Publications

• The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization.

  Thomas J., Cronan J.E.

  The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an en ... >> More

  The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed. << Less

  J. Biol. Chem. 280:34675-34683(2005) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Human fatty acid synthase: assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity.

  Jayakumar A., Chirala S.S., Wakil S.J.

  Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr approximately 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtap ... >> More

  Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr approximately 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 A 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 approximately 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296-2,504; Mr approximately 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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:12326-12330(1997) [PubMed] [EuropePMC]

  This publication is cited by 26 other entries.

• Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases.

  Salas J.J., Ohlrogge J.B.

  The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters ... >> More

  The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases. << Less

  Arch. Biochem. Biophys. 403:25-34(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins.

  Dormann P., Voelker T.A., Ohlrogge J.B.

  An Arabidopsis thaliana partial cDNA was previously identified with a sequence similar to the lauroyl-acyl carrier protein (ACP) thioesterase from Umbellularia california (Grellet et al., 1993, Plant Physiol. Biochem. 31, 599-602). Using this DNA fragment, we isolated a 1.8-kb cDNA coding for a 41 ... >> More

  An Arabidopsis thaliana partial cDNA was previously identified with a sequence similar to the lauroyl-acyl carrier protein (ACP) thioesterase from Umbellularia california (Grellet et al., 1993, Plant Physiol. Biochem. 31, 599-602). Using this DNA fragment, we isolated a 1.8-kb cDNA coding for a 412-amino-acid preprotein. The deduced amino acid sequence is 51% identical to the lauroyl-ACP thioesterase but only 39% identical to safflower oleoyl-ACP thioesterase. The cDNA was expressed in Escherichia coli and the gene product showed thioesterase activity for long-chain acyl-ACPs (14:0, 16:0, 18:0, 18:1 delta 9cis). When expressed in beta-oxidation mutants of E. coli, lipid analysis revealed that cells transformed with the thioesterase produced high amounts of free fatty acids that mostly consisted of 16:0 and some 14:0, 16:1 delta 9cis, and 18:1 delta 11cis. Antibodies were raised to the recombinant protein and used to determine tissue-specific and developmental expression in A. thaliana and Brassica napus. A 40-kDa protein was detected by immunoblots in A. thaliana siliques, leaves, and roots. A maximal expression of the B. napus protein between 18 and 31 days after flowering was found, which correlates with the rapid accumulation of triacylglycerols in the seeds. Based upon these results, we suggest that this long-chain acyl-ACP thioesterase may be a ubiquitous enzyme in plants which is involved in the synthesis of long-chain fatty acids. << Less

  Arch. Biochem. Biophys. 316:612-618(1995) [PubMed] [EuropePMC]

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

  Jayakumar A., Huang W.Y., Raetz B., Chirala S.S., Wakil S.J.

  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 overlap ... >> More

  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 (beta-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and beta-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 (M(r) 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the beta-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and beta-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 beta-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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 93:14509-14514(1996) [PubMed] [EuropePMC]

  This publication is cited by 36 other entries.