Activation of long chain fatty acids with acyl carrier protein: demonstration of a new enzyme, acyl-acyl carrier protein synthetase, in Escherichia coli.

Abstract

A soluble enzyme activity which catalyzes the synthesis of acyl-acyl carrier protein from acyl carrier proteins, a long chain fatty acid, and ATP has been demonstrated in E. coil. The reaction requires high concentrations of both Ca++ and Mg++ for activity, and cleaves ATP to AMP and PP-. The fatty acyl product has been identified as acyl-acyl carrier protein by its solubility, thioester linkage, molecular weight, charge, and biological activity. Several palmitic acid (10,000 cpm/nmol); 2 mg/ml Triton X-100 and crude enzyme (0-0.1 mg) in a total volume of 0.1 ml. After incubation at 370 for 10 min, incorporation of fatty acid into acyl-ACP was assayed by either of two methods. The first method was solvent extraction to remove free fatty acid as described by Mancha et al. (9). A second and less cumbersome method was to pipet the reaction mix onto a filter paper disc (Whatman 3MM, 2.4 cm diameter). The discs were hen washed twice in a beaker containing chloroform-methanol-acetic acid (1:2:0.3, vol/vol) in order to remove free fatty acid (fatty acyl-CoA is also removed). Under these conditions, the enzyme assay gave a linear response with both protein and time. Blank values (minus enzyme, ACP, or ATP) showed <5% of the incorporation given by complete reaction mixtures. A unit of enzyme activity is a nmol of acyl-ACP formed per minute.Separation of ACP-SH and Acyl-ACP. Standard incubation mixtures, in which about 30% of the [3H]pantothenate-ACP-SH (4100 cpm/nmol) was converted to ['4C]palmityl-[3H]pantothenate-ACP by enzyme purified through the ammonium sulfate step, were extracted to remove free fatty acid and then acid precipitated at pH 3.5. The precipitate was resuspended in 1 ml of 0.1 M Tris-HCI buffer at pH 8.0 which contained 1 M KCI (to prevent nonspecific binding), 1 mM NaEDTA, and a 10-fold excess of dithiothreitol. After reduction, the ACP-SH in the mixture was then converted to its thionitrobenzoic acid derivative by addition of a 10-fold excess (over dithiothreitol) of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB). Excess DTNB and the other reaction products were removed from the protein fraction via a Sephadex G-25 column. The ACP derivatives from the void of the G-25 column were then applied to a 5 ml column of DTNB-agarose (10). The DTNB-agarose column was slowly (0.1 ml/min) eluted with the Tris-KCI-EDTA buffer.This treatment removed >95% of the ACP-SH in a parallel sample incubated without ATP. The ratio of palmitate to ACP in the eluate was then determined by dual label scintillation counting, by using the appropriate standards.Electrophoresis. Gels of 12% acrylamide were run in 0.1% sodium dodecyl sulfate (NaDodSO4) as previously described (8,11) except that sample preparation and quantitation were done as described by .Chromatography. The Sephadex G-75 column (1 X 36 cm,

How Bacterial Pathogens Eat Host Lipids: Implications for the Development of Fatty Acid Synthesis Therapeutics

“…This means that the ␤-hydroxyfatty acids produced by FASII for incorporation into lipopolysaccharide via the acyl-ACP-specific acyltransferases cannot be made from extracellular fatty acid sources. One confusing piece of information concerning metabolism is that exogenous fatty acids can be converted into acyl-ACP by the bifunctional 2-acylglycerophosphoethanolamine acyltransferase enzyme, which was called acyl-ACP synthetase (aas) based on the ability to detect this activity in vitro (35) (Fig. 2B).…”

Structural insights into bacterial resistance to cerulenin

“…The AAS activity was assayed using 20 lM purified E. coli holo-ACP and 100 lM sodium [1-14 C]palmitate (55 mCiÁmmol À1 ), 0.1 M Tris-HCl (pH 7.8), 10 mM ATP, 10 mM MgSO 4 , 5 mM dithiothreitol, and 0.2 lg of the AAS enzyme preparation was mixed in a 50-lL reaction system and incubated at 37°C. The reaction mixes were then loaded onto 3MM filter disks (Whatman, Maidstone, UK), which were washed and counted for radioactivity as described by Ray and Cronan [43]. The C6:0 to C10:0 acyl-ACP were synthesized and purified according to Shen et al [44] using essentially the same reaction, except that nonradioactive fatty acids replaced the radioactive substrate.…”

Expression and purification of four different rhizobial acyl carrier proteins The GenBank accession numbers for the nucleotide sequences reported in this paper are AF159244 and AF159243 for the acpP-containing regions of Sinorhizobium meliloti 1021 and of Rhizobium leguminosarum LPR5045, respectively.

“…A chemical method for acylation of ACPs has been described (Cronan & Klages, 1981), but preferably, simpler enzymic methods should be used. Acyl-ACP synthetase (Aas) of E. coli is the enzyme that can acylate E. coli AcpP (Ray & Cronan, 1976). AcpP from S. meliloti functions as effective substrate for E. coli Aas (Platt et al, 1990), whilst NodF is not a good substrate for the E. coli Aas (Ritsema et al, 1998).…”