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Stefan E. H. Alexson - One of the best experts on this subject based on the ideXlab platform.
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characterization of an acyl coa Thioesterase that functions as a major regulator of peroxisomal lipid metabolism
Journal of Biological Chemistry, 2002Co-Authors: Mary C. Hunt, Karianne Solaas, Frode B Kase, Stefan E. H. AlexsonAbstract:Abstract Peroxisomes function in β-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA Thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA Thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA Thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor α. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA Thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acidN-acyltransferase activity, suggesting that this Thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.
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Peroxisome proliferators differentially regulate long-chain acyl-CoA Thioesterases in rat liver
European journal of biochemistry, 1995Co-Authors: L. Thomas Svensson, Mona Wilcke, Stefan E. H. AlexsonAbstract:We have investigated the effects of peroxisome proliferators on rat liver long-chain acyl-CoA Thioesterase activities. Subcellular fractionations of liver homogenates from control, clofibrate- and di(2-ethylhexyl)phthalate-treated rats confirmed earlier studies which demonstrated that peroxisome-proliferating drugs induce long-chain acyl-CoA Thioesterase activity mainly in the mitochondrial and cytosolic fractions. The aim of the present study was to investigate whether the induced activities were due to increases in normally expressed enzymes, or due to induction of novel enzymes. To investigate whether structurally different peroxisome proliferators differentially induced Thioesterase activities, we tested the effects of di(2-ethylhexyl)phthalate (a plastisizer) and the hypolipidemic drug clofibrate. For this purpose, we established an analytical size exclusion chromatography method. Chromatography of solubilised mitochondrial matrix proteins showed that the activity in control mitochondria was mainly due to enzymes with molecular masses of about 50 kDa and 35 kDa. The activity in samples prepared from clofibrate- and di(2-ethylhexyl)phthalate-treated rats eluted as proteins of about 40 kDa and 110 kDa. Highly purified peroxisomes contained two peaks of activity, which were not induced, that corresponded to molecular masses of 40 kDa and 80 kDa. The 80-kDa peak was shown to be due to dimerization by addition of glycerol. Chromatography of cytosolic fractions from control rat livers indicated the presence of long-chain acyl-CoA Thioesterases with molecular masses of approximately 35 kDa and 125 kDa and a broad peak corresponding to a high-molecular-mass protein. The activity in cytosolic fractions from peroxisome-proliferator-treated rats eluted mainly as peaks corresponding to 40, 110 and 150 kDa. In addition, in the 110-kDa peak, a different degree of induction and different chain-length specificities were caused by clofibrate and di(2-ethylhexyl)phthalate, suggesting that these peroxisome proliferators differentially regulate the cytosolic acyl-CoA Thioesterase activities. Western blot analysis showed that enzymes in the 40-kDa peak of the peroxisomal and cytosolic fractions were structurally related, but not identical, to a 40-kDa mitochondrial very-long-chain acyl-CoA Thioesterase. Our data show that the increased acyl-CoA Thioesterase activities in mitochondria and cytosol were mainly due to induction of acyl-CoA Thioesterases which are not, or only weakly, expressed under normal conditions.
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Characterization of acyl-CoA Thioesterase activity in isolated rat liver peroxisomes. Partial purification and characterization of a long-chain acyl-CoA Thioesterase.
European journal of biochemistry, 1994Co-Authors: Mona Wilcke, Stefan E. H. AlexsonAbstract:A common function of peroxisomes in eukaryotic cells is β-oxidation of fatty acids. In animal cells, β-oxidation is compartmentalized to peroxisomes and mitochondria. Although regulation of β-oxidation in mitochondria has been extensively studied, knowledge on its regulation in peroxisomes is still limited. We have considered the possibility that peroxisomes may contain acyl-CoA Thioesterases with different substrate specificities that possibly regulate metabolism of different lipids by regulation of substrate availability. In the present study, we have investigated the presence of short-chain and long-chain acyl-CoA Thioesterase activities in rat liver peroxisomes. Light-mitochondrial fractions, enriched in peroxisomes, were fractionated by Nycodenz density gradient centrifugation and gradient fractions were analyzed for acyl-CoA Thioesterase and marker enzyme distributions. Fractionation of livers from normal rats showed that most of the long-chain acyl-CoA Thioesterase activity was localized in microsomes and mitochondria, and only low activity was found in fractions containing peroxisomes. The gradient distribution of propionyl-CoA Thioesterase activity showed this activity to be localized mainly in mitochondria and in fractions possibly representing lysosomes, with a small peak of activity in peroxisomal fractions. Di(2-ethylhexyl)phthalate treatment induced the specific propionyl-CoA Thioesterase activity approximately threefold in the peak mitochondrial fractions and about onefold in peroxisomal fractions; the activity appeared to be almost exclusively localized to these organelles. The specific activity of myristoyl-CoA Thioesterase was induced 1–2-fold in peroxisomal peak fractions and more than 10-fold in the mitochondrial peak fraction, whereas it was unchanged in microsomes. The chain-length specificity of acyl-CoA Thioesterase activity in isolated peroxisomes suggests that peroxisomes contain an inducible short-chain Thioesterase active on C2-C4 acyl-CoA species (possibly a ‘propionyl-CoA’ Thioesterase). In addition, peroxisomes contain medium-chain to long-chain Thioesterase activity, probably due to separate enzymes based on the different chain-length specificities observed in peroxisomes from normal and di(2-ethylhexyl)phthalate-treated rats. A long-chain acyl-CoA Thioesterase was partially purified from isolated peroxisomes and found to be active only on fatty-acyl-CoA species longer than octanoyl-CoA. The protein is apparently a monomer of about 40 kDa and clearly different from microsomal long-chain acyl-CoA Thioesterase. An induction of this long-chain Thioesterase may explain the observed change in chain-length specificity in peroxisomes isolated from normal and di(2-ethylhexyl)phthalate-treated rats. Possible physiological functions of these Thioesterases are discussed.
Sandra L. Hofmann - One of the best experts on this subject based on the ideXlab platform.
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disruption of ppt1 or ppt2 causes neuronal ceroid lipofuscinosis in knockout mice
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Praveena Gupta, Abigail A. Soyombo, Armita Atashband, Krystyna E Wisniewski, John M Shelton, James A Richardson, Robert E Hammer, Sandra L. HofmannAbstract:PPT1 and PPT2 encode two lysosomal Thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein Thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create “knockout” mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a “clasping” phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.
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Molecular Cloning and Expression of Palmitoyl-protein Thioesterase 2 (PPT2), a Homolog of Lysosomal Palmitoyl-protein Thioesterase with a Distinct Substrate Specificity
The Journal of biological chemistry, 1997Co-Authors: Abigail A. Soyombo, Sandra L. HofmannAbstract:Abstract Palmitoyl-protein Thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal Thioesterase, palmitoyl-protein Thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein Thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein Thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein Thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein Thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal Thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein Thioesterase.
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lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis correction of the defect in lymphoblasts by recombinant palmitoyl protein Thioesterase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jui Yun Lu, Linda A. Verkruyse, Sandra L. HofmannAbstract:Palmitoyl-protein Thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein Thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein Thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein Thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein Thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.
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Lysosomal Targeting of Palmitoyl-protein Thioesterase
The Journal of biological chemistry, 1996Co-Authors: Linda A. Verkruyse, Sandra L. HofmannAbstract:Abstract Palmitoyl-protein Thioesterase is a newly described long chain fatty-acid hydrolase that removes fatty acyl groups from modified cysteines in proteins. We have recently identified palmitoyl-protein Thioesterase as the defective enzyme in the recessive hereditary neurological degenerative disorder infantile neuronal ceroid lipofuscinosis (Vesa, J., Hellsten, E., Verkruyse, L. A., Camp, L. A., Rapola, J., Santavuori, P., Hofmann, S. L., and Peltonen, L. (1995) Nature 376, 584–587). A defect in a lysosomal enzyme had been postulated for the disease, but until recently, the relevant defective lysosomal enzyme had not been identified. In this paper, we present evidence for the lysosomal localization of palmitoyl-protein Thioesterase. We show that COS cells take up exogenously supplied palmitoyl-protein Thioesterase intracellularly and that the cellular uptake is blocked by mannose 6-phosphate, a hallmark of lysosomal enzyme trafficking. The enzyme contains endoglycosidase H-sensitive oligosaccharides that contain phosphate groups. Furthermore, palmitoyl-protein Thioesterase cosediments with lysosomal enzyme markers by Percoll density gradient centrifugation. Interestingly, the pH optimum for the enzyme is in the neutral range, a property shared by two other lysosomal enzymes that remove post-translational protein modifications. These findings suggest that palmitoyl-protein Thioesterase is a lysosomal enzyme and that infantile neuronal ceroid lipofuscinosis is properly classified as a lysosomal storage disorder.
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Molecular cloning and expression of palmitoyl-protein Thioesterase.
The Journal of biological chemistry, 1994Co-Authors: Laura A. Camp, Linda A. Verkruyse, Steven J. Afendis, Clive A. Slaughter, Sandra L. HofmannAbstract:Abstract We have previously reported the purification of a palmitoyl-protein Thioesterase (PPT) from bovine brain that removes palmitate from Ha-Ras (Camp, L. A., and Hofmann, S. L. (1993) J. Biol. Chem. 268, 22566-22574). In the current paper, we have isolated bovine and rat cDNA clones encoding PPT. The deduced amino acid sequence of PPT predicts a protein of 306 amino acids that contains amino acid motifs characteristic of Thioesterases: "Gly-X-Ser-X-Gly" positioned near the NH2 terminus and "Gly-Asp-His" positioned near the COOH terminus of the protein. The identity of the PPT cDNA was further confirmed by expression in simian COS cells and insect Sf9 cells. Comparison of the DNA and protein sequence data suggests that a hydrophobic NH2-terminal sequence of 27 amino acid residues is removed from the primary translation product. Furthermore, the recombinant protein and the native protein purified from bovine brain contain complex asparagine-linked oligosaccharides and a large proportion of the expressed PPT is secreted from COS and Sf9 cells. Thus, while the palmitoyl-protein Thioesterase will deacylate intracellular palmitoylated proteins such as Ha-Ras and the alpha subunits of heterotrimeric G proteins, the physiologic substrates are likely to be externally oriented or secreted proteins.
Hazel M. Holden - One of the best experts on this subject based on the ideXlab platform.
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computational redesign of acyl acp Thioesterase with improved selectivity toward medium chain length fatty acids
ACS Catalysis, 2017Co-Authors: Matthew J Grisewood, Rung-yi Lai, James B. Thoden, Nestor J Hernandezlozada, Nathanael P Gifford, Daniel Mendezperez, Haley Schoenberger, Matthew F Allan, Martha E Floy, Hazel M. HoldenAbstract:Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C8–C12) acyl-ACP Thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of Thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemi...
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The Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. strain SU
The Journal of biological chemistry, 2003Co-Authors: James B. Thoden, Debra Dunaway-mariano, Zhihao Zhuang, Hazel M. HoldenAbstract:Abstract The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a Thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called “hot dog fold” composed of six strands of anti-parallel β-sheet flanked on one side by a rather long α-helix. The dimers come together to form the tetramer with their α-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA Thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their α-helices projecting toward the interfacial region. In the Arthrobacter Thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter Thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial Thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.
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The Three-dimensional Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Pseudomonas sp. Strain CBS-3*
The Journal of biological chemistry, 1998Co-Authors: Matthew M. Benning, Debra Dunaway-mariano, Gary E. Wesenberg, Rui-qin Liu, Kimberly L. Taylor, Hazel M. HoldenAbstract:The soil-dwelling microbe, Pseudomonas sp. strain CBS-3, has attracted recent attention due to its ability to survive on 4-chlorobenzoate as its sole carbon source. The biochemical pathway by which this organism converts 4-chlorobenzoate to 4-hydroxybenzoate consists of three enzymes: 4-chlorobenzoyl-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl-CoA Thioesterase. Here we describe the three-dimensional structure of the Thioesterase determined to 2.0-A resolution. Each subunit of the homotetramer is characterized by a five-stranded anti-parallel beta-sheet and three major alpha-helices. While previous amino acid sequence analyses failed to reveal any similarity between this Thioesterase and other known proteins, the results from this study clearly demonstrate that the molecular architecture of 4-hydroxybenzoyl-CoA Thioesterase is topologically equivalent to that observed for beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli. On the basis of the structural similarity between these two enzymes, the active site of the Thioesterase has been identified and a catalytic mechanism proposed.
David E. Cohen - One of the best experts on this subject based on the ideXlab platform.
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Functional Characterization of Thioesterase Superfamily Member 1/Acyl-CoA Thioesterase 11: Implications for Metabolic Regulation
Journal of lipid research, 2012Co-Authors: Shuxin Han, David E. CohenAbstract:Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA Thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA Thioesterase that comprises tandem hot dog-fold Thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA Thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the Thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA Thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA Thioesterase that could be targeted for the management of metabolic disorders.
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targeted deletion of Thioesterase superfamily member 1 promotes energy expenditure and protects against obesity and insulin resistance
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Yongzhao Zhang, Michele W Niepel, Yuki Kawano, Shuxin Han, Sihao Liu, Alessandro Marsili, Reed P Larsen, Chihhao Lee, David E. CohenAbstract:Mammalian acyl-CoA Thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoAs to form free fatty acids plus CoA, but their metabolic functions remain undefined. Thioesterase superfamily member 1 (Them1; synonyms Acot11, StarD14, and brown fat inducible Thioesterase) is a long-chain fatty acyl-CoA Thioesterase that is highly expressed in brown adipose tissue and is regulated by both ambient temperature and food consumption. Here we show that Them1−/− mice were resistant to diet-induced obesity despite greater food consumption. Them1−/− mice exhibited increased O2 consumption and heat production, which were accompanied by increased rates of fatty acid oxidation in brown adipose tissue and up-regulation of genes that promote energy expenditure. Them1−/− mice were also protected against diet-induced inflammation in white adipose tissue, as well as hepatic steatosis, and demonstrated improved glucose homeostasis. The absence of Them1 expression in vivo and in cell culture led to markedly attenuated diet- or chemically induced endoplasmic reticulum stress responses, providing a mechanism by which Them1 deficiency protects against insulin resistance and lipid deposition. Taken together, these data suggest that Them1 functions to decrease energy consumption and conserve calories. In the setting of nutritional excess, the overproduction of free fatty acids by Them1 provokes insulin resistance that is associated with inflammation and endoplasmic reticulum stress.
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Thioesterase superfamily member 2 (Them2)/acyl-CoA Thioesterase 13 (Acot13): a homotetrameric hotdog fold Thioesterase with selectivity for long-chain fatty acyl-CoAs.
The Biochemical journal, 2009Co-Authors: Jie Wei, Hye Won Kang, David E. CohenAbstract:Them2 (Thioesterase superfamily member 2) is a 140-amino-acid protein of unknown biological function that comprises a single hotdog fold Thioesterase domain. On the basis of its putative association with mitochondria, accentuated expression in oxidative tissues and interaction with StarD2 (also known as phosphatidylcholine-transfer protein, PC-TP), a regulator of fatty acid metabolism, we explored whether Them2 functions as a physiologically relevant fatty acyl-CoA Thioesterase. In solution, Them2 formed a stable homotetramer, which denatured in a single transition at 59.3 °C. Them2 exhibited Thioesterase activity for medium- and long-chain acyl-CoAs, with K m values that decreased exponentially as a function of increasing acyl chain length. Steady-state kinetic parameters for Them2 were characteristic of long-chain mammalian acyl-CoA Thioesterases, with minimal values of K m and maximal values of k cat / K m observed for myristoyl-CoA and palmitoyl-CoA. For these acyl-CoAs, substrate inhibition was observed when concentrations approached their critical micellar concentrations. The acyl-CoA Thioesterase activity of Them2 was optimized at physiological temperature, ionic strength and pH. For both myristoyl-CoA and palmitoyl-CoA, the addition of StarD2 increased the k cat of Them2. Enzymatic activity was decreased by the addition of phosphatidic acid/phosphatidylcholine small unilamellar vesicles. Them2 expression, which was most pronounced in mouse heart, was associated with mitochondria and was induced by activation of PPARα (peroxisome-proliferator-activated receptor α). We conclude that, under biological conditions, Them2 probably functions as a homotetrameric long-chain acyl-CoA Thioesterase. Accordingly, Them2 has been designated as the 13th member of the mammalian acyl-CoA Thioesterase family, Acot13.
Jade K. Forwood - One of the best experts on this subject based on the ideXlab platform.
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Structural insights into GDP-mediated regulation of a bacterial acyl-CoA Thioesterase
The Journal of biological chemistry, 2017Co-Authors: Yogesh B. Khandokar, P. Srivastava, Nathan P. Cowieson, Subir Sarker, David Aragão, Shubhagata Das, Kate Smith, Shane Raidal, Jade K. ForwoodAbstract:Abstract Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 Thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. While many of these enzyme families are well characterized in terms of function and substrate specificity, regulation across most Thioesterase families is poorly understood. Here, we characterized a TE6 Thioesterase from the bacterium Neisseria meningitidis. Structural analysis with X-ray crystallographic diffraction data to 2.0 A revealed that each protein subunit harbors a hot dog fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of Thioesterase activity against a range of acyl-CoA substrates revealed greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn-24 and Asp-39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link Thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg-93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated Thioesterase and of covalent linkage of Thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 Thioesterase.
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structural and functional characterization of the paai Thioesterase from streptococcus pneumoniae reveals a dual specificity for phenylacetyl coa and medium chain fatty acyl coas and a novel coa induced fit mechanism
Journal of Biological Chemistry, 2016Co-Authors: Yogesh B. Khandokar, P. Srivastava, Nathan P. Cowieson, Subir Sarker, David Aragão, Crystall M D Swarbrick, Jade K. ForwoodAbstract:PaaI Thioesterases are members of the TE13 Thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI Thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-A resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of Thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn(37), Asp(52), and Thr(68) are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 A in the N terminus, and a loop region involving Tyr(38)-Tyr(39). This is the first time PaaI Thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.
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structural basis for regulation of the human acetyl coa Thioesterase 12 and interactions with the steroidogenic acute regulatory protein related lipid transfer start domain
Journal of Biological Chemistry, 2014Co-Authors: Crystall M D Swarbrick, Nathan P. Cowieson, Noelia Roman, Edward I Patterson, Jeffrey D Nanson, Marina I Siponen, Helena Berglund, L Lehtio, Jade K. ForwoodAbstract:Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA Thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the Thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the Thioesterase domains (Phe154-Thr178), defined here as RegLoop1, and a second region involving the C terminus of Thioesterase domain 2 (Gln304-Gly326), designated RegLoop2. Mutagenesis confirmed that Arg312 and Arg313 are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic Thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution.
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structural basis for recruitment of tandem hotdog domains in acyl coa Thioesterase 7 and its role in inflammation
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Jade K. Forwood, Anil Thakur, Gregor Guncar, Mary Marfori, Dmitri Mouradov, Weining Meng, Jodie A Robinson, Thomas Huber, Stuart Kellie, Jennifer L MartinAbstract:Acyl-CoA Thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA Thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single Thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.
