Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results.

Cloning, purification, crystallization and preliminary X-ray diffraction crystallographic study of acyl-protein thioesterase 1 from Saccharomyces cerevisiae.

Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme in Saccharomyces cerevisiae that catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Šresolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 146.43, c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.

Phylloquinone (vitamin K(1) ) biosynthesis in plants: two peroxisomal thioesterases of Lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-CoA.

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

An unusual thioesterase promotes isochromanone ring formation in ajudazol biosynthesis.

The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) multienzyme "assembly line" in the myxobacterium Chondromyces crocatus Cm c5. The most striking structural feature of these compounds is an isochromanone ring system; such an aromatic moiety is only known from two other complex polyketides, the electron transport inhibitor stigmatellin and the polyether lasalocid. The cyclization and aromatization reactions in the stigmatellin pathway are presumed to be catalyzed by a cyclase domain located at the end of the PKS, while the origin of the lasalocid benzenoid ring remains obscure. Notably, the ajudazol biosynthetic machinery does not incorporate a terminal cyclase, but instead a variant thioesterase (TE) domain. Here we present detailed phylogenetic and sequence analysis, coupled with experiments both in vitro and in vivo, that suggest that this TE promotes formation of the isochromanone ring, a novel reaction for this type of domain. As the ajudazol TE has homologues in several other secondary-metabolite pathways, these results are likely to be generalizable.

Metabolic engineering of glyoxalase pathway for enhancing stress tolerance in plants.

Glyoxalase system consists of two enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I detoxifies methylglyoxal (MG), a cytotoxic byproduct of glycolysis, to S-lactoylglutathione (SLG) where it uses one molecule of reduced glutathione. Subsequently, SLG is converted to lactate by Gly II and one molecule of reduced glutathione is recycled back into the system. The level of MG, which is produced ubiquitously in all living organisms, is enhanced upon exposure to different abiotic stresses in plants. Overexpression of glyoxalase pathway genes in transgenic plants has been found to keep a check on the MG level under stress conditions, regulate glutathione homeostasis, and the transgenic plants are able to survive and grow under various abiotic stresses.

Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases.

The ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer-dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methyl-gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum.

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k(cat)/K(m), was invariant among the fractions. The value of k(cat)/K(m) governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k(cat)/K(m) on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.

Monitoring enzyme catalysis in the multimeric state: direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry.

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1.

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.

Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function.

The aerobic benzoate metabolism in Azoarcus evansii follows an unusual route. The intermediates of the pathway are processed as coenzyme A (CoA) thioesters and the cleavage of the aromatic ring is non-oxygenolytic. The enzymes of this pathway are encoded by the box gene cluster which harbors a gene, orf1, coding for a putative thioesterase. Benzoyl-CoA thioesterase activity (20 nmol min(-1) mg(-1) protein) was present in cells grown aerobically on benzoate, but was lacking in cells grown on other aromatic or aliphatic substrates under oxic or anoxic conditions. The gene was cloned and overexpressed in Escherichia coli to produce a C-terminal His-tag fusion protein. The recombinant enzyme was a homotetramer of 16 kDa subunits. It catalyzed not only the hydrolysis of benzoyl-CoA, but also of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA, the second intermediate in the pathway. The enzyme exhibited higher activity with mono-substituted derivatives of benzoyl-CoA, showing highest activity with 4-hydroxybenzoyl-CoA. Di-substituted derivatives of benzoyl-CoA, phenylacetyl-CoA, and aliphatic CoA thioesters were not hydrolyzed but some acted as inhibitors. The thioesterase appears to protect the cell from CoA pool depletion. It may constitute the prototype of a new subfamily within the hotdog fold enzyme superfamily.

The unusual macrocycle forming thioesterase of mycolactone.

Mycolactone is a polyketide natural product secreted by Mycobacterium ulcerans, the organism responsible for the tropical skin disease Buruli ulcer. The finding that this small molecule virulence factor is sufficient to reconstitute the necrotic pathology associated with Buruli ulcer suggests that a better understanding of mycolactone biosynthesis, particularly the processes which are distinct from those in human metabolism, may provide a unique avenue for the development of selective therapeutics. In the present study we have cloned, expressed, and biochemically characterized the putative macrocycle forming thioesterase for mycolactone, MLSA2 TE. We have evaluated the enzyme both as the truncated thioesterase domain and as a carrier protein-linked didomain construct. The results of these analyses distinguish MLSA2 TE from traditional fatty acid and polyketide synthase TE-domains in terms of its sequence, kinetic parameters, and susceptibility to traditional active-site directed inhibitors. These findings suggest that MLSA2 TE utilizes a unique biochemical mechanism for macrocycle formation.

Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disease caused by deficiency of palmitoyl protein thioesterase 1 (PPT1). INCL results in dramatic loss of thalamocortical neurons, but the disease mechanism has remained elusive. In the present work we describe the first interaction partner of PPT1, the F(1)-complex of the mitochondrial ATP synthase, by co-purification and in vitro-binding assays. In addition to mitochondria, subunits of F(1)-complex have been reported to localize in the plasma membrane, and to be capable of acting as receptors for various ligands such as apolipoprotein A-1. We verified here the plasma membrane localization of F(1)-subunits on mouse primary neurons and fibroblasts by cell surface biotinylation and TIRF-microscopy. To gain further insight into the Ppt1-mediated properties of the F(1)-complex, we utilized the Ppt1-deficient Ppt1(Delta ex4) mice. While no changes in the mitochondrial function could be detected in the brain of the Ppt1(Delta ex4) mice, the levels of F(1)-subunits alpha and beta on the plasma membrane were specifically increased in the Ppt1(Delta ex4) neurons. Significant changes were also detected in the apolipoprotein A-I uptake by the Ppt1(Delta ex4) neurons and the serum lipid composition in the Ppt1(Delta ex4) mice. These data indicate neuron-specific changes for F(1)-complex in the Ppt1-deficient cells and give clues for a possible link between lipid metabolism and neurodegeneration in INCL.

Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia).

Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.

Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum.

In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222(1) (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 angstroms) and diffract beyond 2.15 angstroms using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.

Flexible metal binding of the metallo-beta-lactamase domain: glyoxalase II incorporates iron, manganese, and zinc in vivo.

Glyoxalase II belongs to the metallo-beta-lactamase superfamily of proteins, possessing the characteristic dinuclear active site. Within this protein family, glyoxalase II from Arabidopsis thaliana is the first member to be isolated with significant amounts of iron, manganese, and zinc when being recombinantly produced in Escherichia coli. Enzyme preparations with different ratios of these three metals all yield k(cat)/K(M) values in the range of 1.5-1.9 s(-1) microM(-1) with the substrate S-d-lactoylglutathione. X-ray absorption spectroscopy reveals binding of all three metals to the dinuclear active site with 5-6-fold coordination consisting of 2.5 +/- 0.5 histidine and 2.5 +/- 0.5 oxygen ligands. This model does not distinguish site-specific or distributed binding. The metal-metal distance is determined to be 3.18 +/- 0.06 A. Electron paramagnetic resonance spectroscopy gives evidence for several different types of dimetal sites, including spin-coupled Fe(III)Fe(II), Fe(III)Zn(II), and Mn(II)Mn(II) centers. The metal-ligand distances measured by X-ray absorption spectroscopy vary depending on the metal type and comply with their element-specific, characteristic values. This reflects a high degree of structural flexibility within the glyoxalase II dinuclear active site, which is considered as the structural basis for its broad metal selectivity.

Cloning and expression of fatty acids biosynthesis key enzymes from sunflower (Helianthus annuus L.) in Escherichia coli.

To further characterize the stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) and the acyl-ACP thioesterase FatB (EC 3.1.2.14) activities from sunflower seeds, we cloned, sequenced and expressed the recombinant genes in Escherichia coli. We obtained two partially purified proteins, His-SAD and His-FATB, each of about 45000 Da. The expression of either proteins produced changes in the E. coli fatty acid profile indicating the functionality of the recombinant proteins. While the expression of His-SAD produced an effect similar to that produced by overexpression of the fabA gene, responsible for the fatty acid desaturation in E. coli, the expression of His-FATB gave rise to an unbalance between unsaturated fatty acids and a toxic effect in E. coli.

Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase.

The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.

Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.

A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the early C. elegans embryo.

In the early Caenorhabditis elegans embryo, establishment of cell polarity and cytokinesis are both dependent upon reorganization of the actin cytoskeleton. Mutations in the cyk-3 gene cause maternal effect embryonic lethality. Embryos produced by homozygous cyk-3 mutant animals become multinucleate. We have further analyzed the cyk-3 mutant phenotype and have found that cyk-3 mutant embryos fail to properly polarize the actin cytoskeleton and fail to segregate germline determinants. In addition, they fail to assemble an intact cleavage furrow. However, we have found that cyk-3 mutant embryos are intrinsically defective in osmotic regulation and that the cytokinesis defects can be partially rescued by providing osmotic support. The cyk-3 gene has been identified and found to encode a ubiquitin C-terminal hydrolase that is active against model substrates. These data indicate that the deubiquitination of certain substrates by CYK-3 is crucial for cellular osmoregulation. Defects in osmoregulation appear to indirectly affect actin-dependent processes.

The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism.

Acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoAs to the free fatty acid and coenzyme A (CoASH), providing the potential to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. These enzymes are localized in almost all cellular compartments such as endoplasmic reticulum, cytosol, mitochondria and peroxisomes. Acyl-CoA thioesterases are highly regulated by peroxisome proliferator-activated receptors (PPARs), and other nutritional factors, which has led to the conclusion that they are involved in lipid metabolism. Although the physiological functions for these enzymes are not yet fully understood, recent cloning and more in-depth characterization of acyl-CoA thioesterases has assisted in discussion of putative functions for specific enzymes. Here we review the acyl-CoA thioesterases characterized to date and also address the diverse putative functions for these enzymes, such as in ligand supply for nuclear receptors, and regulation and termination of fatty acid oxidation in mitochondria and peroxisomes.