Expression in <i>Escherichia coli</i>, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis

Naggert, Jürgen Κ.; Witkowski, Andrzej; Wessa, B; Smith, Stuart

doi:10.1042/bj2730787

Cited by 41 publications

(25 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6C) (28) and E. coli (17). Together, these results from interkingdom switching of fatty acid synthase components further the idea that plant and bacterial type II fatty acid synthases are functionally related and are distinct from type I.…”

mentioning

confidence: 81%

“…Thus, plant and prokaryotic fatty acid synthases resemble each other in structure, and the plant fatty acid synthase resides in the chloroplast (23), which is considered to be of prokaryotic origin. Therefore, the structural similarities may be due to a common ancestry of plant and prokaryotic fatty acid synthases (14,17,20). Currently, it is not known whether type II protein components exist in a dissociated state or whether they are noncovalently combined in a discrete enzyme complex (7,10,20,32).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase

Voelker¹,

Davies²

1994

J Bacteriol

225

203

View full text Add to dashboard Cite

The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component ofE. col, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100o diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.The fatty acid biosynthesis pathway, comprising the sequential condensation of two-carbon units onto the growing fatty acyl chain, is universal. The four enzyme activities necessary for the extension and the acyl carrier protein (ACP) function can reside either as domains in one or two multifunctional polypeptides or as individual entities (16). The multidomain type has been termed the eukaryotic or type I fatty acid synthase and has been isolated from the cytosol of yeast and mammals. In contrast, in plants, Escherichia coli, and many other prokaryotes, each fatty acid synthase reaction is catalyzed by a discrete, monofunctional enzyme. In these organisms, the growing acyl chain is bound to ACP, which is itself a soluble polypeptide. This fatty acid synthase version has been termed prokaryotic, or type II. Thus, plant and prokaryotic fatty acid synthases resemble each other in structure, and the plant fatty acid synthase resides in the chloroplast (23), which is considered to be of prokaryotic origin. Therefore, the structural similarities may be due to a common ancestry of plant and prokaryotic fatty acid synthases (14,17,20). Currently, it is not known whether type II protein components exist in a dissociated state or whether they are no...

show abstract

mentioning

confidence: 81%

mentioning

confidence: 99%

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase

Voelker¹,

Davies²

1994

J Bacteriol

225

203

View full text Add to dashboard Cite

show abstract

“…These results suggest that Spot14 might augment release of fatty acids via the resident thioesterase domain of FASN. Naggert et al ( 65 ) showed that the resident thioesterase domain from FASN relies entirely on its covalent linkage to FASN for function, as the recombinant "free" thioesterase domain had no activity toward S-acyl-FASN. We speculate that Spot14 could foster a more productive interaction of the resident thioesterase domain with the acyl carrier domain of FASN to stimulate release of its fatty acid products.…”

Section: Spot14 Increases Fatty Acid Synthase Activity In Mecsmentioning

confidence: 99%

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Rudolph

Wellberg

Lewis

et al. 2014

Journal of Lipid Research

View full text Add to dashboard Cite

This article is available online at http://www.jlr.orgMammalian cells manufacture fatty acids de novo using a distinct pathway to synthesize fatty acids from acetyl and malonyl esters of CoA catalyzed by the dimer of FASN (EC 2.3.1.85) ( 1 ). FASN is absolutely essential for production of de novo fatty acids in mammals ( 2 ). The biological requirement for FASN is highlighted by the fact that FASN is detected in most normal human tissues and that homozygous deletion of FASN in the mouse results in embryonic lethality ( 3, 4 ). The importance of the de novo pathway is underscored by the complex biological functions of its fatty acid products, including biosynthesis of phospholipids, energy storage via esterifi cation into triglycerides (TAGs), use as energy substrates for ␤ -oxidation, providing both endocrine and nuclear hormone signaling ligands, and for critical posttranslational modifi cations of proteins ( 5-12 ). Perhaps the most demanding setting for FASN catalysis is during lactation, when extremely high quantities of de novo fatty acids are transmitted to the offspring as substrate for the growth of the young ( 13-15 ).Regulation of the principle enzymes of de novo fatty acid synthesis, ATP citrate lyase (ACLY) (EC 2.3.3.8), acetyl-CoA carboxylase (ACC) (EC 6.4.1.2), and FASN, has been shown to occur at the level of enzyme gene expression ( 16 ), translation/degradation ( 17-19 ), phosphorylation ( 20-22 ), assembly into multimeric complexes ( 23-25 ), and allosteric activation/inhibition ( 18,23,26,27 ). Recently, evidence for the control of de novo fatty acid synthesis by small effector proteins has emerged. Thyroid hormone responsive protein "Spot 14" (THRSP) (Spot14, S14) and only family Abstract Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefi ned during lactation. Spot14-null mice produce milk defi cient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When

show abstract

“…Such is the case for the multifunctional animal fatty acid synthase (8,(12)(13)(14)(15)(16). Additionally, the thioesterase domain of the fatty acid synthase has been expressed as an independent catalytically active recombinant protein in E. coli (3,17) demonstrating that domains of the fatty acid synthase may be capable of folding correctly into catalytically competent proteins out of context of the full-length polypeptide. In this study, for the first time, a domain of the fatty acid synthase has been expressed as a recombinant The results of this study provide strong evidence implicating His-683 in the catalytic mechanism of the malonyl-/acetyltransferase.…”

mentioning

confidence: 99%

“…Thus, each of the catalytic components of the fatty acid synthase is envisioned to be an independently folded domain. However, as yet, only one of the components of the animal fatty acid synthase, the thioesterase, has been expressed as an independent, catalytically active recombinant protein (3). In this study we sought to develop a system for expression of the malonyl-/acetyltransferase domain of the animal fatty acid synthase that would allow us to define the boundaries of this domain and ultimately would facilitate a detailed analysis of the catalytic mechanism of the enzyme through the construction and characterization of specific mutants.…”

mentioning

confidence: 99%

Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

Rangan¹,

Smith²

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

A cDNA encoding residues 429 -815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase. Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein. Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl-and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate. The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 3 Ala mutant. These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity.The animal fatty acid synthase is a multifunctional protein consisting of two identical head-to-tail oriented subunits, each carrying seven functional domains (1). It is generally believed that multifunctional proteins such as the fatty acid synthase have evolved by the fusion of genes encoding their monofunctional counterparts (2). Thus, each of the catalytic components of the fatty acid synthase is envisioned to be an independently folded domain. However, as yet, only one of the components of the animal fatty acid synthase, the thioesterase, has been expressed as an independent, catalytically active recombinant protein (3). In this study we sought to develop a system for expression of the malonyl-/acetyltransferase domain of the animal fatty acid synthase that would allow us to define the boundaries of this domain and ultimately would facilitate a detailed analysis of the catalytic mechanism of the enzyme through the construction and characterization of specific mutants. EXPERIMENTAL PROCEDURESMaterials-The pET17b and pET23a expression vectors were obtained from Novagen, Madison, WI. Rabbit anti-(rat)-fatty acid synthase antibodies were prepared as described previously (4) and purified by affinity chromatography on Sepharose-antigen columns, essentially as recommended by Pharmacia Biotech Inc. Alkaline phosphatasecoupled goat anti-rabbit IgG antibodies and SDS-PAGE 1 standards were purchased from Bio-Rad. Acetyl-[1-14 C]CoA (54 Ci/mol) and malonyl-[2-14 C]CoA (57 Ci/mol) were obtained from Moravek Biochemicals (Brea, CA). Baker-flex cellulose thin layer chromatography sheets were obtained from J. T. Baker Inc., and DEPC was purchased from SERVA feinbiochemica (New York). Synthe...

show abstract

Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis

Cited by 41 publications

References 20 publications

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

Contact Info

Product

Resources

About