Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.     

Computational screening of fatty acid synthase inhibitors against thioesterase domain

Thioesterase (TE) domain of fatty acid synthase (FAS) is an attractive therapeutic target for design and development of anticancer drugs. In this present work, we search for the potential FAS inhibitors of TE domain from the ZINC database based on similarity search using three natural compounds as templates, including flavonoids, terpenoids, and phenylpropanoids. Molecular docking was used to predict the interaction energy of each screened ligand compared to the reference compound, which is methyl γ-linolenylfluorophosphonate (MGLFP). Based on this computational technique, rosmarinic acid and its eight analogs were observed as a new series of potential FAS inhibitors, which showed a stronger binding affinity than MGLFP. Afterward, nine docked complexes were studied by molecular dynamics simulations for investigating protein–ligand interactions and binding free energies using MM-PB(GB)SA, MM-3DRISM-KH, and QM/MM-GBSA methods. The binding free energy calculation indicated that the ZINC85948835 (R34) displayed the strongest binding efficiency against the TE domain of FAS. There are eight residues (S2308, I2250, E2251, Y2347, Y2351, F2370, L2427, and E2431) mainly contributed for the R34 binding. Moreover, R34 could directly form hydrogen bonds with S2308, which is one of the catalytic triad of TE domain. Therefore, our finding suggested that R34 could be a potential candidate as a novel FAS-TE inhibitor for further drug design.     

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 a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by Orlistat

Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-A-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.     

Stereochemistry of the reactions catalyzed by chicken liver fatty acid synthase

The stereochemistry of the four partial reactions catalyzed by chicken liver fatty acid synthase that lead to the synthesis of palmitic acid has been determined. The reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH proceeds with the transfer of the pro-4S hydrogen of NADPH to form D-3-hydroxybutyryl-CoA. During the subsequent dehydration of D-3-hydroxybutyryl-CoA the pro-2S hydrogen and the 3-hydroxyl group are removed in a syn elimination to form crotonyl-CoA. Crotonyl-CoA is reduced to butyryl-CoA by NADPH, with the transfer of the pro-4R hydrogen of NADPH to the pro-3R position in butyryl-CoA and the transfer of a solvent hydrogen to the pro-2S position. The occurrence of the syn dehydration, when combined with the results of a previous study [ Sedgwick , B., & Cornforth , J. W. (1977) Eur. J. Biochem. 75, 465-479], implies that the condensation of the enzyme-bound malonyl moiety with the enzyme-bound saturated fatty acid to form a 3-keto intermediate proceeds with inversion at C-2 of the malonyl. The stereochemistry of the hydration was derived from an analysis of the spin-spin coupling constant of 3-hydroxy[2-2H]butyric acid benzylamides obtained from 3-hydroxy[2-2H]butyryl-CoA synthesized by fatty acid synthase. The elucidation of the stereochemistry of the reduction of crotonyl-CoA relied on the previously established stereochemistry of pork liver acyl-CoA dehydrogenase. The source of all 28 prochiral hydrogens of the palmitic acid synthesized by chicken liver fatty acid synthase was inferred from the results of this work.     

Distribution of reaction intermediates on chicken liver fatty acid synthase

The distributions of covalent intermediates in the reaction cycle catalyzed by chicken liver fatty acid synthase were studied. In isotope-trapping experiments, 30% of [1-14C] acetyl-enzyme and 6.7% of [2-14C]malonyl-enzyme are converted to long-chain fatty acids, indicating the initiation reaction is partially random. The 3-hydroxybutyryl intermediate is located on the tryptic peptide which contains the 4'-phosphopantetheine (greater than 90%), while the C4-C18 saturated intermediates are distributed both on this peptide and on the peptide that contains the active cysteine. The ratio of intermediates on the two peptides is about unity for chain lengths less than C14, but the amount on the active cysteine progressively decreases for chain lengths of C14, C16, and C18 with trypsinized enzyme. The distributions of carbon chain lengths for the saturated or 3-keto intermediates when acetoacetyl-labeled trypsinized enzyme is incubated with limiting malonyl coenzyme A or NADPH, respectively, show large fractions both of unreacted enzyme and of C16 or longer intermediates. A detailed analysis suggests that the initial condensation and reduction steps are slower than the analogous reactions with longer chain length intermediates. The 3-keto intermediate comprises over 70% of each chain length intermediate detected when NADPH is the limiting substrate, indicating the reduction of the 3-keto intermediates is at least 2 times slower than the reduction of the unsaturated intermediates.     

Complete amino acid sequence of subunit e of rat liver mitochondrial H(+)-ATP synthase.

Subunit e of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high-performance liquid chromatography. The amino acid sequence of the subunit was determined by automated Edman degradation of the whole protein and derived peptides. The nucleotide sequence of the import precursor of subunit e of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 289 nucleotides including a coding region for the import precursor of subunit e and noncoding regions on the 5'- and 3'-sides. The possible import precursor of subunit e and its mature polypeptide deduced from the open reading frame consisted of 71 and 70 amino acid residues with molecular weights of 8254 and 8123, respectively. Subunit e is a basic hydrophilic protein with an isoelectric point of 9.78. The sequence of the rat subunit e is highly homologous with that of subunit e of bovine heart, but has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase. The function of subunit e is unknown. However, a homology search in the database of the National Biomedical Research Foundation revealed that residues 34-65 of subunit e are homologous with residues 90-117 of troponin T, and with residues 529-561 of h-caldesmon and residues 289-319 of l-caldesmon, which are the homologous sequences corresponding to the Ca(2+)-dependent tropomyosin-binding region of troponin T.     

Amino acid sequences of substrate-binding sites in chicken liver fatty acid synthase

The amino acid sequences of three essential regions of chicken liver fatty acid synthase have been determined: that around 4'-phosphopantetheine ("carrier" site), the substrate "loading" site containing serine, and a "waiting" site for the growing fatty acid containing cysteine. The amino acid sequence of the 4'-phosphopantetheine region was determined for the acetyl-, malonyl-, hydroxybutyryl-, and butyryl-enzyme with peptides obtained by hydrolysis of the enzyme with trypsin and Staphylococcus aureus (V8) protease. The sequence region around the essential serine was obtained for the acetyl- and malonyl-enzyme. The N-terminus of the tryptic peptide was blocked. However, the same sequence is obtained for the acetyl- and malonyl-peptide after S. aureus protease digestion, suggesting that the enzyme contains a single acyl transferase rather than two separate transacylases. The sequence around the cysteine was obtained by use of a radioactive iodoacetamide label. An unusual sequence of three serines adjacent to the cysteine was found. The strong similarities between peptides from different species for all three of the regions suggest that the multifunctional polypeptides from yeast and animals have evolved from the monofunctional enzymes of lower species.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Purification and amino acid sequence of chicken liver cathepsin L

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.     

Effects of osmolytes on unfolding of chicken liver fatty acid synthase

Urea-induced aggregation of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating,oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85 ] was studied. The aggregation was facilitated at increased ionic strength. Methyl--cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during fatty acid synthase unfolding. The osmolytes also protected the enzyme from inactivation.     

Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence

Alcohol dehydrogenase (ADH) catalyzes the rate-determining reaction in the metabolism of ethanol. We report here the complete nucleotide sequence of a cDNA encoding rat liver ADH, and the deduced amino acid (aa) sequence of the protein. The rat enzyme contains a cluster of aa substitutions and an aa insertion in the region between aa residues 111 and 118, which is near the intron-exon junction reported for the human ADH gene. It also contains an additional cysteine in the highly variable region from aa residues 108-125 which may account for the unusual lability of rat ADH compared with ADH from other species.     

Molecular docking study of the interactions between the thioesterase domain of human fatty acid synthase and its ligands

Human fatty acid synthase (hFAS) thioesterase domain (TE) is an attractive drug target to treat obesity and cancer. On the basis of the recently published crystal structure of TE domain of hFAS, we performed molecular surface analysis and docking study to characterize the molecular interactions between the enzyme and its various ligands. Surface analysis identified the ligand-binding pocket of TE domain that encompasses the catalytic triad of Ser2308, His2481, Asp2338. Docking of palmitate, the main biological product of hFAS, into this pocket revealed the ligand-binding mode, in which the hydrophobic interactions are the dominant driving forces. The catalytic mechanism of TE domain can also be well explained based on the generated TE-palmitate complex structure. Moreover, the comparison of the binding modes of five fatty acids with chain lengths ranging from 12 to 20 carbons confirmed that the ligand binding pocket of TE domain is a decisive factor in chain length specificity. In addition, docking of two known TE inhibitors, c75 and orlistat revealed the pharmacophore of these hFAS TE inhibitors, which will prove useful in structure-based drug design against this important target.     

Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.     

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.     

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.     

Inhibitive effect of zinc ion on fatty acid synthase from chicken liver

Fatty acid synthase (FAS; acyl-CoA:malonyl-CoA C-acyltransferase [decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing], EC 2.3.1.85) is an important enzyme participating in energy metabolism in vivo which is related to adiposis and cancer [Cancer Lett. 167 (1) (2001) 99; Nat. Med. 8 (4) (2002) 335]. Tests of fast- and slow-binding inhibitions showed that fatty acid synthase of chicken liver is rapidly and irreversibly inactivated by low Zn(2+) concentrations. Electrophoresis and FPLC results showed that FAS cross-links occurred in the presence of high Zn(2+) concentrations (>4 microM) which may be another reason that FAS lost its activity. The modification velocity of FAS by DTNB decreased with increasing Zn(2+) concentration, which confirmed that Zn(2+) interacted with SH groups. Substrate protective experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all three substrates tested had some protective effects on FAS in the presence of Zn(2+), and malonyl-CoA was the most effective of the three substrates. In the presence of malonyl-CoA, the activity loss of FAS decreased sharply and almost no cross-link was observed in SDS-PAGE. This suggests that the phosphopantetheine SH group is the critical group in the cross-link and inhibition of FAS in the presence of Zn(2+).