Transacylation Reactions Research Articles

PNPLA-mediated lipid hydrolysis and transacylation – At the intersection of catabolism and anabolism

Patatin-like phospholipase domain containing proteins (PNPLAs) play diverse roles in lipid metabolism. In this review, we focus on the enzymatic properties and predicted 3D structures of PNPLA1-5. PNPLA2-4 exert both catabolic and anabolic functions. Whereas PNPLA1 is predominantly expressed in the epidermis and involved in sphingolipid biosynthesis, PNPLA2 and 4 are ubiquitously expressed and exhibit several enzymatic activities, including hydrolysis and transacylation of various (glycero-)lipid species. This review summarizes known biological roles for PNPLA-mediated hydrolysis and transacylation reactions and highlights open questions concerning their physiological function.

Recent Insights and Future Perspectives on Promiscuous Hydrolases/Acyltransferases

Promiscuous hydrolases/acyltransferases have attracted attention for their ability to efficiently catalyze selective transacylation reactions in water to produce esters, thioesters, amides, carbona...

Cyanide Anions as Nucleophilic Catalysts in Organic Synthesis

AbstractThe nucleophilic addition of a cyanide anion to a carbonyl group is the basis for several cyanide-catalyzed organic reactions, which are summarized in this review. Since cyanide is also a good leaving group, it is an excellent catalyst for transacylation reactions. As an electron-withdrawing group, it also stabilizes a negative charge in its α-position, thus allowing the umpolung of aldehydes to formyl anion equivalents. The two leading examples are the benzoin condensation and the Michael–Stetter reaction furnishing α-hydroxy ketones and 1,4-dicarbonyl compounds, which are both catalyzed by cyanides. The review also covers variants like the silyl-benzoin coupling, the aldimine coupling and the imino-Stetter reaction. Moreover, some cyanide-catalyzed heterocyclic syntheses are reviewed.1 Introduction2 Nucleophilic Additions2.1 Cyanohydrin Formation2.2 Corey–Gilman–Ganem and Related Oxidation Reactions2.3 Conjugate Addition2.4 Intramolecular Carbocyanation3 Transacylation Reactions3.1 Ester Hydrolysis and Transesterification3.2 Formation of Amides3.3 Ketones from Esters3.4 Esters from Ketones4 Transformations Involving an Umpolung4.1 Benzoin Condensation4.2 Aldimine Coupling4.3 Michael–Stetter Reaction4.4 Imino-Stetter Reaction5 Formation of Heterocycles5.1 Oxazolines from Isocyanoacetates5.2 Imidazoles from TosMIC via Oxazolines5.3 Bargellini Reaction6 Conclusion

Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling.

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.

Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomyces tsukubaensis YN06, we found that AT4FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.

Oxalyltransferase, a plant cell-wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates.

SummaryIn the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O‐oxalyl esters [oxalyl‐l‐threonate (OxT) and cyclic oxalyl‐l‐threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl‐polysaccharides, potentially modifying the cell wall. [oxalyl‐14C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell‐suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [14C]OxT and [14C]cOxT were incubated in vitro with cell‐wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell‐wall polymers via ester bonds. When sugars were added, [14C]oxalyl‐sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [14C]oxalyl‐glucose, was relatively stable in vivo (half‐life >24 h), whereas [14C]OxT underwent rapid turnover (half‐life ~6 h). Ionically wall‐bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O‐oxalylated, not N‐oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long‐lived O‐oxalyl‐carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl‐sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties.

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering.

Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

Expanding the Scope of Sortase-Mediated Ligations by Using Sortase Homologues.

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

Essential Role for Ethanolamine Plasmalogen Hydrolysis in Bacterial Lipopolysaccharide Priming of Macrophages for Enhanced Arachidonic Acid Release.

Due to their high content in esterified arachidonic acid (AA), macrophages provide large amounts of eicosanoids during innate immune reactions. Bacterial lipopolysaccharide (LPS) is a poor trigger of AA mobilization in macrophages but does have the capacity to prime these cells for greatly increased AA release upon subsequent stimulation. In this work, we have studied molecular mechanisms underlying this phenomenon. By using mass spectrometry-based lipidomic analyses, we show in this work that LPS-primed zymosan-stimulated macrophages exhibit an elevated consumption of a particular phospholipid species, i.e., the ethanolamine plasmalogens, which results from reduced remodeling of phospholipids via coenzyme A-independent transacylation reactions. Importantly however, LPS-primed macrophages show no changes in their capacity to directly incorporate AA into phospholipids via CoA-dependent acylation reactions. The essential role for ethanolamine plasmalogen hydrolysis in LPS priming is further demonstrated by the use of plasmalogen-deficient cells. These cells, while responding normally to zymosan by releasing quantities of AA similar to those released by cells expressing normal plasmalogen levels under the same conditions, fail to show an LPS-primed response to the same stimulus, thus unambiguously demonstrating a cause–effect relationship between LPS priming and plasmalogen hydrolysis. Collectively, these results suggest a hitherto unrecognized role for ethanolamine plasmalogen hydrolysis and CoA-independent transacylation reactions in modulating the eicosanoid biosynthetic response.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units.

Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. Nucleotide sequence data have been submitted to the GenBank database under accession numbers KJ000382 and KJ000383.

Biochemical characterization of a malonyl-specific acyltransferase domain of FK506 biosynthetic polyketide synthase.

Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions.

Novel reactions in acyl editing of phosphatidylcholine by lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities in microsomal preparations of plant tissues

Main conclusion Plants have lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities. The combined action of LPCT and GPCAT provides a novel route of PC re-synthesis after its deacylation. Phosphatidylcholine (PC) is the major lipid in eukaryotic membranes and has a central role in overall plant lipid metabolism. It is also the site of production of polyunsaturated fatty acids in plants. The recently discovered acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activity in yeast provides a novel route of re-synthesising PC via lysophosphatidylcholine (LPC) after its deacylation. This route does not require the degradation of the glycerophosphocholine (GPC) into free choline, the activation of choline to CDP-choline, nor the utilization of CDP-choline by the CDP-choline:diacylglycerol cholinephosphotransferase. We show here that GPCAT activities also are present in membrane preparations from developing oil seeds of safflower and other species as well as in membrane preparations of roots and leaves of Arabidopsis, indicating that GPCAT activity plays a ubiquitous role in plant lipid metabolism. The last step in formation of GPC, the substrate for GPCAT, is the deacylation of LPC. Microsomal membranes of developing safflower seeds utilized LPC in LPC:LPC transacylation reactions (LPCT activities) creating PC and GPC. The results demonstrate that safflower membranes have LPCT and GPCAT activities that represent novel reactions for PC acyl editing. The physiological relevance of these reactions probably has to await identification of the enzymes catalysing these reactions.

ChemInform Abstract: Chemical Drug Stability in Lipids, Modified Lipids, and Polyethylene Oxide‐Containing Formulations

AbstractReview: 44 refs.

Chemical Drug Stability in Lipids, Modified Lipids, and Polyethylene Oxide-Containing Formulations

To critique the stability complications seen in formulating poorly water-soluble, problematic drugs in lipids, modified lipids, and polyethylene oxide solvents and surfactants in hard and soft gelatin capsules as well as some parenterals, a literature search was performed and personal experiences, and those of colleagues, collated. The literature is replete with examples of molecules undergoing rapid oxidative degradation in the presence of polyethylene oxide based solvents and surfactants as well as in the presence of unsaturated lipids. More recently appreciated is instability caused by the reaction of amine and amide drugs, with formaldehyde, formic acid found in many of these solvents as impurities and other degradation byproducts of the solvents themselves. One would expect acylation and transacylation reactions to be more common than reported but the literature has some good examples. An added complexity is occasionally seen with the use of hard and soft gelatin capsules with these solvents. The chemical stability of drugs in liquid and semi-solid formulations in the presence of lipids, modified lipids, and polyoxyethylene oxide-based solvents and surfactants can be complex, further exacerbated by the use of gelatin capsules, and can lead to a plethora of degradation pathways often not seen when the same drugs are formulated in solid dosage forms.

Selective Monoacylation of Diols by Substrate Assisted Catalysis in T40A Candida antarctica Lipase B

AbstractThe selectivity towards diols over monoesters in the esterification of diols catalysed by lipase B from Candida antarctica (CALB) was improved by the single point mutation T40A in the enzyme’s oxyanion hole. Substrate‐assisted catalysis was suggested from molecular modelling of the tetrahedral intermediate in esterification of 1,2‐ethanediol catalysed by T40A CALB. The non‐reacting hydroxyl group of the diol forms a hydrogen bond to the oxyanion in the transition state, replacing that deleted in mutation. Monoester yields in transacylation reactions were monitored over time to compare the selectivities for wild‐type and T40A CALB. The results showed increased selectivities towards the diols tested over their corresponding monoesters as a result of the T40A mutation with substrate‐assisted catalysis as a plausible explanation.

Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.

ChemInform Abstract: Environmentally Friendly Transesterification and Transacylation Reactions under LiBr Catalysis.

A room-temperature, convenient transesterification of various esters, induced by catalytic amounts of lithium bromide and diethylamine in a solvent-free environment, is described. Products are obtained in good purity by simple filtration to remove the catalyst and evaporation of the volatile portion. The procedure has also been successfully applied to transacetylation and aminolysis of esters.

High‐performance liquid chromatography/mass spectrometric and proton nuclear magnetic resonance spectroscopic studies of the transacylation and hydrolysis of the acyl glucuronides of a series of phenylacetic acids in buffer and human plasma

The use of high-performance liquid chromatography/mass spectrometry (HPLC/MS) and proton nuclear magnetic resonance ((1)H NMR) spectroscopy for the kinetic analysis of acyl glucuronide (AG) isomerisation and hydrolysis of the 1-β-O-acyl glucuronides (1-β-O-AG) of phenylacetic acid, (R)- and (S)-α-methylphenylacetic acid and α,α-dimethylphenylacetic acid is described and compared. Each AG was incubated in both aqueous buffer, at pH 7.4, and control human plasma at 37°C. Aliquots of these incubations, taken throughout the reaction time-course, were analysed by HPLC/MS and (1)H NMR spectroscopy. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the calculated rates of reaction were much faster than for buffer and, in contrast to the observations in buffer, hydrolysis to the free aglycone was a significant contributor to the overall reaction.A diagnostic analytical methodology based on differential mass spectrometric fragmentation of 1-β-O-AGs compared to the 2-, 3- and 4-positional isomers, which enables selective determination of the former, was confirmed and applied. These findings show that HPLC/MS offers a viable alternative to the more commonly used NMR spectroscopic approach for the determination of the transacylation and hydrolysis reactions of these AGs, with the major advantage of having the capability to do so in a complex biological matrix such as plasma.

Environmentally friendly transesterification and transacylation reactions under LiBr catalysis

A room-temperature, convenient transesterification of various esters, induced by catalytic amounts of lithium bromide and diethylamine in a solvent-free environment, is described. Products are obtained in good purity by simple filtration to remove the catalyst and evaporation of the volatile portion. The procedure has also been successfully applied to transacetylation and aminolysis of esters.