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.     

Specificity and site of action of a mammary gland thioesterase which releases acyl moieties from thioester linkage to the fatty acid synthetase

The lactone (I) of 2-hydroxy-1-naphthaleneacetic acid was developed as a reagent for novel, highly efficient, covalent attachment of an excited-state proton transfer fluorescence probe (i.e. 2-naphthol) to protein amino groups. The lactone (I) was shown to react with amines faster than it was hydrolyzed. Reaction of the lactone (I) with bovine serum albumin (BSA) was faster than its reaction with corresponding concentrations of small organic amines, which suggested that the lactone (I) first adsorbed to BSA and subsequently reacted covalently; data are presented which suggest that this covalent binding occurs at a unique, single site on BSA (i.e., affinity labeling). Equilibrium studies involving instantaneous and time-dependent fluorescence changes were interpreted in terms of a 1:1 lactone:BSA labeling ratio at neutral pH. Steady-state fluorescence spectroscopy of the 1:1 lactone:BSA conjugate and of the conjugate of the lactone with glycine ethyl ester were recorded, compared and interpreted in terms of the microenvironment of the protein-bound fluorescence probe. Applications are suggested for use of this new and powerful procedure for study of the conformational changes and molecular interactions in other proteins.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of greater than 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.     

The isolation of acyl carrier protein from the pigeon liver fatty acid synthetase complex1 II

A low molecular weight protein of less than 10, 000 Daltons has been isolated from Subunit I (β-ketoacyl thioester reductase) of the pigeon liver fatty acid synthetase complex and purified to homogeneity. This protein contains all of the [¹⁴C]-labeled pantetheine incorporated into the fatty acid synthetase on injection of [¹⁴C]-labeled pantetheine into pigeons. It also has one β-alanine and one sulfhydryl group. This protein is an acceptor of an acetyl group from acetyl-CoA and a malonyl group from malonyl-CoA in the presence of Subunit II (transacylase). In these respects it is very similar to $\text{E. coli}$ acyl carrier protein.     

Binding site of cerulenin in fatty acid synthetase

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.     

Identification of borrelidin binding site on threonyl-tRNA synthetase

Borrelidin exhibits a wide spectrum of biological activities and has been considered as a non-competitive inhibitor of threonyl-tRNA synthetase (ThrRS). However, the detailed mechanisms of borrelidin against ThrRS, especially borrelidin binding site on ThrRS, are still unclear, which limits the development of novel borrelidin derivatives and rational design of structure-based ThrRS inhibitors. In this study, the binding site of borrelidin on Escherichia coli ThrRS was predicted by molecular docking. To validate our speculations, the ThrRS mutants of E. coli (P424K, E458Δ, and G459Δ) were constructed and their sensitivity to borrelidin was compared to that of the wild-type ThrRS by enzyme kinetics and stopped-flow fluorescence analysis. The docking results showed that borrelidin binds the pocket outside but adjacent to the active site of ThrRS, consisting of residue Y313, R363, R375, P424, E458, G459, and K465. Site-directed mutagenesis results showed that sensitivities of P424K, E458Δ, and G459Δ ThrRSs to borrelidin were reduced markedly. All the results showed that residue Y313, P424, E458, and G459 play vital roles in the binding of borrelidin to ThrRS. It indicated that borrelidin may induce the cleft closure, which blocks the release of Thr-AMP and PPi, to inhibit activity of ThrRS rather than inhibit the binding of ATP and threonine. This study provides new insight into inhibitory mechanisms of borrelidin against ThrRS.     

Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase

A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4'-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4'-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.     

Active site organization of bacterial type I fatty acid synthetase

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.     

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.     

Enzymatic transfer of the acyl group from acyl dihydroyxacetone phosphate to different substrates

The acyl group of acyl dihydroxyacetone phosphate was shown to be enzymatically transferred in guinea pig liver mitochondria to various acceptors such as lysolecithin, lysophosphatidyl ethanolamine and $\text{sn}$-glycerol-3-phosphate to form lecithin, phosphatidyl ethanolamine and phosphatidate, respectively. Coenzyme A and Mg⁺⁺, but not ATP, were required for this reaction. A rapid exchange of acyl group between acyl dihydroxyacetone phosphate and dihydroxyacetone phosphate was also observed.     

Interaction of rat mammary gland thioesterase II with fatty acid synthetase is dependent on the presence of acyl chains on the synthetase

The interaction between rat mammary gland thioesterase II and fatty acid synthetase has been studied by a variety of physicochemical techniques. Pyrene-labeled thioesterase II does not exhibit increased fluorescence anisotropy when mixed with fatty acid synthetase, suggesting that the enzymes do not readily form a complex. Nevertheless, the functional interaction between the enzymes can be easily demonstrated by observing the hydrolysis, by unmodified thioesterase II, of acyl chains from their thioester linkage to the 4-phosphopantetheine of the fatty acid synthetase. This hydrolytic reaction is not inhibited even in the presence of a large excess of fatty acid synthetase with vacant 4'-phosphopantetheine thiols, indicating that interaction occurs only between thioesterase and fatty acid synthetase species which carry acyl chains on the 4'-phosphopantetheine thiols. A novel model system was devised which allowed us to explore the nature of the physical interaction between the two enzymes under conditions where the synthetase was actively engaged in acyl chain assembly. Fatty acid synthetase was treated with phenylmethanesulfonyl fluoride to inhibit its resident thioesterase activity, immobilized via a specific antibody to a column of Sepharose 4B, and exposed to the substrates required for acyl-enzyme assembly. When thioesterase II was introduced to the column, it passed through unretarded even though it efficiently catalyzed hydrolysis of the immobilized S-acyl synthetase en route. These results indicate that the two enzymes associate when an acyl chain is present on the synthetase and that they dissociate rapidly following completion of the catalytic process. Thus, the mammary system differs from that of the avian uropygial gland in which the two enzymes associate to form a stable complex even in the absence of substrates.     

Identification of a long-chain polyunsaturated fatty acid acyl-coenzyme A synthetase from the diatom Thalassiosira pseudonana

The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast.     

Identification of bacteria from single colonies by fatty acid analysis.

While a commercially available system for microbial identification by fatty acid analysis (Microbial Identification System, MIDI, Newark, DE, USA) is available, it requires approximately 40-mg wet weight of biomass. Various authors have published methods for fatty acid analysis of single colonies, but no database of organisms has been developed for those methods. A modification of the MIDI system to increase sensitivity while maintaining relative retention times and peak areas allows the standard peak naming tables and libraries of organisms to be used with single colonies. Samples are evaporated down before injection to concentrate the fatty acids, while splitless injection is used to allow more sample to enter the gas chromatograph. Several known bacteria were correctly identified by this method.