Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

哈氏弧菌脂酰-ACP合成酶的体外功能

【目的】系统鉴定哈氏弧菌脂酰-ACP合成酶(Acyl-ACP synthetase,Aas S)以不同链长游离脂肪酸和非脂肪链羧酸作为底物的体外催化反应。【方法】利用非变性蛋白质凝胶电泳和紫外分光光度计法从定性和定量两个方面分析了Aas S的体外催化功能与活性。【结果】Aas S能够催化不同链长直链的自由脂肪酸合成脂酰-ACP,其中以C6–C12作为底物时活性最高;以羟基脂肪酸作为底物的情况下,Aas S催化C8–C14的羟基脂肪酸有较高的活性。非脂肪链羧酸类作为底物的反应中,20种蛋白质氨基酸、苯甲酸和水杨酸均可以作为Aas S的底物,合成相应的脂酰-ACP。【结论】本研究系统地证明了哈氏弧菌脂酰-ACP合成酶(Aas S)对不同底物的不同催化活性,为生物体内氨基酸代谢和菌黄素合成代谢的研究提供了可行性的分析依据。     

Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis.

We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II.     

Fat metabolism in higher plants. The determination of acyl-acyl carrier protein and acyl coenzyme A in a complex lipid mixture 1,2.

A method has been developed that permits the quantitative analysis of [¹⁴C]acyl-acyl carrier proteins and [¹⁴C]acyl CoAs from a typical reaction mixture. The method is based on (a) the initial extraction of free fatty acids and the less polar lipids into petroleum ether from aqueous isopropanol; (b) the precipitation of [¹⁴C]acyl-acyl carrier proteins in the presence of ammonium sulfate and chloroform-methanol; and (c) the final separation of acyl CoAs from the more polar lipids by selective adsorption on neutral alumina gel. All fractions can then be analyzed for the composition of complex lipids and ¹⁴C-labeled fatty acids by the usual methods.     

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.     

Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form

Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d-thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body.     

Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl-acyl carrier protein desaturase and ferredoxin.

Expression of a plant delta 6-palmitoyl (16:0)-acyl carrier protein desaturase in Escherichia coli resulted in the accumulation of the novel monounsaturated fatty acids delta 6-hexadecenoic acid (16:1 delta 6) and delta 8-octadecenoic acid. Amounts of 16:1 delta 6 accumulated by E. coli were increased more than twofold by the expression of a plant ferredoxin together with the delta 6-16:0-acyl carrier protein desaturase.     

A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L. (English ivy) can synthesize 16- and 18-carbon monoene and diene products

A desaturase with 83% sequence identity to the coriander delta(4)-16:0-ACP desaturase was isolated from developing seeds of Hedera helix (English ivy). Expression of the ivy desaturase in Arabidopsis resulted in the accumulation of 16:1delta(4) and its expected elongation product 18:1delta(6) (petroselinic acid). Expression in Escherichia coli resulted in the accumulation of soluble, active protein that was purified to apparent homogeneity. In vitro assays confirmed delta(4) desaturation with 16:0-ACP; however, with 18:0-acyl acyl carrier protein (ACP) desaturation occurred at the delta(9) position. The ivy desaturase also converted 16:1delta(9)-ACP and 18:1delta(9)-ACP to the corresponding delta(4,9) dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, delta(4) desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that delta(4) desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity.     

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (相似文献   

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Occurrence of apoAcyl carrier protein and an S-alkylation-resistant form of holoAcyl carrier protein in Escherichia coli.

The 4′-phosphopantetheine prosthetic group of holoacyl carrier protein (holoACP) in Escherichia coli turns over independently of the apoprotein, due to the activities of holoACP hydrolase and holoACP synthetase. There is no measurable pool of apoACP in pantothenate-supplemented cells of a pantothenate-requiring mutant, but extended incubation on deficient medium, with exhaustion of cellular coenzyme A (CoA), leads to slow accumulation of the apoprotein. It is concluded that, although the activities of the synthetase and hydrolase are about equal in crude extracts, in the cells an excess synthetase activity maintains ACP completely as holoACP unless cells are artifically depleted of CoA, the donor of the 4′-phosphopantetheine group. About 20% of the holoACP in normal cells was designated as “holoACP esters,” being resistant to S-alkylation unless first treated with neutral hydroxylamine; this proportion increased to about 80% in pantothenate starvation. A preliminary attempt to identify acyl portions from this material was unsuccessful. The proportion of this material was not elevated in other strains under conditions which show feedback inhibition of fatty acid biosynthesis in vivo.     

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.     

Rhodanese can partially refold in its GroEL-GroES-ADP complex and can be released to give a homogeneous product

Molecular chaperones GroEL and GroES facilitate reactivation of denatured rhodanese which folds poorly unless the process is assisted. The present work tests the hypothesis that more extensively unfolded forms of rhodanese bind tighter than those forms that appear later in the folding pathway. The study of the interaction of different urea-induced forms of rhodanese with GroEL suggests that species preceding the domain folded form bind directly and productively to GroEL. Rhodanese partially folds while in the GroEL-GroES-ADP complex, but it does not significantly reach an active state. Partially folded rhodanese can be released from the GroEL-GroES-ADP complex by subdenaturing concentrations of urea as a homogeneous species that is committed to fold to the native conformation with little or no partitioning to the aggregated state. Dilution of denatured rhodanese to the same final concentration gives less active enzyme and significant aggregation. Urea denaturation studies show that active rhodanese released from complexes behaves identically to native enzyme, while spontaneously folded rhodanese has a different stability. These results are interpreted using a previously proposed model based on studies of unassisted rhodanese folding [Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120-128. Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63-70].     

RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.