protein Domains of Both Subunits

A double-tagging, dual affinity chromatographic procedure, which permits isolation of dimers independently mutated in each subunit, has been exploited to probe the functional topology of the animal fatty acid synthase. Dimers were engineered in which the chain-terminating thioesterase reaction was compromised by mutation of the (active-site) serine residue in both subunits; these dimers assembled two long-chain fatty acyl moieties, which remained covalently linked to the 4'-phosphopantetheine residues of the two acyl carrier protein domains. Significantly, dimers that contained an additional mutation that compromised the activity of either the beta-ketoacyl synthase or malonyl/acetyltransferase activity in only one subunit also assembled two long-chain acyl moieties. In contrast, in a control experiment, introduction of an additional mutation that compromised the function of the acyl carrier protein domain in only one subunit resulted in the assembly of only one long-chain acyl moiety per dimer. Because the beta-ketoacyl synthase and malonyl/acetyltransferase domains are located near the amino terminus of the polypeptide and the acyl carrier protein domain near the carboxyl terminus, these results support a modified model for the animal fatty acid synthase in which head-to-tail functional contacts are possible both within as well as between subunits.

A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.

The regulation of proinsulin biosynthesis in pancreatic beta-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate proinsulin biosynthesis, but since insulin exocytosis and proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells. In MIN6 cells, glucose-induced proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both proinsulin synthesis (threshold 0.13-0.2 mM) and insulin release (threshold 0.2-0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM alpha-cyano-4-hydroxycinnamate). A combination of alpha-oxoisohexanoate and glutamine also stimulated proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the beta-cell. A consequence of increased anaplerosis in beta-cells is a marked increase in malonyl-CoA, which in turn inhibits beta-oxidation and elevates cytosolic fatty acyl-CoA levels. In the beta-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same beta-cells, these fatty acids in contrast inhibited glucose-induced proinsulin biosynthesis. This implies that neither fatty acyl moieties nor beta-oxidation are required for the metabolic stimulus-response coupling pathway specific for proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the beta-cell, as reflected by changes in the ratio of alpha-glycerophosphate to dihydroxyacetone phosphate. Although 16.7 mM glucose produced a significant rise in the alpha-glycerophosphate/dihydroxyacetone phosphate ratio, 1 mM pyruvate did not. It follows that the cytosolic redox state and fatty acyl moieties are not necessarily involved as secondary metabolic stimulus-coupling factors for regulation of proinsulin biosynthesis. However, the results indicate that glycolysis and the subsequent increase in anaplerosis are indeed necessary for this signalling pathway, and therefore an extramitochondrial product of beta-cell pyruvate metabolism (that is upstream of the increased cytosolic fatty acyl-CoA) acts as a key intracellular secondary signal for specific control of proinsulin biosynthesis by glucose at the level of translation.

Mitochondrial carnitine-acylcarnitine translocase deficiency presenting as sudden neonatal death: Postmortem diagnosis, prenatal exclusion in subsequent pregnancies, and biochemical specificity of the mitochondrial translocase

A breast-fed female infant died suddenly in the neonatal period at 31 hours of age with profound macrovesicular fatty infiltration of liver, kidney, and muscle on postmortem examination, suggestive of a defect in fatty acid β-oxidation. Fatty acid and palmitoylcarnitine oxidation studies and direct enzyme study of cultured skin fibroblasts suggested a deficiency in the oxidation of long-chain fatty acids distal to carnitine palmitoyltransferase I and before long-chain acyl–coenzyme A dehydrogenases. Deficient activity of carnitine-acylcarnitine translocase was demonstrated with intermediate levels of activity in the infant’s parents, consistent with autosomal recessive inheritance. Fatty acid oxidation studies showed deficient oxidation of fatty acids at all chain lengths from C 10:0 to C 24:0 , with partially reduced oxidation of C 26:0 fatty acid, indicating the occurrence of a single mitochondrial carnitine-acylcarnitine translocase and demonstrating the requirement in vivo for l -carnitine for mitochondrial transport of all medium- and long-chain fatty acyl moieties. The disorder may have been precipitated in this breast-fed infant by poor initial feeding, fasting stress, and the long-chain triglycerides of human milk. The severity of the disorder prompted prenatal diagnosis, and affected siblings were excluded in two subsequent pregnancies by fatty acid oxidation in cultured chorionic villus cells and amniocytes. (J Pediatr 1997;131:220-5)

Lipids of fish parasites and their hosts: fatty acids of phospholipids of Paratenuisentis ambiguus and its host eel ( Anguilla anguilla)

The fatty acid composition of various phospholipid classes, e.g. phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and sphingomyelins, of the fish endoparasite Paratenuisentis ambiguus and of intestinal tissue of its host, eel ( Anguilla anguilla), were investigated. Phospholipids from parasite and infected host intestine show considerable differences in the fatty acid compositions. High proportions of saturated long-chain and very long-chain acyl moieties were found in phosphatidylcholines, ethanolamines and serines of the parasite. Membrane phospholipids of P. ambiguus contain remarkably high proportions of polyunsaturated acyl moieties, in particular eicosapentaenoyl moieties (20:5 n-3), which undoubtedly originate from aquatic feed or host intestine. It is suggested that the parasite's membranes are stabilized by incorporation of high proportions of saturated long-chain and very long-chain fatty acids to counterbalance the destabilizing effects of the polyunsaturated fatty acids.

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.

Analysis of triacylglycerols containing cyclopropane fatty acids in Sterculia fostida (Linn.) seed lipids

The distribution of cyclopropene acyl moieties in triacylglycerols of Sterculiia foetida (Linn.) seed lipids was determined after conversion into oxo derivatives which are stable under GLC conditions and readily separated. The triacylglycerols after derivatization were analyzed by spectroscopic and chromatographic techniques. It was found that the triacylglycerols of S. foetida seed lipids are composed of four types of molecular species I-IV. The minor molecular species I contains common long-chain acyl moieties without a cyclopropene ring

The importance of hydroperoxide activation for the detection and assay of mammalian 5-lipoxygenase

Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1–5 μM) during the reaction which effected a 10–20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.

Pyruvate carboxylase from Saccharomyces cerevisiae. Quaternary structure, effects of allosteric ligands and binding of avidin.

The quaternary structure of pyruvate carboxylase purified from Saccharomyces cerevisiae was investigated by electron microscopic examination of negatively stained samples. In the most frequently observed projection form four intensity maxima were arranged at the corners of a rhombus; a cleft along the longitudinal axis of individual protomers could often be discerned. The observation of occasional triangular and dual-intensity projections and the interconversion of all three projection forms in tilting studies indicates that this tetrameric enzyme has a structure very similar to the tetrahedron-like configuration previously proposed for pyruvate carboxylases from vertebrate sources [Mayer, F., Wallace, J. C. and Keech, D. B. (1980) Eur. J. Biochem. 112, 265-272] and Aspergillus nidulans [Osmani, S. A., Mayer, F., Marston, F. A. O., Selmes, I. P. and Scrutton, M. C. (1984) Eur. J. Biochem. 139, 509-518]. An improved structural preservation of the enzyme was observed in the presence of either of the activators acetyl-CoA (250 microM) and palmitoyl-CoA (1-5 microM). At higher than 5 microM palmitoyl-CoA, although activity was further increased, dissociation of enzyme tetramers was evident, presumably because of the detergent effect of the long-chain acyl moiety. Two inhibitors of yeast pyruvate carboxylase, L-aspartate (10 mM) and 2-oxoglutarate (40 mM), added alone or together decreased significantly the proportion of intact tetramers even in the presence of acetyl-CoA or palmitoyl-CoA. When yeast pyruvate carboxylase was incubated with avidin, the formation of unbranched linear concatemers occurred at avidin:enzyme ratios between 2:1 and 1:2. Avidin molecules were sometimes bound asymmetrically to the enzyme, appearing to complex only one biotin group on each side of the enzyme. This appeared to permit kinking and circularization of some concatemers.

Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer.

We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.