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.

• 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.

• Roles of the FabA and FabZ beta-hydroxyacyl-acyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis.

  Heath R.J., Rock C.O.

  There are two genes, fabA and fabZ, encoding beta-hydroxyacyl-acyl carrier protein (ACP) dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting ... >> More

  There are two genes, fabA and fabZ, encoding beta-hydroxyacyl-acyl carrier protein (ACP) dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins. FabA and FabZ exhibited broad, overlapping chain length specificities. The FabZ dehydratase efficiently catalyzed the dehydration of short chain beta-hydroxyacyl-ACPs and long chain saturated and unsaturated beta-hydroxyacyl-ACPs. FabA was most active on intermediate chain length beta-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated beta-hydroxyacyl-ACPs. Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated beta-hydroxyacyl-ACP. The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of beta-ketoacyl-ACP synthase I (FabB). A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP. The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated beta-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two dehydratases. << Less

  J. Biol. Chem. 271:27795-27801(1996) [PubMed] [EuropePMC]

  This publication is cited by 23 other entries.

• 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.

• Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

  Pasta S., Witkowski A., Joshi A.K., Smith S.

  Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regi ... >> More

  Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain. << Less

  Chem. Biol. 14:1377-1385(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 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.