Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro

Rangan, Vangipuram S.; Serre, Laurence; Witkowska, H. Ewa; Bari, Alfa Umaro; Smith, Steven S.

doi:10.1093/protein/10.5.561

Cited by 15 publications

(10 citation statements)

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“…The boundaries of the constructs were based on sequence conservations between FAS of various organisms, sequence homologies with the bacterial type II counterparts, and biochemical evidence on production of soluble fragments. [27][28][29][30][31] Protein production in E. coli was either unsuccessful (His 6 -tagged hFAS) or yielded aggregated material (MBP-fused hFAS). Two hFAS fragments (1-963 and 1-1077) could be produced in a soluble form via baculovirus-infected insect cells, using a modified pFASTBAC1 vector ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure of the Human Fatty Acid Synthase KS–MAT Didomain as a Framework for Inhibitor Design

Pappenberger

Benz

Gsell

et al. 2010

Journal of Molecular Biology

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Section: Resultsmentioning

confidence: 99%

Structure of the Human Fatty Acid Synthase KS–MAT Didomain as a Framework for Inhibitor Design

Pappenberger

Benz

Gsell

et al. 2010

Journal of Molecular Biology

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“…Plasmids carrying the mutations were used to transform E. coli BL21(DE3) cells, and expression of the recombinant proteins was induced by 1 mM isopropyl-␤-D-thiogalactoside. The recombinant transacylases were recovered from inclusion bodies and refolded in vitro (10,12).…”

Section: Methodsmentioning

confidence: 99%

Alteration of the Substrate Specificity of the Malonyl-CoA/Acetyl-CoA:Acyl Carrier ProteinS-Acyltransferase Domain of the Multifunctional Fatty Acid Synthase by Mutation of a Single Arginine Residue

Rangan¹,

Smith²

1997

Journal of Biological Chemistry

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The structural basis for the dual specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase associated with the multifunctional animal fatty acid synthase has been investigated by mutagenesis. Arginine 606, which is positionally conserved in the transacylase domains of all multifunctional fatty acid and polyketide synthases, was replaced by alanine or lysine in the context of the isolated transacylase domain, and the mutant proteins were expressed in Escherichia coli. Malonyl transacylase activity of the Arg-606 3 Ala and Arg-606 3 Lys mutant enzymes was reduced by 100-and 10-fold, respectively. In contrast, acetyl transacylase activity was increased 6.6-fold in the Arg-606 3 Ala mutant and 1.7-fold in the Arg-606 3 Lys mutant. Kinetic studies revealed that selectivity of the enzyme for acetyl-CoA was increased >16,000-fold by the Ala mutation and 16-fold by the Lys mutation. Activity toward medium chain length acyl thioesters was also increased >3 orders of magnitude by mutation of Arg-606, so that the Ala-606 enzyme is an effective medium chain length fatty acyl transacylase. These results indicate that Arg-606 plays an important role in the binding of malonyl moieties to the transacylase domain but is not required for binding of acetyl moieties; these results are also consistent with a mechanism whereby interaction between the positively charged guanidinium group of Arg-606 and the free carboxylate anion of the malonyl moiety serves to position this substrate in the active site of the enzyme.The animal FAS 1 consists of two identical polypeptides, each carrying six enzymes and an acyl carrier protein, that are juxtaposed to form two centers for the synthesis of palmitic acid from acetyl-and malonyl-CoA (1-3). The iterative condensation of an acetyl moiety with successive malonyl moieties and reduction of the ␤-keto intermediates normally results in the formation of palmitic acid as the major product. Initiation of the series of condensation reactions necessitates the translocation of an acetyl moiety and subsequently seven malonyl moieties, from CoA thioester to the 4Ј-phosphopantetheine of the acyl carrier protein domain of the FAS. A single transacylase enzyme is responsible for translocation of both substrates, and intermediate formation of acyl-O-serine intermediates occurs at the same site (4). The employment of a common enzyme for the loading of both substrates has important consequences in terms of the kinetics of the FAS reaction sequence, since substrate binding to the transacylase domain is random and each substrate is a competitive inhibitor for the other. Thus, for synthesis to proceed efficiently, inappropriately bound substrates must be rapidly removed by transfer back to the CoA acceptor (5). This substrate sorting or "self-editing" process occurs extremely rapidly, with turnover numbers Ͼ100 s Ϫ1 , and does not limit the rate of acyl chain assembly, provided an appropriate concentration of free CoA is available (5, 6).The chain initiation mechanism for FASs that consist ...

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“…Details are described elsewhere (24). The malonyl/acetyltransferase domain of the rat cytosolic FAS (amino acid residues 488 -809) was expressed in E. coli inclusion bodies, refolded in vitro, and purified as described earlier (27).…”

Section: Expression and Purification Of The Malonyl/acetyltransferasementioning

confidence: 99%

“…1A). In addition, all of these sequences contained three positionally conserved features that are characteristic of malonyl and malonyl/acetyltransferases: a Gly-Xaa-Ser-XaaGly serine esterase active site motif, a conserved Arg residue that interacts with the 3-carboxylate of the malonyl substrate, and a conserved His residue that is required for activation of the Ser nucleophile (27)(28)(29)(30). On the basis of this analysis, we identified expressed sequence tag cDNA clones encoding the putative human mitochondrial MT protein for further study.…”

Section: Identification Of Sequences Of the Putative Mitochondrialmentioning

confidence: 99%

Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase

Zhang¹,

Joshi²,

Smith³

2003

Journal of Biological Chemistry

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The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonylCoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/ acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonylCoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.The existence of a mitochondrial system for the biosynthesis of fatty acids in animals was first reported 40 years ago and postulated to function by a reversal of the process of fatty acid ␤-oxidation (1-3). Indeed one laboratory reported that short and medium chain-length fatty acids could be produced using the partially purified mitochondrial ␤-oxidation enzymes, ␤-ketoacyl thiolase, ␤-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase (4), and another found that inhibitory antibodies to pig heart 3-ketoacyl-CoA thiolase inhibited, in a parallel fashion, the fatty acid elongation system of pig heart mitochondria (5). The picture was complicated by reports that liver and heart mitochondria have two distinct fatty acid-synthesizing systems, one acetyl-CoA-dependent, possibly involving some of the ␤-oxidation enzymes, the other malonyl-CoA-dependent and apparently resembling the cytosolic FAS 1 system. Most ...

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Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro

Cited by 15 publications

References 1 publication

Structure of the Human Fatty Acid Synthase KS–MAT Didomain as a Framework for Inhibitor Design

Structure of the Human Fatty Acid Synthase KS–MAT Didomain as a Framework for Inhibitor Design

Alteration of the Substrate Specificity of the Malonyl-CoA/Acetyl-CoA:Acyl Carrier ProteinS-Acyltransferase Domain of the Multifunctional Fatty Acid Synthase by Mutation of a Single Arginine Residue

Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase

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