Publications

• Human fatty acid synthase: properties and molecular cloning.

  Jayakumar A., Tai M.-H., Huang W.-Y., Al-Feel W., Hsu M., Abu-Elheiga L., Chirala S.S., Wakil S.J.

  Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable ... >> More

  Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS. << Less

  Proc. Natl. Acad. Sci. U.S.A. 92:8695-8699(1995) [PubMed] [EuropePMC]

  This publication is cited by 38 other entries.

• The architecture of the animal fatty acid synthetase. III. Isolation and characterization of beta-ketoacyl reductase.

  Wong H., Mattick J.S., Wakil S.J.

  Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column ... >> More

  Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column permitted the isolation of a polypeptide (Mr = 94,000) containing the beta-ketoacyl reductase activity but no other partial activities normally associated with the synthetase. The specific activity of the beta-ketoacyl reductase increased 2 to 3 times in this fraction, an increase that is within the expected range based on relative molecular weight. The kinetic parameters of this fraction towards NADPH and N-acetyl-S-acetoacetyl cysteamine were essentially the same as the beta-ketoacyl reductase component of the intact synthetase. However, the purified fragment did not catalyze the reduction of acetoacetyl-S-CoA derivative, a substrate that is readily reduced by the intact synthetase. Fluorescence measurements with etheno-NADP+ indicate the binding of about 1 mol of NADP+/94,000 daltons, a value which is in agreement with the results obtained from fluorescence measurements with NADPH and the binding of a radiolabeled photoaffinity analog of NADP+. Phenylglyoxal inhibits the beta-ketoacyl reductase activity of either the intact synthetase or the isolated fragment, suggesting the involvement of an essential arginine at or near the active site. Another fragment (Mr 36,000) containing beta-ketoacyl reductase activity was isolated from the synthetase after kallikrein/subtilisin double digestion. Previous mapping studies had shown that this fragment lies adjacent to the COOH-terminal thioesterase domain and overlaps the tryptic Mr 94,000 peptide by approximately 21 daltons. This fragment, but not the Mr 94,000 fragment, was found to contain the phosphopantetheine prosthetic group, indicating that the acyl carrier protein moiety is located in the 15,000-dalton segment that separates the beta-ketoacyl reductase from the thioesterase domain. << Less

  J. Biol. Chem. 258:15305-15311(1983) [PubMed] [EuropePMC]

• Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis.

  Choi K.-H., Heath R.J., Rock C.O.

  A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram- ... >> More

  A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the beta-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. coli enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production. << Less

  J. Bacteriol. 182:365-370(2000) [PubMed] [EuropePMC]

  This publication is cited by 22 other entries.

• 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.

• Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli.

  Heath R.J., Rock C.O.

  The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl ... >> More

  The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl-CoA to butyryl-ACP in vitro were purified. These proteins were malonyl-CoA:ACP transacylase (fabD), beta-ketoacyl-ACP synthase III (fabH), beta-ketoacyl-ACP reductase (fabG), beta-hydroxydecanoyl-ACP dehydrase (fabA), and enoyl-ACP reductase (fabI). Unlike the other enzymes in the cycle, FabA did not efficiently convert its substrate beta-hydroxybutyryl-ACP to crotonyl-ACP, but rather the equilibrium favored formation of beta-hydroxybutyryl-ACP over crotonyl-ACP by a ratio of 9:1. The amount of butyryl-ACP formed depended on the amount of FabI protein added to the assay. Extracts from fabI(Ts) mutants accumulated beta-hydroxybutyryl-ACP, and the addition of FabI protein to the fabI(Ts) extract restored both butyryl-ACP and long-chain acyl-ACP synthesis. FabI was verified to be the only enoyl-ACP reductase required for the synthesis of fatty acids by demonstrating that purified FabI was required for the elongation of both long-chain saturated and unsaturated fatty acids. These results were corroborated by analysis of the intracellular ACP pool composition in fabI(Ts) mutants that showed beta-hydroxybutyryl-ACP and crotonyl-ACP accumulated at the nonpermissive temperature in the same ratio found in the fabI(Ts) extracts and in the in vitro reconstruction experiments that lacked FabI. We conclude that FabI is the only enoyl-ACP reductase involved in fatty acid synthesis in E. coli and that the activity of this enzyme plays a determinant role in completing cycles of fatty acid biosynthesis. << Less

  J. Biol. Chem. 270:26538-26542(1995) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.