Abstract
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.
Highlights
Thioesterase domain junction sequence at exactly the The thioesterase domainof fatty acid synthasecatalyzes the psacirsneiacormdvomimdieticeebalcaaiilonntersem,adittntoiephtatnhosatdaeshtwciiectpyiitaorltnhtehnciaesenateoxginrnfvpraceiprtteeheieavresnroestzpfpieyraudo0fmlrat.ie6eptpftiiaarnyaemocnndidttoddeoorlitemonhtscfheayohaninm8ntayt-.ashblaiaTialtnactsshifnoaeoeonin.snlnotfdeefoTiplnrtmrhrmhgeoeoestaieulntrailieotntomofs-ninoycardreeaso3lairmesbc3atow-asiknioeinuDtnohnacfioataffpsn-arcbtfohtaetylyteotcmiaynlncigop(ia,d4tpcrs)eaiy.ddwnpTdsfhhsirtyenehonnmuiatsshtnohetldehasotieacesncadlsyn(yyb1tlhen0cpit,hioahsaer1oatis1slintacee)btil.repyeaTdanshltgeaieem,tnsthaihdinstiiestposhadueyearslpebitafderoisoerutwatdtesio1eathl6sfunctionally active domains is independent of the re- fatty acyl-CoA as a substrate, shows the same specificity for maining domains of the multifunctional synthase sub- long chain fatty acyl-CoA (c16, C18)as does the intact thioesunit
Fatty acid synthesis in animal tissues is catalyzed by two uropygial gland of goose [15, 16] and is able to interact with multifunctional enzymes: acetyl-coA carboxylase and fatty the fattyacid synthase torelease shorter chain acids
Acid synthase [1].Acetyl-coA carboxylase catalyzes the con- to release the fatty acids from the synthase, the thioesterase
Summary
ACP components of the chicken liver fatty thioesteras eand acid synthase ha ously [4].Cleavage of the recombinant ACP-thioesterase protein with vTae-hcehycmonocterynptrsaitniownaosfctahreriepduroifuitedbyAeCssPe-nthtiiaolelystethraesesapmroeteminethwoads [4]. adbeen determined [17, 18]T. hese sequences were confirmed by justed to 1 mg/ml in 0.1 M Tris HC1 (pH 7.6), 1 mM dithiothreitol, sequencing the cDNA coding for these two domains of the and 1mM EDTA and treated with a-chymotrypsin at a ratio of 10001 fatty acid synthase [19, 20]. The expressed ACP-thioesterase protein was Amino Acid Analysis and Gas-phaseProtein Sequencing-The proisolated and foundbetopartially pantothenylatedand to have thioesterase activity comparable to its native counterpartof the chicken liver fatty acid synthase. Land BioLabs and Bethesda Research Laboratories; [14C]pantothenic Determination of the amino acid sequence at the NHp-terminal acid was from Du Pont-New England Nuclear; DEAE-Bio-Gel, hy- end of the protein was performed using an Applied Biosystems model droxyapatite, Western blot reagents, and protein assay reagents were 470A gas-phase protein sequenator [26]with an on-line PTH analyzer from Bio-Rad. All materials were used according to the manufactur- 120A,equipped with a reverse-phase column This fragment was blunt endligated to thePOTS vector [21], which had been digested with BamHI and filled in with the
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