Human fatty acid synthase: role of interdomain in the formation of catalytically active synthase dimer.

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (M(r) 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

The subcellular localization of acetyl-CoA carboxylase 2.

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC ), ACC1 (M(r) = 265 kDa) and ACC2 (M(r) = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter.

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5'-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides -870 to -650) and TRE2 (nucleotides -272 to -40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter-luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides -716 to -731) represents TRE1 and that the sequence GGGTCC (nucleotides -117 to -112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

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

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.

Animal fatty acid synthase: functional mapping and cloning and expression of the domain I constituent activities.

Animal fatty acid synthase (FAS; EC 2.3.1.85) is a homodimer of a multifunctional subunit protein and catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH. The subunit (Mr approximately 270,000) carries seven distinct component activities and a site for the prosthetic group 4'-phosphopantetheine (acyl carrier protein). Based on proteolytic mapping, the organization of the activity domains along the subunit polypeptide from the N terminus is as follows: beta-ketoacyl synthase, acetyl and malonyl transacylases, beta-hydroxyacyl dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase. By comparing the amino acid sequences of the chicken, rat, and human synthases, we found that kallikrein cleavage sites occur in the least conserved regions of the FAS polypeptide subunit. Determining the amino acid sequences of the N-terminal end of the major kallikrein cleavage peptides helped delineate the most likely boundaries of the component activities in the cDNA-derived amino acid sequence. To confirm this organization, we cloned the chicken FAS cDNA coding for domain I and expressed it in Escherichia coli as a maltose-binding fusion protein. The isolated recombinant protein contained the activities of the acetyl and malonyl transacylases and the beta-hydroxyacyl dehydratase. Based on the boundaries of the acetyl and malonyl transacylases and the beta-hydroxyacyl dehydratase, we also cloned the appropriate cDNA fragments encoding the domains that contain the transacylases and the dehydratase in pET vectors and expressed them in E. coli as thioredoxin-6xHis fusion proteins. The purified recombinant proteins contained, respectively, the activities of the acetyl and malonyl transacylases and the dehydratase. These results not only confirmed the order of the component activities in domain I, but also paved the way for successful expression and characterization of the remaining activities.

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

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.

Human fatty-acid synthase gene. Evidence for the presence of two promoters and their functional interaction.

We have isolated and sequenced a genomic clone coding for the first three exons and the 5'-flanking region of the human fatty-acid synthase gene. The translation initiation site, ATG, is located in exon II. Primer extension and S1 nuclease analyses showed the presence of three transcription initiation (Ti) sites: Ti I, Ti II, and Ti III. The Ti I site is mapped to the beginning of the untranslated exon I and preceded by a promoter with recognizable TATA and CAAT boxes. The Ti II and Ti III sites are located in intron I, at 60 and 49 nucleotides upstream of the translation initiation site ATG in exon II, respectively. These two Ti sites are preceded by four putative Sp1 boxes, but lack TATA and CAAT boxes. Analysis of luciferase reporter gene expression in transient transfection assays confirmed the existence of two promoters. A 200-base pair 5'-flanking region, which has strong promoter activity comparable with that of the CMV promoter, is considered human fatty-acid synthase promoter I. In a wild-type human fatty-acid synthase-luciferase construct, in which promoter I and intron I are present in their natural configuration, the reporter gene activity is only 1% of that of promoter I. Deletion analysis showed the existence of promoter II, which is located in intron I immediately upstream of the Ti II site. The strength of promoter II is approximately th of that of promoter I in transient transfection assays. Further analysis of reporter gene constructs showed that promoter II inhibited the reporter gene activity of the wild-type construct that contained promoter I and intron I and that the spatial separation of the two promoters is important for this inhibition. A model is proposed based on the possibility that the assembly of transcription complexes on promoter II creates a "roadblock" and reduces the overall expression of the fatty-acid synthase gene by interfering with the progression of transcription from promoter I.

Human fatty acid synthase: properties and molecular cloning.

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.

Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms.

We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4.1.2). The sequence has an open reading frame of 7038 bp that encode 2346 amino acids (M(r), 264,737). The C-terminal 2.6-kb sequence is very different from that recently reported for human ACC (Ha, J., Daniel, S., Kong, I.-S., Park, C.-K., Tae, H.-J. & Kim, K.-H. [1994] Eur. J. Biochem. 219, 297-306). Northern blot analysis revealed that the ACC mRNA is approximately 10 kb in size and that its level varies among the tissues tested. Evidence is presented to show that the human ACC gene is 200-480 kbp in size and maps to chromosome 17q12. We also provide evidence for the presence of another ACC-like gene with similarly sized mRNA but tissue-specific expression different from that of the ACC gene reported herein. That this second ACC-like gene encodes the 280-kDa carboxylase is not ruled out.

Amino-terminal blocking group and sequence of the animal fatty acid synthase.

We have cloned and sequenced the cDNA encoding the chicken fatty acid synthase. Based on the nucleotide-derived amino acid sequence of the chicken synthase, the N-terminal sequences are highly conserved among animal species, suggesting that translation of the animal synthases initiates with the same ATG codon. Like other fatty acid synthases, the NH2-terminal sequence of the chicken enzyme is blocked. We have isolated and purified the blocked NH2-terminal peptide from a tryptic digest of chicken synthase and have established that the blocking group is an acetyl group. The sequence of the native tryptic peptide confirmed the cDNA-derived amino acid sequence and suggested that all animal synthases begin with this homologous sequence. We developed simple procedures that can be used to isolate and characterize any blocked NH2-terminal peptide.

Isolation and chromosomal mapping of genomic clones encoding the human fatty acid synthase gene.

We have isolated and sequenced 0.5- and 3.6-kb cDNA clones that cover the N-terminal and carboxy-terminal regions, respectively, of the human fatty acid synthase. To localize the fatty acid synthase gene and to define its genomic structure, we have also isolated overlapping genomic clones by screening two human YAC libraries with PCR primers derived from the fatty acid synthase cDNA sequences. The DNA inserts in these human fatty acid synthase YACs hybridized with human synthase-specific cDNA probes. Using biotin-labeled Alu-PCR products of the human synthase YACs as probes for fluorescence in situ hybridization, we mapped the fatty acid synthase gene to chromosome 17q25. We also screened a chromosome 17-specific cosmid library with human synthase cDNA probes and isolated 12 cosmids, all of which had EcoRI fragments in common. DNA sequencing of an amplified PCR product from the fatty acid synthase cosmids confirmed that these genomic clones contained expressed fatty acid synthase sequences. Furthermore, the results of Southern analyses suggested that a single 40-kb cosmid clone encompasses the entire coding region of the fatty acid synthase gene. The synthase gene is located on chromosome 17 near the q25 band, which is close to the telomere and could serve as an important marker in analysis of this chromosome.

Analysis of FAS3/ACC regulatory region of Saccharomyces cerevisiae: identification of a functional UASINO and sequences responsible for fatty acid mediated repression.

We have determined the sequence of the FAS3/ACC regulatory region and mapped the transcription initiation site. In this sequence, there are two putative UASINO sequences. Deletion and mutation analyses revealed that the UASINO sequence at nucleotides -719 to -710 is functional. The expression of FAS3-lacZ reporter genes and the measurement of mRNA levels in regulatory mutants of phospholipid biosynthesis clearly indicated that FAS3 is regulated by inositol and choline. Previous studies have shown that the genes coding for fatty acid synthase, FAS1 and FAS2, are regulated by inositol (Chirala, S.S. [1992] Proc. Natl. Acad. Sci. USA 89, 10232-10236). Thus all three genes involved in saturated fatty acid biosynthesis are coordinately regulated with phospholipid biosynthesis. Comparison of the UASINO sequences present in FAS1, FAS2, and FAS3 suggested that the functional sequence of this UAS element is YTTCACATG. However, even when the functional UASINO was mutated, substantial expression of the FAS3-lacZ reporter gene was observed. Deletion analysis, electrophoretic mobility shift assays, and expression using a heterologous reporter gene showed that the region between nucleotides -840 and -736 has two UAS elements. The same sequence seems to be responsible for fatty acid-mediated repression of FAS3. The presence of these additional UAS sequences explains why yeast does not require fatty acids even when repressing amounts of inositol and choline are present in the medium.

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.

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.

Coordinated regulation and inositol-mediated and fatty acid-mediated repression of fatty acid synthase genes in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, FAS1, FAS2, and FAS3 are the genes involved in saturated fatty acid biosynthesis. The enzymatic activities of both fatty acid synthase (FAS) and acetyl-CoA carboxylase are reduced 2- to 3-fold when yeast cells are grown in the presence of exogenous fatty acids. The mRNA levels of the FAS genes are correspondingly lower under repressive conditions. Expression of the FAS-lacZ reporter gene is also regulated by fatty acids. When a FAS2 multicopy plasmid is present in the cells, expression of both FAS1 and FAS3 increases. Thus, the FAS genes are coordinately regulated. Deletion analyses of the regulatory regions of FAS1 and FAS2 revealed common regulatory sequences. These include the GGCCAAAAAC and AGCCAAGCA sequences that have a common GCCAA core sequence and the UASINO (upstream activation sequence). Derepression of the FAS genes in the absence of exogenous inositol is not observed when UASINO is mutated, indicating that this cis element is a positive regulator of these genes. The GCCAA elements and UASINO act synergistically for optimal expression of the FAS genes.

Cloning of the yeast FAS3 gene and primary structure of yeast acetyl-CoA carboxylase.

We have isolated and determined the nucleotide sequence of the yeast FAS3 gene, which encodes acetyl-CoA carboxylase (EC 6.4.1.2). The sequence has an open reading frame of 6711 bases coding for a protein of 2237 amino acids with a calculated molecular weight of 250,593. The presence of the unique biotin-binding site, Met-Lys-Met, and the known CNBr peptide and COOH-terminal sequences confirmed the nucleotide-derived amino acid sequence. The yeast, chicken, and rat carboxylases have an overall sequence identity of 34%, suggesting that the eukaryotic carboxylase evolved from a single ancestral gene. The amino acid sequences of yeast fatty acid synthase subunits are least homologous with the animal synthase sequences, whereas carboxylase sequences are highly conserved. The sequences of the ATP, HCO3-, and CoA binding sites of the carboxylases are also well conserved (approximately 50% identical). The sequences surrounding the biotin binding site are poorly conserved, suggesting that this sequence may not be critical as long as the biotin is available for carboxylase reactions. On the basis of this sequence identity, we have defined the putative biotin carboxylase and transcarboxylase domains.

Site-directed mutagenesis studies on the recombinant thioesterase domain of chicken fatty acid synthase expressed in Escherichia coli.

The animal fatty acid synthase is a multifunctional protein with a subunit molecular weight of 260,000. We recently reported the expression and characterization of the acyl carrier protein and thioesterase domains of the chicken liver fatty acid synthase in Escherichia coli. In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site serine 101 with alanine and cysteine and the conserved histidine 274 with alanine by site-directed mutagenesis. While both the Ser101----Ala and His274----Ala mutant proteins were inactive, the Ser101----Cys mutant enzyme (thiol-thioesterase) retained considerable activity, but the properties of the enzyme were changed from an active serine esterase to an active cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile. In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations. The double-mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the proton from the Cys-SH in the thiol-thioesterase or Ser101 in the wild-type enzyme. Incubation of the recombinant thioesterases with [1-14C] palmitoyl-CoA resulted in the incorporation of [1-14C] palmitoyl into the enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-enzyme intermediate. This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl enzyme. These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the serine proteases and lipases, which employ a serine-histidine-aspartic acid catalytic triad as part of their catalytic mechanism.

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli.

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones.

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.

Primary structure of the multifunctional alpha subunit protein of yeast fatty acid synthase derived from FAS2 gene sequence.

The yeast fatty acid synthase consists of two multifunctional proteins, alpha and beta, arranged in an alpha 6 beta 6 complex with a molecular weight of 2.4 x 10(6). Five of the seven enzymatic activities reside in the beta subunit, while the remaining two activities, beta-ketoacyl synthase and beta-ketoacyl reductase, and the domain of the acyl carrier protein, with its prosthetic group, 4'-phosphopantetheine, are in the alpha subunit. The genes FAS1 and FAS2 coding for beta and alpha subunits, respectively, have been cloned and the sequence of FAS1 has been reported (Chirala, S. S., Kuziora, M. A., Spector, D. M., and Wakil, S. J. (1987) J. Biol. Chem. 262, 4231-4240). In this study, we present the nucleotide sequence of the FAS2 gene. The sequence has an open reading frame, coding for a protein of 1894 amino acids with a calculated molecular weight of 207,863. The location of the serine site of attachment of the prosthetic group of the acyl carrier protein domain and the active cysteine-SH site of beta-ketoacyl synthase have been identified at residues 180 and 1312, respectively, in the deduced amino acid sequence. A putative NADPH binding site of beta-ketoacyl reductase has been suggested at residue 1038 based on the similarities to the consensus amino acid sequences -Gly-Ser-Ala- of the pyridine nucleotide enzymes. We could not find any sequence homology in the 5' flanking sequence of the FAS1 and FAS2 genes that would suggest common regulatory function. However, in the sequence of these two genes there is an identical eight-base pair sequence TCATTATG at the translational initiation site suggesting that the subunit stoichiometry probably results from equal translational efficiency of the mRNAs of both FAS1 and FAS2 genes. The S1 endonuclease mapping suggests that there is a transcriptional initiation site at about 40 nucleotides upstream of the first ATG codon and a transcriptional termination site about 300 nucleotides downstream of the TAG stop codon. The gene does not contain introns as no intron consensus TACTAAC have been found in the sequence.