Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Light-induced expression of fatty acid desaturase genes

In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases.

The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes

Fatty acid binding proteins (FABPs) exhibit a β-barrel topology, comprising 10 antiparallel β-sheets capped by two short α-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the α-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of α-eleostearic (18:3Δ9cis,11trans,13trans) and α-parinaric (18:4Δ9cis,11trans,13trans,15cis) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of α-eleostearic and α-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Δ12-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of α-eleostearic and α-parinaric acids, neither of which is present in untransformed soybean embryos. α-Eleostearic and α-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Δ12-oleic acid desaturase-type enzymes that we have termed “conjugase.”

Lipoprotein lipase controls fatty acid entry into adipose tissue, but fat mass is preserved by endogenous synthesis in mice deficient in adipose tissue lipoprotein lipase

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the import of triglyceride-derived fatty acids by muscle, for utilization, and adipose tissue (AT), for storage. Relative ratios of LPL expression in these two tissues have therefore been suggested to determine body mass composition as well as play a role in the initiation and/or development of obesity. To test this, LPL knockout mice were mated to transgenics expressing LPL under the control of a muscle-specific promoter (MCK) to generate induced mutants with either relative (L2-MCK) or absolute AT LPL deficiency (L0-MCK). L0-MCK mice had normal weight gain and body mass composition. However, AT chemical composition indicated that LPL deficiency was compensated for by large increases in endogenous AT fatty acid synthesis. Histological analysis confirmed that such up-regulation of de novo fatty acid synthesis in L0-MCK mice could produce normal amounts of AT as early as 20 h after birth. To assess the role of AT LPL during times of profound weight gain, L0-MCK and L2-MCK genotypes were compared on the obese ob/ob background. ob/ob mice rendered deficient in AT LPL (L0-MCK-ob/ob) also demonstrated increased endogenous fatty acid synthesis but had diminished weight and fat mass. These findings reveal marked alterations in AT metabolism that occur during LPL deficiency and provide strong evidence for a role of AT LPL in one type of genetic obesity.

Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a β-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ6 or Δ9 positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ9-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Human fatty acid synthase: Assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity

Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr ≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297; Mr ≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504; Mr ≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.

Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (Mr 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and β-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thioredoxin and six in-frame histidine residues. The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and β-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities.

Aspergillus has distinct fatty acid synthases for primary and secondary metabolism

Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.