Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE

Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid-lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N-terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two-step binding profile.     

Lipid-modified proteins as biomarkers for cardiovascular disease: a review

Lipid-modified proteins are classified based on the identity of the attached lipid, a post- or co-translational modification required for their biological function. At least five different lipid modifications of cysteines, glycines and other residues on the COOH- and NH₂-terminal domains have been described. Cysteine residues may be modified by the addition of a 16-carbon saturated fatty acyl group by a labile thioester bond (palmitoylation) or by prenylation processes that catalyze the formation of thioether bond with mevalonate derived isoprenoids, farnesol and geranylgeraniol. The NH₂-terminal glycine residues may undergo a quite distinct process involving the formation of an amide bond with a 14-carbon saturated acyl group (myristoylation), while glycine residues in the COOH-terminal may be covalently attached with a cholesterol moiety by an ester bond. Finally, cell surface proteins can be anchored to the membrane through the addition of glycosylphosphatidylinositol moiety. Several lines of evidence suggest that lipid-modified proteins are directly involved in different steps of the development of lesions of atherosclerosis, from leukocyte recruitment to plaque rupture, and their expression or lipid modification are likely altered during atherogenesis. This review will briefly summarize the different enzymatic pathways of lipid modification and propose a series of lipid-modified proteins that can be used as biomarkers for cardiovascular disease.     

Probing the role of C-1 ester group in Naja naja phospholipase A2-phospholipid interactions using butanetriol-containing phosphatidylcholine analogues.

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions.     

Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein

Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.     

N-acylphosphatidylethanolamines: effect of the N-acyl chain length on its orientation

N-Acylphosphatidylethanolamines, or NAPEs, are found in tissues involved in degenerating processes, such as dehydrated endosperm of seeds, erythrocyte membranes, or cell injury. To determine the conformation and orientation of the acyl chains of these phospholipids, NAPEs with deuterated N-acyl chains of 6 and 16 carbon atoms were synthesized and studied by transmission and attenuated total reflectance (ATR) infrared spectroscopy. For N-C16d-DPPE, the ATR measurements show that the N-acyl chain has the same orientation as the two acyl chains attached to the glycerol moiety, while the N-acyl chain of N-C6d-DPPE is randomly oriented. These results demonstrate that for N-C16d-DPPE, the N-acyl chain is embedded into the hydrophobic core of the bilayer, while for the short chain derivative the N-acyl chain remains in the lipid headgroup region. The analysis of the carbonyl stretching band and of the amide I band suggests that, for the long N-acyl chain lipid, the ester C=O and the N-H groups are linked by intermolecular hydrogen bonds.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Conformational differences between sn-3-phospholipids and sn-2-phospholipids. A neutron and x-ray diffraction investigation

Extensive work has been reported on the conformation in membranes of sn-3-phosphatidylcholines, -ethanolamines, -glycerols and -serines (sn-3-phospholipids), where the headgroup is linked to the third carbon atom in the glycerol backbone. One important feature common to all these naturally occurring phospholipids is that the glycerol moiety is oriented almost perpendicular to the membrane surface, with the sn-1 chain continuing in this direction, whereas the sn-2 chain starts first in a direction parallel to the layer and then bends sharply at the second carbon atom. We present here neutron diffraction results on 1,3-dipalmitoyl-glycero-2-phosphocholine (1,3-DPPC) in which the headgroup is attached to the second carbon atom in the middle of the glycerol part and the two other adjacent carbon atoms are linked to the paraffin chains. Two 1,3-DPPC samples. ²H-labelled at different positions, were studied. One sample was deuterated at the first methylene segment in each fatty acyl chain, and the other at the first segment in one chain and at the second segment in the other chain. The Patterson maps as well as the neutron density profiles show that both fatty acyl chains in 1,3-DPPC have the same conformation introduced by this symmetric chemical bond pattern. It is concluded from this that the C₍₁₎C₍₃₎ vector of the glycerol backbone part is oriented parallel to the bilayer surface and the ²H nuclear magnetic resonance signals indicate that both chains have a conformation similar to that observed for the bent chain in sn-3-phospholipids. These findings indirectly confirm the idea that an intramolecular energy minimum, together with the packing geometry of the lipids in the membrane, produce the characteristic conformation around the glycerol backbone as is found for all naturally occurring phospholipids that have been studied so far.     

A method to search for similar protein local structures at ligand-binding sites and its application to adenine recognition

We have developed a method of searching for similar spatial arrangements of atoms around a given chemical moiety in proteins that bind a common ligand. The first step in this method is to consider a set of atoms that closely surround a given chemical moiety. Then, to compare the spatial arrangements of such surrounding atoms in different proteins, they are translated and rotated so that the chemical moieties are superposed on each other. Spatial arrangements of surrounding atoms in a pair of proteins are judged to be similar, when there are many corresponding atoms occupying similar spatial positions. Because the method focuses on the arrangements of surrounding atoms, it can detect structural similarities of binding sites in proteins that are dissimilar in their amino acid sequences or in their chain folds. We have applied this method to identify modes of nucleotide base recognition by proteins. An all-against-all comparison of the arrangements of atoms surrounding adenine moieties revealed an unexpected structural similarity between protein kinases, cAMP-dependent protein kinase (cAPK), and casein kinase-1 (CK1), and D-Ala:D-Ala ligase (DD-ligase) at their adenine-binding sites, despite a lack of similarity in their chain folds. The similar local structure consists of a four-residue segment and three sequentially separated residues. In particular the four-residue segments of these enzymes were found to have nearly identical conformations in their backbone parts, which are involved in the recognition of adenine. This common local structure was also found in substrate-free three-dimensional structures of other proteins that are similar to DD-ligase in the chain fold and of other protein kinases. As the proteins with different folds were found to share a common local structure, these proteins seem to constitute a remarkable example of convergent evolution for the same recognition mechanism. Received: 9 December 1996 / Accepted: 7 February 1997     

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway: THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE*

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C_α and C_β of Ser-37 in tetradecanoyl-ACP. ¹³C,¹⁵N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C₈)- and dodecanoyl (C₁₂)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C₁₄)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK_a value for the carboxylate, ∼1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.In the malarial parasite Plasmodium falciparum, fatty acid biosynthesis occurs by a pathway distinct from the host. A number of enzymes involved in the process viz. β-ketoacyl acyl carrier protein (ACP)⁵ synthase III, β-hydroxy acyl-ACP hydratase, and enoyl-ACP reductase are targets for drug design (1, 2). An indispensable component, crucial for each step of the pathway, is a small acidic protein, the ACP. ACP plays a pivotal role in a range of biochemical processes, like fatty acid biosynthesis (3), polyketide synthesis (4, 5), oligosaccharides (6), biotin, and nonribosomal peptide synthesis (7, 8). Thus, the knowledge of structural features, which dictate ACP function, could offer new avenues for inhibitor design to disable several pathways of the parasite in parallel.Acyl carrier protein differs structurally in the host and the parasite. It exists as an independent protein in type II fatty acid synthesis pathway, observed in P. falciparum, Escherichia coli, spinach, and most prokaryotes. In the type II pathway, fatty acids are synthesized by multiple enzymes catalyzing different reactions. Conversely, mammalian ACP (malarial host) is an integral domain of one single multidomain, multifunctional fatty acid synthase (FAS) (type I pathway), each domain catalyzing a particular reaction. Interestingly, ACPs of type I and II pathway share a similar fold, the ACP molecule of type II pathway can be substituted with the ACP domain of type I pathway in some cases, and the latter is recognized as a substrate in vitro by key enzymes of type II pathway (9).The primary function of ACP is to shuttle the lengthening acyl chains to the catalytic site of FAS enzymes. It is expressed as an apoprotein (inactive) and modified to holo-ACP (active) by the transfer of a 4′-phosphopantetheine moiety from coenzyme A (CoA) to a conserved serine residue, Ser-36/37, with ACP synthase acting as a catalyst. The acyl chain gets covalently tethered to the terminal cysteamine thiol of the 4′-phosphopantetheine prosthetic group, which in turn transfers the acyl chain to the respective enzymes during elongation. Biosynthesis of fatty acid(s) is initiated by the carboxylation of acetyl-CoA to malonyl-CoA, which is transacylated to malonyl-ACP. Malonyl-ACP condenses with acetyl-CoA, resulting in the formation of enoyl-/butyryl-ACP (C₄-) which enters the elongation cycle. Two carbon atoms are added per elongation cycle, resulting in acyl-ACPs C₆-, C₈-, C₁₀-, C₁₂-, C₁₄-, and C₁₆-ACP. Palmitate (C₁₆) is the most common product of type I pathway, whereas in the type II pathway, products range from saturated to unsaturated, branched, unbranched, or variable chain lengths.Structurally, ACP is a four-helix bundle protein, with the helices enclosing a central hydrophobic cavity (10–17). In the type II pathway, the hydrophobic cavity accommodates the growing acyl chain and the β-mercaptoethyl moiety of the 4′-phosphopantetheine arm. The acyl chain remains embedded in the cavity, which expands with increasing length of the acyl chain as observed in E. coli and spinach (12). The mechanism of acyl chain interaction with the ACP molecule is remarkably different in rat, which belongs to the type I fatty acid pathway. Insignificant interactions between the ACP molecule and the acyl chain are observed, suggesting that the ACP molecule does not sequester the acyl chain, and therefore, the acyl chain in type I pathway is protected in a way different from the type II pathway (18).Despite the availability of structural data for a number of acyl-ACPs e.g. E. coli and spinach (12, 14, 19, 20), molecular details pertaining to acyl chain carriage and its presentation to the FAS enzymes of type II pathway is still an enigma. The general consensus is that the ACP molecule can accommodate 10 carbon atoms only. In spinach, the hydrophobic cavity of ACP expands to accommodate acyl chain lengths ranging from C_10:0 to C_18:0. However, chains longer than 10 carbon units are not fully protected (14). In E. coli, 10 carbon atoms have been observed to be accommodated in the hydrophobic core (19). A molecular dynamics study on E. coli published recently also shows that the hydrophobic core of ACP can hold a maximum of 10 carbon atoms only (21). Here, we demonstrate that P. falciparum ACP (PfACP) can protect more than 10-carbon-atom-long acyl chains, with a maximum of 12 carbon atoms. An in silico study on PfACP published recently proposes the possible mechanism of substrate delivery based on steered molecular dynamics simulations using E. coli acyl-ACPs as the starting model (22). There are no experimental data (x-ray or NMR) available to date on the acyl-ACPs of P. falciparum. Present work for the first time provides structural insights into the acyl-PfACP intermediates using NMR as a primary tool. The precision and sensitivity of NMR allowed identification of key interactions between the acyl chain and the ACP molecule, leading to the proposal of a model unraveling the sequence of structural changes accompanying acyl chain insertion. The molecular basis of ACP expansion in PfACP upon acyl chain elongation has also been deciphered.     

Lipid-modified proteins as biomarkers for cardiovascular disease: a review.

Lipid-modified proteins are classified based on the identity of the attached lipid, a post- or co-translational modification required for their biological function. At least five different lipid modifications of cysteines, glycines and other residues on the COOH- and NH(2)-terminal domains have been described. Cysteine residues may be modified by the addition of a 16-carbon saturated fatty acyl group by a labile thioester bond (palmitoylation) or by prenylation processes that catalyze the formation of thioether bond with mevalonate derived isoprenoids, farnesol and geranylgeraniol. The NH(2)-terminal glycine residues may undergo a quite distinct process involving the formation of an amide bond with a 14-carbon saturated acyl group (myristoylation), while glycine residues in the COOH-terminal may be covalently attached with a cholesterol moiety by an ester bond. Finally, cell surface proteins can be anchored to the membrane through the addition of glycosylphosphatidylinositol moiety. Several lines of evidence suggest that lipid-modified proteins are directly involved in different steps of the development of lesions of atherosclerosis, from leukocyte recruitment to plaque rupture, and their expression or lipid modification are likely altered during atherogenesis. This review will briefly summarize the different enzymatic pathways of lipid modification and propose a series of lipid-modified proteins that can be used as biomarkers for cardiovascular disease.     

Solution structure of B. subtilis acyl carrier protein

BACKGROUND: Acyl carrier protein (ACP) is a fundamental component of fatty acid biosynthesis in which the fatty acid chain is elongated by the fatty acid synthetase system while attached to the 4'-phosphopantetheine prosthetic group (4'-PP) of ACP. Activation of ACP is mediated by holo-acyl carrier protein synthase (ACPS) when ACPS transfers the 4'-PP moiety from coenzyme A (CoA) to Ser36 of apo-ACP. Both ACP and ACPS have been identified as essential for E. coli viability and potential targets for development of antibiotics. RESULTS: The solution structure of B. subtilis ACP (9 kDa) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 22 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 1,050 experimental NMR restraints. The atomic rmsd about the mean coordinate positions for the 22 structures is 0.45 +/- 0.08 A for the backbone atoms and 0.93 +/- 0.07 A for all atoms. The overall ACP structure consists of a four alpha-helical bundle in which 4'-PP is attached to the conserved Ser36 that is located in alpha helix II. CONCLUSIONS: Structural data were collected for both the apo and holo forms of ACP that suggest that the two forms of ACP are essentially identical. Comparison of the published structures for E. coli ACP and actinorhodin polyketide synthase acyl carrier protein (act apo-ACP) from Streptomyces coelicolor A3(2) with B. subtilis ACP indicates similar secondary structure elements but an extremely large rmsd between the three ACP structures (>4.3 A). The structural difference between B. subtilis ACP and both E. coli and act apo-ACP is not attributed to an inherent difference in the proteins, but is probably a result of a limitation in the methodology available for the analysis for E. coli and act apo-ACP. Comparison of the structure of free ACP with the bound form of ACP in the ACP-ACPS complex reveals a displacement of helix II in the vicinity of Ser36. The induced perturbation of ACP by ACPS positions Ser36 proximal to coenzyme A and aligns the dipole of helix II to initiate transfer of 4'-PP to ACP.     

The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization

The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed.     

Shape change of human erythrocytes induced by phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths

Intact human erythrocytes were treated, under non-haemolytic conditions at 37 degrees C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8-18 even-numbered carbon atoms (C8-C18-PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8-18 carbon atoms (C8-C18-lysoPC). The C8-C14-PC and C12-C18-lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16-PC, C18-PC, C8-lysoPC and C10-lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.     

Antibodies to long-chain acyl-CoAs: A new tool for lipid biochemistry

Antibodies directed against long chain acyl-CoAs (having 16 and 18 carbon atoms) have been prepared and are reported for the first time. A modified ELISA procedure adapted to these amphiphilic molecules has been developed: it is a rapid, simple and sensitive test permitting to detect as little as 3 pmol of acyl-CoA. These antibodies represent a new tool for studying long-chain acyl-CoAs. Their use in an immunochemical approach for the study of protein-acyl-CoA interactions is presented.     

The critical micelle concentration of some physiologically important fatty acyl-coenzyme A's as a function of chain length

The critical micelle concentration (CMC) of a series of saturated fatty acyl-CoAs have been determined using a fluorescent titration method with 2-toluidinylnaphthalene-6-sulfonate as a probe. The CMC was found to be a function of the number of carbon atoms in the acyl chain over the range tested (12 to 18). A double bond has the effect on the CMC of decreasing the acyl chain by 1.6 carbon atoms. Knowledge that the CMC of each of these fatty acyl-CoAs is a function of the acyl chain length and the availability of a simple and appropriate method for the estimation of this property under other conditions should be of importance in designing and interpreting in vitro experiments with these compounds.     

Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-¹³C₂]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [¹³C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-¹³C₂]hexadecane-derived fatty acids contained either two ¹³C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Structure of biologically active and inactive cerebrosides prepared from Schizophyllum commune

A cerebroside fraction prepared from the mycelia of Schizophyllum commune was further fractionated into five components (I-V) by reverse-phase high-performance liquid chromatography. Fruiting-inducing activity was found in I-IV but not in V. By gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses it was shown that these fractions contained: I, a mixture of N-2'-hydroxypentadecanoyl-1-O-glucosyl-nonadecasphingadienine++ + and N-2'-hydroxyhexadecanoyl-1-O-glucosyl-sphingadienine; II, (4E,8E)-N-D-2'-hydroxyhexadecanoyl-1-O-beta-D-glucopyr anosyl-9-methyl-4,8- sphingadienine (Kawai and Ikeda. 1983. Biochim. Biophys. Acta. 754: 243-248); III, N-2'-hydroxyheptadecanoyl-1-O-glucosyl-nonadecasphingadienine++ +; IV, N-2'-hydroxyoctadecanoyl-1-O-glucosyl-nonadecasphinadienine; V, (4E,8E)-N-2'-hydroxytetracosanoyl-1-O-beta-glucopyrano syl-9-methyl-4,8- sphingadienine. The only structural difference observed between biologically active and inactive cerebrosides was the chain length of acyl moiety; the cerebroside having an acyl chain of 24 carbon atoms was inactive.     

Lipase-catalyzed preparation of biologically active esters of dehydroepiandrosterone

A series of acyl esters derivatives of dehydroepiandrosterone have been prepared by an enzymatic methodology. The acyl chain had a length that varied from two to eighteen carbon atoms. The C₁₈ derivative could be saturated or unsaturated. Following this biocatalytic approach we have also obtained a chloropropionyl derivative. We have observed that several lipases catalyzed esterification and transesterification reactions of dehydroepiandrosterone with carboxylic acids or alkyl carboxylates. The advantages presented by this methodology such as mild reaction conditions, economy and low environmental impact, make biocatalysis a convenient way to prepare acyl derivatives of DHEA with biological activity.     

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.