Acetyl Acyl Carrier Protein Research Articles

Dissecting the Component Reactions Catalyzed by the Actinorhodin Minimal Polyketide Synthase

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k approximately 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.

Different elongation pathways in the biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous plants.

Two common pathways are known for elongation of aliphatic acids via acetate in biological organisms: the fatty acid synthase (FAS) and the alpha-ketoacid elongation (alphaKAE) pathways. The alphaKAE route is utilized in many biosynthetic pathways, including the tricarboxylic acid cycle, leucine biosynthesis, and in formation of coenzyme B, glucosinolates, alpha-ketoadipate, sugar-ester acyl acids, short-chain alcohols of yeast and Clostridium species, 2-amino-4-methylhex-4-enoic acid, and l-gamma-phenyl butyrine. In the FAS route, both carbons from acetyl-acyl carrier protein are retained per elongation cycle, while in the alphaKAE route only one carbon from acetyl-coenzyme A is retained. Available evidence indicates that different members of the family Solanaceae may use one or the other of these elongation mechanisms in the synthesis of acyl groups of trichome-exuded sugar esters. In both, precursors for elongation are derived from branched-chain amino acid metabolism. Here we compared radiolabeling patterns in sugar-ester acyl groups from trichomes (the specific tissue in which sugar esters are synthesized) of the tobaccos, Nicotiana benthamiana, N. gossei, N. glutinosa, of Petunia x hybrida cv. Falcon Red & White, and Datura metel, and epidermal peels of Lycopsersicon pennellii after their synthesis from [2-(14)C]-, [1-(14)C]- and [1,2-(14)C]acetate. Recovered acyl acids were purified and then degraded to determine label distribution between the carboxyl termini and the remainder of the molecules. Six- and 20-h incubations were studied, and membrane fatty acids were monitored as internal controls for FAS-mediated elongation. Results are consistent with participation of alphaKAE in synthesis of sugar-ester acyl groups of tobaccos and petunia, but apparently FAS is utilized in the formation of these groups in L. pennellii and D. metel.

Fatty Acid and Lipoic Acid Biosynthesis in Higher Plant Mitochondria

Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP). We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl(C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors. Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria.

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 Km 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 Ki 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.

Acetyl-acyl carrier protein is not a major intermediate in fatty acid biosynthesis in spinach.

The extent to which acetyl-acyl carrier protein (acetyl-ACP) is an intermediate in fatty acid biosynthesis was examined. Acetyl-ACP was the least effective primer of fatty acid synthesis by spinach extracts when compared to acetyl-CoA, butyryl-ACP or hexanoyl-ACP. Furthermore, the rate of acetyl-ACP-primed fatty acid synthesis was inhibited significantly by cerulenin, indicating that the slow utilization of acetyl-ACP was predominantly by 3-oxoacyl-ACP synthase I. In light-incubated isolated chloroplasts with high rates of fatty acid synthesis (greater than 800 nmol.h-1.mg chlorophyll-1), the rate of acetyl-ACP metabolism was at least 10-30-fold slower than the rate of butyryl-ACP metabolism. The relatively slow metabolism of acetyl-ACP provided in situ evidence that (a) butyryl-ACP was formed principally from condensation of malonyl-ACP with acetyl-CoA and (b) acetyl-ACP was a minor participant in fatty acid biosynthesis.

Acetyl-acyl carrier protein is not a major intermediate in fatty acid biosynthesis in spinach

Acetyl-acyl carrier protein is not a major intermediate in fatty acid biosynthesis in spinach

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C(18:0) and C(16:0) were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K(m) values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that beta-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

Fat metabolism in higher plants: The nonenzymatic acylation of dithiothreitol by acyl coenzyme A

A compound soluble in organic solvents and synthesized from [ 14C]acetate by isolated spinach chloroplasts incubated in the dark in the presence of dithiothreitol was shown to be O-acetyl dithiothreitol. The chloroplast system was required for the activation of acetate to acetyl CoA, but the transfer of the acetyl moiety to dithiothreitol was nonenzymatic. The first product of the reaction was shown to be S-acetyl dithiothreitol, but in the presence of an oxidant, simultaneous ring closure and migration of the acetyl group from the thiol to an adjacent hydroxyl group occurred to form an O-acetyl dithiothreitol. The acetyl transfer reaction involving acetyl CoA and dithiothreitol showed a marked pH dependence, being most active at about pH 9 and inoperative below pH 6. All acyl CoAs tested (C 2-C 18) rapidly labeled dithiothreitol; acetyl acyl carrier protein, and palmityl acyl carrier protein were much less reactive and free fatty acids were unreactive. The thiol reagents dithioerythritol, glutathione, and cysteine, in addition to dithiothreitol, reacted rapidly with acetyl CoA to form the corresponding acetyl mercaptans. 2-Mercaptoethanol was much less reactive; oxidized dithiothreitol was unreactive. The second-order rate constant for acetyl dithiothreitol synthesis was 12.3 m −1 min −1 at pH 8.5 and 30 °C.

The Acyl‐Carrier Protein of Citrate Lyase

When [14C]acetyl citrate lyase was dissociated in sodium dodecylsulfate and the products were separated by gel electrophoresis, all of the radioactive label appeared in a small protein. This had a molecular weight of about 10000 and contained phosphopantetheine. It was concluded, therefore, that this protein component represents the acyl carrier protein of citrate lyase. [14C]Acetylated acyl carrier protein was purified by conventional methods to homogeneity as judged from the results of gel electrophoretic analysis. The well‐known dissociation of citrate lyase into subunits and their recombination were studied in detail for optimal conditions. It was found that inactivated, deacetylated citrate lyase could also undergo the dissociation‐association process. Recombination of this inactivated and dissociated lyase species in presence of the purified [14C]acetyl acyl carrier protein produced enzymatically active citrate lyase. The biological activity of the lyase acyl carrier protein thus demonstrated was confirmed by correlating enzymic activity and radioactive label of the reconstituted complex. An acyl carrier protein present in crude extracts of Klebsiella aerogenes, but not the deacetylated, purified lyase acyl carrier protein, was active in the malonyl‐CoA‐CO2 exchange reaction catalyzed by an Escherichia coli fatty acid synthetase preparation lacking acyl carrier protein. It was concluded, therefore, that two different acyl carrier proteins are present in K. aerogenes, one for fatty acid synthetase, the other for citrate lyase. Based on experimental results a model is proposed for the construction of citrate lyase. It was assumed that four moles of acyl carrier protein are present in one mole of the native complex which is additionally composed of 16 protein subunits of identical or nearly identical size. The basic functional unit appears to be a pentamer containing one acyl carrier protein and four of these protein subunits. Hence the native complex would consist of a tetramer of the functional basic pentamers. Some of the properties of the lyase acyl carrier protein are described. A remarkable feature is the presence of two sulfhydryl groups, one contributed by a cysteamine, the other by a cysteine residue. Both groups were shown to be acetylated in the isolated lyase acyl carrier protein by labelling experiments performed with iodo[14C]acetate. Whereas these results amply demonstrate the thioester linkage of the acetyl group to acyl carrier protein, the question whether both of the thioesters are enzymically active has not yet been clarified.

Comparative aspects of fatty acid synthesis in Bacillus subtilis and Escherichia coli.

Fatty acid synthesis in B. subtilis and E. coli is compared. Cell free fatty acid synthesizing systems from both organisms require malonyl-CoA, NADPH and a “priming” substrate. In the case of E. coli the priming substrate is acetyl-CoA and the products of fatty acid synthesis are saturated and unsaturated straight-chain fatty acids. B. subtilis synthesizes branched-chain fatty acids predominantly and uses branched-chain acyl-CoA esters as priming substrates. The fundamental difference between the fatty acid synthesizing systems of these two organisms lies in the specificity of the initial acyl-CoA:acyl carrier protein transacylase and the role this enzyme plays in dictating the nature of the fatty acids formed. 1 The utilisation of various acyl-CoA primers by a B. subtilis cell-free fatty acid synthesizing system was studied. This system was unable to utilize acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the initial acyl-CoA:acyl carrier protein transacylase was by-passed and acetate was rapidly incorporated into straight-chain fatty acids. 2 Similar experiments using a cell-free system from E. coli indicated that the substrate specificity of the E. coli acyl-CoA:acyl carrier protein transacylase was much less critical. The branched-chain CoA esters and intermediates were, as a whole, poorly utilized by the E. coli system.

Acyl Carrier Protein: IX. ACYL CARRIER PROTEIN HYDROLASE

Abstract Acyl carrier protein hydrolase, a phosphodiesterase which catalyzes the hydrolysis of Escherichia coli acyl carrier protein to yield the prosthetic group and the apoprotein, was purified 30-fold from extracts of E. coli. Hydrolysis of acyl carrier protein by this enzyme requires manganese (or other divalent cations) and it is stimulated by sulfhydryl compounds such as dithiothreitol or 2-mercaptoethanol. The enzymatic reaction products are 4'-phosphopantetheine and the apoprotein which was isolated and identified by comparison of its molecular weight and amino acid composition to intact acyl carrier protein. Acyl carrier protein hydrolase is very specific in regard to substrate since it does not catalyze the hydrolysis of 4'-phosphopantetheine from large peptides of E. coli acyl carrier protein. It does, however, catalyze the cleavage of 4'-phosphopantetheine from E. coli acetyl acyl carrier protein and from Clostridium butyricum acyl carrier protein.