Publications

• Functional screening and in vitro analysis reveal thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli.

  McMahon M.D., Prather K.L.

  Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse ... >> More

  Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent. << Less

  Appl. Environ. Microbiol. 80:1042-1050(2014) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Analysis of the mouse and human acyl-CoA thioesterase (ACOT) gene clusters shows that convergent, functional evolution results in a reduced number of human peroxisomal ACOTs.

  Hunt M.C., Rautanen A., Westin M.A.K., Svensson L.T., Alexson S.E.H.

  The maintenance of cellular levels of free fatty acids and acyl-CoAs, the activated form of free fatty acids, is extremely important, as imbalances in lipid metabolism have serious consequences for human health. Acyl-coenzyme A (CoA) thioesterases (ACOTs) hydrolyze acyl-CoAs to the free fatty acid ... >> More

  The maintenance of cellular levels of free fatty acids and acyl-CoAs, the activated form of free fatty acids, is extremely important, as imbalances in lipid metabolism have serious consequences for human health. Acyl-coenzyme A (CoA) thioesterases (ACOTs) hydrolyze acyl-CoAs to the free fatty acid and CoASH, and thereby have the potential to regulate intracellular levels of these compounds. We previously identified and characterized a mouse ACOT gene cluster comprised of six genes that apparently arose by gene duplications encoding acyl-CoA thioesterases with localizations in cytosol (ACOT1), mitochondria (ACOT2), and peroxisomes (ACOT3-6). However, the corresponding human gene cluster contains only three genes (ACOT1, ACOT2, and ACOT4) coding for full-length thioesterase proteins, of which only one is peroxisomal (ACOT4). We therefore set out to characterize the human genes, and we show here that the human ACOT4 protein catalyzes the activities of three mouse peroxisomal ACOTs (ACOT3, 4, and 5), being active on succinyl-CoA and medium to long chain acyl-CoAs, while ACOT1 and ACOT2 carry out similar functions to the corresponding mouse genes. These data strongly suggest that the human ACOT4 gene has acquired the functions of three mouse genes by a functional convergent evolution that also provides an explanation for the unexpectedly low number of human genes. << Less

  FASEB J. 20:1855-1864(2006) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.

• Purification and properties of fatty acyl thioesterase I from Escherichia coli.

  Bonner W.M., Bloch K.

  J. Biol. Chem. 247:3123-3133(1972) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Peroxisome proliferator-induced acyl-CoA thioesterase from rat liver cytosol: molecular cloning and functional expression in Chinese hamster ovary cells.

  Engberg S.T., Aoyama T., Alexson S.E.H., Hashimoto T., Svensson L.T.

  We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established con ... >> More

  We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established consensus sequences suggested that this enzyme represents a novel kind of esterase with an incomplete lipase serine active site motif. Analyses of mRNA and its expression indicated that the enzyme is significantly expressed in liver only after peroxisome proliferator treatment, but isoenzymes are constitutively expressed at high levels in testis and brain. The reported cDNA sequence is highly homologous to the recently cloned brain acyl-CoA thioesterase [Broustas, Larkins, Uhler and Hajra (1996) J. Biol. Chem. 271, 10470-10476], but subtle differences throughout the sequence, and distinct differences close to the resulting C-termini, suggest that they are different enzymes, regulated in different manners. A full-length cDNA clone was expressed in Chinese hamster ovary cells and the expressed enzyme was characterized. The palmitoyl-CoA hydrolysing activity (Vmax) was induced approx. 9-fold to 1 micromol/min per mg of cell protein, which was estimated to correspond to a specific activity of 250 micromol/min per mg of cDNA-expressed enzyme. Both the specific activity and the acyl-CoA chain length specificity were very similar to those of the purified rat liver enzyme. << Less

  Biochem. J. 323:525-531(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Purification and properties of long-chain acyl-CoA hydrolases from the liver cytosol of rats treated with peroxisome proliferator.

  Yamada J., Matsumoto I., Furihata T., Sakuma M., Suga T.

  Two long-chain acyl-CoA hydrolases, referred to as ACH1 and ACH2, were purified from the liver cytosol of rats fed a diet containing di(2-ethylhexyl)phthalate, a peroxisome proliferator. The molecular mass of ACH1 was estimated to be 73 kDa by gel filtration, and that of the subunits, 36 kDa by so ... >> More

  Two long-chain acyl-CoA hydrolases, referred to as ACH1 and ACH2, were purified from the liver cytosol of rats fed a diet containing di(2-ethylhexyl)phthalate, a peroxisome proliferator. The molecular mass of ACH1 was estimated to be 73 kDa by gel filtration, and that of the subunits, 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The corresponding values of ACH2 were 42 and 43 kDa, respectively. Both enzymes were active toward fatty acyl-CoAs with chain-lengths of C12-16, but ACH1 had relatively broad specificity as acyl-CoAs with C8-18 were good substrates. A marked difference in their catalytic properties was found in the maximal velocity; for palmitoyl-CoA, 553 and 4.23 mumol/min/mg with Km values of 5.9 and 5.4 microM for ACH1 and ACH2, respectively. ACH2 underwent severe substrate inhibition with high concentrations of long-chain acyl-CoAs, whereas ACH1 did not. Examination with various reagents including divalent cations, sulfhydryl-blocking reagent, nucleotides, and hypolipidemic drugs, characterized ACH1 and ACH2 with several properties distinct from those of mitochondrial and microsomal hydrolases. ACH1 and ACH2 were also discernible in that the former, but not the latter, was inhibited by ATP. In the liver cytosol of rats treated with di(2-ethylhexyl)phthalate, about 90% of palmitoyl-CoA hydrolase activity was titrated with anti-ACH1 and anti-ACH2 antibodies. Immunoblot analysis suggested the presence of the enzymes also in extrahepatic tissues, especially in the brain and testis (ACH1), and in the heart and kidney (ACH2). << Less

  Arch. Biochem. Biophys. 308:118-125(1994) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.

  Ofman R., el Mrabet L., Dacremont G., Spijer D., Wanders R.J.

  Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic ... >> More

  Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science. << Less

  Biochem. Biophys. Res. Commun. 290:629-634(2002) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases.

  Westin M.A.K., Alexson S.E.H., Hunt M.C.

  Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mai ... >> More

  Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression. << Less

  J. Biol. Chem. 279:21841-21848(2004) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• The Akt C-terminal modulator protein is an acyl-CoA thioesterase of the Hotdog-Fold family.

  Zhao H., Martin B.M., Bisoffi M., Dunaway-Mariano D.

  Herein, we report on an in vitro kinetic activity analysis that demonstrates that the protein known as the Akt C-terminal modulator protein is a broad-range, high-activity acyl-CoA thioesterase. In vitro tests of possible activity regulation by product inhibition or by Akt1 binding gave negative r ... >> More

  Herein, we report on an in vitro kinetic activity analysis that demonstrates that the protein known as the Akt C-terminal modulator protein is a broad-range, high-activity acyl-CoA thioesterase. In vitro tests of possible activity regulation by product inhibition or by Akt1 binding gave negative results. Truncation mutants confined the thioesterase activity to the C-terminal domain, consistent with our threading model. The N-terminal domain of unknown fold and function was found to contribute to solubility. << Less

  Biochemistry 48:5507-5509(2009) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Mycobacterium tuberculosis Rv3802c encodes a phospholipase/thioesterase and is inhibited by the antimycobacterial agent tetrahydrolipstatin.

  Parker S.K., Barkley R.M., Rino J.G., Vasil M.L.

  The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the alpha and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme en ... >> More

  The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the alpha and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications. << Less

  PLoS ONE 4:e4281-e4281(2009) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase.

  Tai M.H., Chirala S.S., Wakil S.J.

  Thioesterase II (TE II), present in specialized tissues, catalyzes the chain termination and release of medium-chain fatty acids from fatty acid synthase [FAS; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85]. We have exp ... >> More

  Thioesterase II (TE II), present in specialized tissues, catalyzes the chain termination and release of medium-chain fatty acids from fatty acid synthase [FAS; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85]. We have expressed rat mammary gland TE II in Escherichia coli and created several site-directed mutants. Replacing both Ser101 and His237 with Ala yielded inactive proteins, suggesting that these residues are part of the catalytic triad as in FAS thioesterase (TE I). Mutating the conserved Asp236 or modifying it with Woodward's reagent K caused partial loss (40%) of TE II activity and reduced reactivity of Ser101 and His237 toward their specific inhibitors, phenylmethylsulfonyl fluoride and diethylpyrocarbonate, respectively. These results suggested that Asp236 enhances, but is not essential for, the reactivity of Ser101 and His237. Mutation analyses revealed that, at the C terminus, Leu262 is critical for TE II to interact with FAS. Hydrophobic interactions seem to play a role, since the interaction of TE II with FAS is enhanced by polyethylene glycol but reduced by salt. The Ser101 and His237 mutants and a synthetic C-terminal decapeptide did not compete in the interaction. These results suggest that a TE II-acyl FAS complex forms first, which then is stabilized by the interaction of the hydrophobic C terminus of TE II with FAS, leading ultimately to hydrolysis and release of fatty acid. << Less

  Proc. Natl. Acad. Sci. U.S.A. 90:1852-1856(1993) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Characterization of an acyl-CoA thioesterase that functions as a major regulator of peroxisomal lipid metabolism.

  Hunt M.C., Solaas K., Kase B.F., Alexson S.E.H.

  Peroxisomes function in beta-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 ac ... >> More

  Peroxisomes function in beta-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 alpha. 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 acid N-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. << Less

  J. Biol. Chem. 277:1128-1138(2002) [PubMed] [EuropePMC]

  This publication is cited by 22 other entries.