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

1991 ◽  
Vol 273 (3) ◽  
pp. 787-790 ◽  
Author(s):  
J Naggert ◽  
A Witkowski ◽  
B Wessa ◽  
S Smith
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Lambda Repressor ◽  
Thioesterase Ii ◽  
Chain Lengths

Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively. The enzymes have been cloned and expressed in Escherichia coli under the control of the temperature-sensitive lambda repressor. The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes.

scholarly journals Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

2004 ◽  
Vol 186 (6) ◽  
pp. 1869-1878 ◽  
Author(s):  
Chiou-Yan Lai ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Salmonella Enterica ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Serovar Typhimurium

ABSTRACT FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

scholarly journals Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

2006 ◽  
Vol 188 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Nicholas R. De Lay ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Lipid Synthesis ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Overlap Extension

ABSTRACT Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage λ Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.

scholarly journals Fatty acid synthase is required for mammary gland development and milk production during lactation

2014 ◽  
Vol 306 (10) ◽  
pp. E1132-E1143 ◽  
Author(s):  
Janel Suburu ◽  
Lihong Shi ◽  
Jiansheng Wu ◽  
Shihua Wang ◽  
Michael Samuel ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Milk Production ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Mammary Gland Development ◽  
De Novo ◽  
Acid Synthesis ◽  
Lactating Mammary Gland ◽  
Gland Development

The mammary gland is one of the few adult tissues that strongly induce de novo fatty acid synthesis upon physiological stimulation, suggesting that fatty acid is important for milk production during lactation. The committed enzyme to perform this function is fatty acid synthase (FASN). To determine whether de novo fatty acid synthesis is obligatory or dietary fat is sufficient for mammary gland development and function during lactation, Fasn was specifically knocked out in mouse mammary epithelial cells. We found that deletion of Fasn hindered the development and induced the premature involution of the lactating mammary gland and significantly decreased medium- and long-chain fatty acids and total fatty acid contents in the milk. Consequently, pups nursing from Fasn knockout mothers experienced growth retardation and preweanling death, which was rescued by cross-fostering pups to a lactating wild-type mother. These results demonstrate that FASN is essential for the development, functional competence, and maintenance of the lactating mammary gland.

scholarly journals Synthesis of fatty acids in the perfused mouse liver

1974 ◽  
Vol 142 (3) ◽  
pp. 611-618 ◽  
Author(s):  
D. Michael W. Salmon ◽  
Neil L. Bowen ◽  
Douglas A. Hems
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Saturated Fatty Acids ◽  
Carbon Sources ◽  
Acid Synthesis ◽  
Hepatic Glycogen ◽  
Total Rate ◽  
Glucose Carbon

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of3H from3H2O (1–7μmol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-14C]lactic acid and [U-14C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of3H2O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12–16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with3H2O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.

scholarly journals A low-protein, high-carbohydrate diet increases de novo fatty acid synthesis from glycerol and glycerokinase content in the liver of growing rats

2013 ◽  
Vol 33 (6) ◽  
pp. 494-502 ◽  
Author(s):  
Andreza Lúcia Menezes ◽  
Mayara Peron Pereira ◽  
Samyra Lopes Buzelle ◽  
Maísa Pavani dos Santos ◽  
Suélem Aparecida de França ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Acid Synthesis ◽  
High Carbohydrate Diet ◽  
Carbohydrate Diet ◽  
High Carbohydrate ◽  
Growing Rats ◽  
Low Protein
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