Structure and function of lecithin cholesterol acyl transferase: new insights from structural predictions and animal models

The enzyme lecithin cholesterol acyl transferase is responsible for the synthesis of most of the cholesteryl esters in plasma, and therefore plays a key role in lipoprotein metabolism. The relationship between the structure and function of lecithin cholesterol acyl transferase has been extensively studied in the past years, and new data appeared in 1999 documenting the substrate specificity and physiological role of lecithin cholesterol acyl transferase. The discovery of natural mutants, together with the proposal of a three-dimensional model for the enzyme, has provided new tools to unravel the function of specific residues of lecithin cholesterol acyl transferase. The use of transgenic animals and the production of knock-out lecithin cholesterol acyl transferase mice has further contributed to the understanding of the lecithin cholesterol acyl transferase 'in vivo' function. Evidence for a protective role of lecithin cholesterol acyl transferase against the development of atherosclerosis through the hydrolysis of oxidized lipids was recently proposed. Lecithin cholesterol acyl transferase patterns in several pathologies were further clarified. These newer developments are reviewed here.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Separation and functions of two acyl CoA transferases from Ascaris lumbricoides mitochondria

Many invertebrates accumulate propionate, or products derived from propionate, as products of fermentation. Evidence has been reported that the nematode, Ascaris suum, the cestode, Spirometra mansonoides, and the trematode, Fasciola hepatica, accumulate propionate by means of an adenosine triphosphate (ATP)-generating decarboxylation of succinate. To generate energy, an acyl coenzyme A (CoA) transferase that would transfer CoA to succinate is required as one component of the sequence of reactions. Recently, an acyl CoA transferase was isolated from Ascaris mitochondria and purified to electrophoretic homogeneity. However, upon examination of the substrate specificities of this enzyme, it was found essentially to lack the ability to use succinate or succinyl CoA as an acceptor or donor of CoA, respectively. Therefore, this transferase could not serve to activate succinate. This article describes the isolation of an additional acyl CoA transferase from Ascaris mitochondria that appears to be unique in its substrate specificity and that could easily account not only for the activation of succinate but also for the regulation of succinate metabolism primarily in the direction of decarboxylation to propionate. This is in contrast with mammalian tissues, which act in the opposite direction by catalyzing the fixation of CO2 into propionate, thereby forming succinate and accounting for the glycogenic nature of dietary propionate. Possible functions of the two acyl CoA transferases are discussed.     

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl CoA:lysolecithin acyl transferase

Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-¹⁴C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.     

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O-ester intermediates after incubation with [¹⁴C]acetyl- or malonyl-CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl-Ser---Leu---Gly---Glu---Val---Ala) was obtained after labelling with acetyl- or malonyl-CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl- and malonyl-groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.     

Normal cholesterol absorption in rats deficient in intestinal acyl coenzyme A:cholesterol acyltransferase activity

Acyl coenzyme A:cholesterol acyl transferase and/or cholesterol esterase may regulate the esterification and absorption of exogenous cholesterol. To assess this, mucosal acyl coenzyme A:cholesterol acyl transferase activity was inhibited selectively with three different drugs [Sandoz #58-035, inhibitor 1; Lederle inhibitor 2 and inhibitor 3] and the effect upon the absorption of a [4-14C]cholesterol meal was studied in the lymph fistula rat. Compared to control rats, ACAT activity measured in mucosal homogenates from the drug-treated rats was reduced 80-90%, 40%, and 30%, respectively, during the predicted time-frame for maximum mucosal esterification of cholesterol (i.e., after cholesterol is fed and before it appears in lymph). In contrast, [14C]cholesterol absorption in the drug-treated animals was unchanged from controls [5.7 +/- 1.2 (inhibitor 1) vs. 5.4 +/- 1.6 mumol/6 hr (control); 6.1 +/- 2.1 (inhibitor 2) and 5.2 +/- 1.5 (inhibitor 3) vs. 4.1 +/- 1.3 mumol/6 hr (control)]. Of the absorbed [14C]cholesterol, approximately 75% was esterified in all groups. Cholesterol esterase activity measured in the drug-treated rats was unchanged compared to controls nor did the drugs inhibit this enzyme in vitro. Under the conditions of this study, drugs causing substantial inhibition of acyl coenzyme A:cholesterol acyl transferase activity had no effect on the absorption of exogenous cholesterol.     

Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains

Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.     

Palmitoyl transferase activity of lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.     

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.     

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A₂ (PLA₂) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were K_m 18 μM and V_max 30 nmol/min/mg protein; the V_max was increased 25-fold by phosphorylation of the protein while K_m was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA₂ activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA₂ and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.     

Stereochemistry of the acyl dihydroxyacetone phosphate acyl exchange reaction

The fatty acid of acyl dihydroxyacetone phosphate can be exchanged enzymatically for another fatty acid. It has been shown that this reaction proceeds by cleavage of the oxygen bound to C-1 of the dihydroxyacetone phosphate (DHAP) moiety rather than by the more common cleavage at the acyl to oxygen bond. In the present study, the stereochemistry of this reaction was defined further; using deuterated substrates and fast atom bombardment-mass spectrometry, it was shown that the fatty acid exchange involves the stereospecific labilization of the pro-R hydrogen at C-1 of the DHAP moiety of acyl DHAP. The mechanism of ether bond formation, in which acyl DHAP is converted to O-alkyl DHAP, also proceeds via labilization of the pro-R hydrogen and cleavage of the fatty acid at the C-1 to oxygen bond. In addition, other workers have provided evidence that the enzyme responsible for the exchange reaction is O-alkyl DHAP synthetase. Therefore, the present results support the hypothesis that the acyl exchange is the reverse reaction of the first step in O-alkyl DHAP synthesis; in both of these reactions the pro-R hydrogen of C-1 of the DHAP moiety of acyl DHAP and the fatty acid moiety are labilized with cleavage of the fatty acid at the DHAP C-1 to oxygen bond.     

Two histidine residues are essential for catalysis by lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.     

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.     

Facile syntheses of acyl dihydroxyacetone phosphates and lysophosphatidic acids having different acyl groups

In this study, we report novel and simple chemical syntheses of acyl dihydroxyacetone phosphate (DHAP) and 1-acyl glycero-3-phosphate [lysophosphatidic acid (LPA)], key intermediaries in the formation of glycerolipids containing ester and ether bonds. The synthesis of acyl DHAPs involved acylating the dimethyl ketal of DHAP by acid anhydride using 4-pyrrolidinopyridine as the catalyst, and the resulting product was deketalized by HClO(4) in acetone to produce acyl DHAP. The acid anhydride was either added directly or generated in the reaction mixture from the corresponding fatty acid using dicyclohexylcarbodiimide as the condensing agent. Using these methods, a number of acyl DHAPs having short-, medium-, and long-chain saturated and unsaturated acyl groups were synthesized, with overall yields from 37% to 75%. The activities of these acyl DHAPs as substrates for guinea pig liver peroxisomal acyl DHAP:NADPH reductase and alkyl DHAP synthase were then determined. Next, starting from these acyl DHAPs, a variety of LPAs were synthesized by chemical reduction of the ketone group. Biological activities of these LPAs were determined by measuring their relative abilities to release intracellular Ca(2+) via the LPA receptor. A combined chemical-enzymatic method is also described to prepare the natural LPA from the racemic mixture.     

Evidence for the association of acyl transferases with the production of nuclear evaginations in maturing eggs of the fern Dryopteris filix-mas.

Utilizing the ability of free CoA to reduce ferricyanide, evidence is presented that acyl transferase activity is associated with the nuclear envelope in the maturing egg of the fern Dryopteris filix-mas. This activity is particularly marked in the nuclear evaginations. This strengthens the view that the evaginations are distinct structures formed by localized growth of the envelope, and not transient extensions of the nucleus.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis