Acyl Exchange Research Articles

Mlg1, a yeast acyltransferase located in ER membranes associated with mitochondria (MAMs), is involved in denovo synthesis and remodelling of phospholipids.

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids.

Efficiently and directly produce triacetylglycerol from oils and fats over mesoporous polymeric solid acid catalysts

A series of mesoporous solid acid catalysts (PDS-x) with large BET surface area, superior thermal stability, strong sulfonic acid groups and tunable Brønsted acidity were synthesized by the copolymerization of divinylbenzene (DVB) and sodium p-styrenesulfonate (SPSS). The PDS-x catalysts showed high activity on the direct and efficient production of triacetylglycerol (TAG) via acyl exchange reaction of oils (fats) and acetic acid. The highest TAG yield of 93.0 mol% was obtained on the PDS-x catalysts at a mild condition of 200 °C. By combining SEM, TEM, FT-IR/DRIFTS, BET, XRD, XPS, EA, 31P MAS NMR and TG-DTG, the key factors that affected the TAG yield were clarified on the PDS-x catalysts. Moreover, the origin of the slow deactivation on these catalysts was elucidated. The large BET surface area and abundant mesopores of catalysts allowed the contact of large-sized oils (fats) with the strong sulfonic acid groups, accounting for the high TAG yield.

Amphiphilic catalyst for decomposition of unsaturated polyester resins to valuable chemicals with 100% atom utilization efficiency

Plastic waste is increasingly aggravating environmental pollution problems. Unsaturated polyester resin is one kind of thermoset resins. Its three-dimensional network endows it good mechanical and chemical properties, but made its degradation and recovery challenging. Here we report a novel and efficient reaction system consisting of amphiphilic p-toluenesulfonic acid/acetic acid aqueous solution for decomposition of unsaturated polyester resin by acyl exchange reaction. Amphiphilic p-toluenesulfonic acid facilitated the entrance of itself and solvent into the resin and consequently promoted catalytic cleavage of ester bonds in unsaturated polyester resin. Water, as a co-catalyst, stabilized the key reaction intermediate species, which reduced largely the overall reaction activation energy and thus accelerated the degradation of the resins. Unsaturated polyester resin was completely degraded and converted to the valuable chemicals at 180 °C, which can be then recycled by a new separation process. The degradation products were mainly ethylene glycol diacetate, phthalic acid, and copolymer products of styrene and maleic anhydride, with the mass ratio of 43.18%, 24.81% and 44.16% to raw unsaturated polyester resin waste, respectively. The atoms of unsaturated polyester resin were completely incorporated into the high-value degradation products with zero production of waste. Hence, this work provides a green, economical, practical strategy to degrade waste unsaturated polyester resin and recycle high-value chemicals with an 100% atom utilization efficiency.

Techniques for the Measurement of Molecular Species of Acyl-CoA in Plants and Microalgae.

The acyl-CoA pool is pivotal in cellular metabolism. The ability to provide reliable estimates of acyl-CoA abundance and distribution between molecular species in plant tissues and microalgae is essential to our understanding of lipid metabolism and acyl exchange. Acyl-CoAs are typically found in low abundance and require specific methods for extraction, separation and detection. Here we describe methods for acyl-CoA extraction and measurement in plant tissues and microalgae, with a focus on liquid chromatography hyphenated to detection techniques including ultraviolet (UV), fluorescence and mass spectrometry (MS). We address the resolution of isobaric species and the selection of columns needed to achieve this, including the analysis of branched chain acyl-CoA thioesters. For MS analyses, we describe diagnostic ions for the identification of acyl-CoA species and how these can be used for both discovery of new species (data dependent acquisition) and routine quantitation (triple quadrupole MS with multiple reaction monitoring).

Efficiently and Directly Produce Triacetylglycerol from Oils and Fats Over Mesoporous Polymer Solid Acid Catalysts

A series of mesoporous solid acid catalysts (PDS-x) with large BET surface area, superior thermal stability, strong sulfonic acid groups and tunable Bronsted acidity were synthesized by copolymerization of divinylbenzene (DVB) and p-styrenesulfonate hydrate (SPSS). The PDS-x catalysts showed high activity on the direct and efficient production of triacetylglycerol (TAG) by acyl exchange reaction of oils (fats) and acetic acid. The highest TAG yield of 92.2 mol% was obtained on the PDS-x catalysts at a mild condition of 200 o C. By combining SEM, TEM, FT-IR/DRIFT, BET, XRD, and TG-DTG, the key factors that affected the TAG yield were clarified on the PDS-x catalysts. Moreover, the origin of the slow deactivation on these catalysts was elucidated. The large BET surface area and abundant mesopores allowed the contact of large-sized glycerol tristearate with the strong sulfonic acid groups, accounting for the high TAG yield.

Interactions of the Diazabicyclooctane Serine β-Lactamase Inhibitor ETX1317 with Target Enzymes

ETX0282 is an orally bioavailable prodrug of the diazabicyclooctane serine β-lactamase inhibitor ETX1317. The combination of ETX0282 with cefpodoxime proxetil is in clinical trials as an oral therapy for complicated urinary tract infections caused by Enterobacterales. Earlier diazabicyclooctane β-lactamase inhibitors, such as avibactam and durlobactam, contain a sulfate moiety as the essential anionic group and are administered intravenously. In contrast, ETX1317 contains a fluoroacetate moiety, which is esterified with an isopropyl group in ETX0282 to provide high oral bioavailability. Previous studies of avibactam and durlobactam showed that covalent inhibition of certain β-lactamases is reversible due to the ability of the ring-opened inhibitors to recyclize and dissociate in their original form. We investigated the interaction of ETX1317 with several β-lactamases commonly found in relevant bacterial pathogens, including CTX-M-15, KPC-2, SHV-5, and TEM-1 from Ambler Class A; Pseudomonas aeruginosa AmpC and Enterobacter cloacae P99 from Class C, and OXA-48 from Class D. The second-order rate constants for inhibition (kinact/Ki) of these enzymes show that ETX1317 is intermediate in potency between durlobactam and avibactam. The partition ratios were all approximately 1, indicating that the inhibitor is not also a substrate of these enzymes. The rate constants for dissociation of the covalent complex (koff) were similar to those for durlobactam and avibactam. Acylation exchange experiments demonstrated that ETX1317 dissociated in its original form. No loss of mass from the inhibitor was observed in the covalent inhibitor-enzyme complexes.

Assembly of the complexes of oxidative phosphorylation triggers the remodeling of cardiolipin

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis.

The lipid composition of thylakoid membranes inside chloroplasts is conserved from leaves to developing embryos. A finely tuned lipid assembly machinery is required to build these membranes during Arabidopsis thaliana development. Contrary to thylakoid lipid biosynthetic enzymes, the functions of most predicted chloroplast lipid-degrading enzymes remain to be elucidated. Here, we explore the biochemistry and physiological function of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A1 In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific phosphatidylglycerol that contains 16:1 Δ3trans as its second acyl group. Thus far, a specific function of this 16:1 Δ3trans -containing phosphatidylglycerol in chloroplasts has remained elusive. The PLIP1 gene is highly expressed in seeds, and plip1 mutant seeds contain less oil and exhibit delayed germination compared with the wild type. Acyl groups released by PLIP1 are exported from the chloroplast, reincorporated into phosphatidylcholine, and ultimately enter seed triacylglycerol. Thus, 16:1 Δ3trans uniquely labels a small but biochemically active plastid phosphatidylglycerol pool in developing Arabidopsis embryos, which is subject to PLIP1 activity, thereby contributing a small fraction of the polyunsaturated fatty acids present in seed oil. We propose that acyl exchange involving thylakoid lipids functions in acyl export from plastids and seed oil biosynthesis.

ChemInform Abstract: K2CO3‐Promoted Formation of Aryl Esters from Primary Aryl Amides by the Acyl—Acyl Exchange Process

AbstractThe metal‐free reaction is not applicable to secondary or tertiary aryl amides, to alkyl amides or alkyl esters.

ChemInform Abstract: Potassium tert‐Butoxide‐Mediated Amine Acyl Exchange Reaction of N,N‐Disubstituted Formamides with Aromatic Carbonyl Derivatives via Sequential C—N Bond Cleavage/Formation: An Approach to Aromatic Amides.

ChemInform Abstract: Potassium tert‐Butoxide‐Mediated Amine Acyl Exchange Reaction of N,N‐Disubstituted Formamides with Aromatic Carbonyl Derivatives via Sequential C—N Bond Cleavage/Formation: An Approach to Aromatic Amides.

Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiaeale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-015-4102-0) contains supplementary material, which is available to authorized users.

Potassium tert‐Butoxide‐Mediated Amine Acyl Exchange Reaction of N,N‐Disubstituted Formamides with Aromatic Carbonyl Derivatives via Sequential CN Bond Cleavage/Formation: an Approach to Aromatic Amides

AbstractA novel potassium tert‐butoxide‐mediated amine acyl exchange of N,N‐disubstituted formamides with aromatic carbonyl derivatives in a sequential CN bond cleavage/formation process leading to aromatic amides is described. This methodology tolerates a wide range of aromatic carbonyl compounds, including aromatic aldehydes, acyl chlorides, unactivated esters, and acid anhydrides. The usage of inexpensive and readily available reagents, broad substrate scope, and the simple, mild (50 °C) and transition metal‐free conditions make this protocol very practical. In addition, a plausible reaction mechanism is proposed on the basis of experimental observations.magnified image

Scent Transmutation: A New Way To Teach on Chemical Equilibrium, Distillation, and Dynamic Combinatorial Chemistry

Esters are volatile and pleasantly smelling compounds, commonly used as food additives. Using Ti(OBu)4-catalyzed acyl exchange, we demonstrate a scent transmutation experiment, in which two fragrant esters swap their acyl and alkoxy substituents and are, during the course of a reactive distillation, quantitatively converted into two different esters with distinct fragrance properties. We view this simple and appealing experiment as highly suitable for instructional use, since it can be used to teach the concepts of chemical equilibrium, distillation, transesterification, dynamic combinatorial chemistry, and the industrially relevant process of reactive distillation.

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega-3 (also called n-3) long-chain polyunsaturated fatty acids (≥C20; LC-PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega-3 LC-PUFAs, i.e. eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6 n-3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega-3 LC-PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non-native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.

Distillative Self-Sorting of Dynamic Ester Libraries

Metal alkoxides, such as NaOt-Bu or Ti(OBu)4, can initiate acyl exchange within complex ester libraries. Reactive distillation of such dynamic combinatorial libraries (DCLs) isolates the most volatile ester at the expense of the less volatile library members that share a constituent with it. This process can be iteratively repeated to yield up to four industrially relevant esters as pure products from a single reaction setup. An algorithm has been developed to predict reactive distillation products in DCLs of as many as 121 members.

Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites.

The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.

Enzymatic Amine Acyl Exchange in Peptides on Gold Surfaces

Reversible as well as stereo- and chemoselective: various proteases such as thermolysin and chymotrypsin catalyze amine acyl exchange in peptides. This acyl exchange can be used to modify amino-functionalized surfaces under physiological reaction conditions and provides an alternative mechanism for posttranslational transpeptidation reactions such as peptide-splicing reactions in the proteasome.

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Identification of Brassica napus lysophosphatidylcholine acyltransferase genes through yeast functional screening

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which acylates lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), is a key enzyme in the Lands cycle. There is evidence that acyl exchange involving LPCAT is a prevailing metabolic process during triacylglycerol (TAG) synthesis in seeds. In this study, by complementing the yeast lca1Δ mutant deficient in LPCAT activity with an Arabidopsis seedling cDNA library, it was found that the previously reported lysophospholipid acyltransferases (LPLATs), At1g12640 and At1g63050, were the only two acyltransferase genes that restored hyposensitivity of the lca1Δ mutant to lyso-platelet-activating factor (lyso-PAF). A developing seed cDNA library from Brassica napus L. cv Hero was constructed to further explore the heterologous yeast complementation approach. Three B. napusLPCAT homologs were identified, of which BnLPCAT1-1 and BnLPCAT1-2 are orthologous to ArabidopsisAtLPLAT1 (At1g12640) while BnLPCAT2 is an ortholog of AtLPLAT2 (At1g63050). The proteins encoded by BnLPCAT1-1 and BnLPCAT2 were chosen for further study. Enzymatic assays demonstrated that both proteins exhibited a substrate preference for LPCs and unsaturated fatty acyl-CoAs. In addition to the enzymatic properties of plant lysophosphatidylcholine acyltransferases uncovered in this study, this report describes a useful technique that facilitates subsequent analyses into the role of LPCATs in PC turnover and seed oil biosynthesis.

A 10‐kDa acyl‐CoA‐binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl‐CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.