Structural and functional studies of fatty acyl adenylate ligases from E. coli and L. pneumophila

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 Å, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.     

Molecular basis of the functional divergence of fatty acyl-AMP ligase biosynthetic enzymes of Mycobacterium tuberculosis

Activation of fatty acids as acyl-adenylates by fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis is a variant of a classical theme that involves formation of acyl-CoA (coenzyme A) by fatty acyl-CoA ligases (FACLs). Here, we show that FAALs and FACLs possess similar structural fold and substrate specificity determinants, and the key difference is the absence of a unique insertion sequence in FACL13 structure. A systematic analysis shows a conserved hydrophobic anchorage of the insertion motif across several FAALs. Strikingly, mutagenesis of two phenylalanine residues, which are part of the anchorage, to alanine converts FAAL32 to FACL32. This insertion-based in silico analysis suggests the presence of FAAL homologues in several other non-mycobacterial genomes including eukaryotes. The work presented here establishes an elegant mechanism wherein an insertion sequence drives the functional divergence of FAALs from canonical FACLs.     

Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC ) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif ((213)YTGGTTGVAKGA(224) and (356)GYGLTE(361)) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD(G216A), fadD(T217A), fadD(G219A), and fadD(K222A)) and 2) those with 10% or less wild-type activity (fadD(Y213A), fadD(T214A), and fadD(E361A)). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.     

Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys⁴⁸⁷ resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys¹⁷² and Ala³⁰² to be involved in the binding of ATP and Ser⁴⁰⁴ in the binding of CoenzymeA. The influence of mutations of the residues Val²⁰⁹ and Trp³⁷⁷ emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C₂₆/C₂₄ fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys¹⁷², Ala³⁰² and Lys⁴⁸⁷ and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs.     

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.     

Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase

Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.     

Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.     

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B₁₂-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.     

Some properties of diacylglycerol acyltransferase in a particulate fraction from maturing safflower seeds

The activity of diacylglycerol acyltransferase of a subcellular particulate fraction from maturing safflower seeds was remarkably stimulated by the addition of 1, 2-diacylglycerols which were previously emulsified in a gelatin solution by sonication. Metal ions were inhibitory to the reaction. Deoxycholate and diisopropyl fluorophosphate were the most effective inhibitors. Sulfhydryl groups seemed to be of limited significance in the enzyme. Both 1, 2-dioleoyl-sn-glycerol and 2, 3-dioleoyl-sn-glycerol were good substrates of diacylglycerol acyltransferase, but the 1, 3-isomer did not serve as an acyl acceptor. The enzyme showed broad specificity for synthetic rac-1, 2-diacylglycerols containing various fatty acids. However, rac-1, 2-diacetylglycerol and rac-1, 2-dibutyrylglycerol, which are soluble in water, were ineffective. The enzyme exhibited no significant specificity for saturated and unsaturated fatty acyl-CoA thioesters as acyl donors. This suggests that the fatty acid composition at the 3-position of the glycerol molecule of safflower triacylglycerols may depend on the composition of the endogenous acyl-CoA pool.     

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.     

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.     

Involvement of acyl exchange between acyl-CoA and phosphatidylcholine in the remodelling of phosphatidylcholine in microsomal preparations of rat lung

Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant.     

Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding

Although each of the five mammalian long-chain acyl-CoA synthetases (ACSL) can bind saturated and unsaturated fatty acids ranging from 12 to 22 carbons, ACSL4 prefers longer chain polyunsaturated fatty acids. In order to gain a better understanding of ACSL4 fatty acid binding, we based a mutagenesis approach on sequence alignments related to ttLC-FACS crystallized from Thermus thermophilus HB8. Four residues selected for mutagenesis corresponded to residues in ttLC-FACS that comprise the fatty acid binding pocket; the fifth residue aligned with a region thought to be involved in fatty acid selectivity of the Escherichia coli acyl-CoA synthetase, FadD. Changing an amino acid at the entry of the putative fatty acid binding pocket, G401L, resulted in an inactive enzyme. Mutating a residue near the pocket entry, L399M, did not significantly alter enzyme activity, but mutating a residue at the hydrophobic terminus of the pocket, S291Y, altered ACSL4's preference for 20:5 and 22:6 and increased its apparent K(m) for ATP. Mutating a site in a region previously identified as important for fatty acid binding also altered activation of 20:4 and 20:5. These studies suggested that the preference of ACSL4 for long-chain polyunsaturated fatty acids can be modified by altering specific amino acid residues.     

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k_cat and K_m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3ΔfadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3ΔfadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3ΔfadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid:CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg²⁺-dependent enzymes that activate alkanoic acids to CoA esters for β oxidation (Fig. ​(Fig.11 ) (2, 17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (34). FadD is a cytoplasmic membrane-associated FACS (7), with sizes ranging from 47 kDa to 62 kDa (2, 14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.Open in a separate windowFIG. 1.FadD activation of fatty acids to their CoA derivatives proceeds through ATP-dependent covalent binding of AMP to fatty acid with the release of inorganic pyrophosphate, followed by C-S bond formation to obtain fatty acyl-CoA ester and subsequent release of AMP. FadD requires the presence of Mg²⁺ ions to be active (2, 17).It is known that bacteria such as Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (5, 6, 42, 45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in P. putida U with a high homology to long-chain fadD products from Escherichia coli and Pseudomonas fragi (6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (6). In a follow-up study, Olivera et al. showed that the fadD mutants reverted to wild-type characteristics within 3 days of incubation, indicating that fadD could be replaced by the activity of a second enzyme (25). Indeed, two fadD gene homologues have been identified in P. putida U, namely, fadD1 and fadD2, with fadD2 being expressed only when fadD1 is inactivated (25). A putative FadD in P. putida KT2440 is encoded by PP_4549 (24), but the protein has not been studied nor has the effect of fadD (PP_4549) expression/disruption been examined. In the current study the knockout and complementation of fadD from P. putida CA-3 demonstrated that its activity is critical for growth and PHA accumulation with long-chain aromatic and aliphatic alkanoic acids and that the activity is not replaced by a second enzyme. While reports have shown that PHA polymerase greatly affects PHA monomer composition (30, 40), no evidence of the specific effect of FACS on PHA accumulation so far exists.We describe here the purification, kinetic characterization, gene deletion, and homologous expression of FadD from P. putida CA-3. This is a fundamental study of the activity and physiological role of FACS activity in aromatic and aliphatic alkanoic acid activation and PHA accumulation.     

Mechanism and Regulation of Mycobactin Fatty Acyl-AMP Ligase FadD33

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ϵ-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD⁺-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.     

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.     

FadD is required for utilization of endogenous fatty acids released from membrane lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.     

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.