Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

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.

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

Journal of Bacteriology ◽

10.1128/jb.188.1.287-296.2006 ◽

2006 ◽

Vol 188 (1) ◽

pp. 287-296 ◽

Cited By ~ 23

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.

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Characterization of a Malonyl-Enzyme Intermediate and Identification of the Malonyl Binding Site in Malonyl Coenzyme A-Acyl Carrier Protein Transacylase of Escherichia coli

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43198-0 ◽

1973 ◽

Vol 248 (23) ◽

pp. 8095-8106 ◽

Cited By ~ 1

Author(s):

Frank E. Ruch ◽

P. Roy Vagelos

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Coenzyme A ◽

Acyl Carrier Protein ◽

Carrier Protein ◽

Enzyme Intermediate

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Mixed disulfides of acyl carrier protein and coenzyme A with specific soluble proteins in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.3.1298-1300.1982 ◽

1982 ◽

Vol 152 (3) ◽

pp. 1298-1300

Author(s):

C O Rock

Keyword(s):

Escherichia Coli ◽

Coenzyme A ◽

Acyl Carrier Protein ◽

Soluble Proteins ◽

The Other ◽

Carrier Protein ◽

Mixed Disulfides

Three soluble proteins in Escherichia coli specifically from mixed disulfides with either acyl carrier protein or coenzyme A. Coenzyme A was attached to one of these proteins, and the amount bound depended on the cellular coenzyme A concentration. The other two proteins were mixed disulfides between acyl carrier protein and each of the two 3-ketoacyl-acyl carrier protein synthases.

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The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

Journal of Bacteriology ◽

10.1128/jb.185.5.1582-1589.2003 ◽

2003 ◽

Vol 185 (5) ◽

pp. 1582-1589 ◽

Cited By ~ 64

Author(s):

Sean W. Jordan ◽

John E. Cronan,

Keyword(s):

Escherichia Coli ◽

Lipoic Acid ◽

Pyruvate Dehydrogenase ◽

Octanoic Acid ◽

Acyl Carrier Protein ◽

Temperature Sensitive ◽

Carrier Protein ◽

Genomic Databases ◽

E Coli ◽

Lipoyl Domain

ABSTRACT In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.

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Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds

Plant Molecular Biology ◽

10.1007/bf00039531 ◽

1994 ◽

Vol 26 (1) ◽

pp. 189-202 ◽

Cited By ~ 14

Author(s):

Ira I. G. S. Verwoert ◽

Karin H. van der Linden ◽

H. John J. Nijkamp ◽

Antoine R. Stuitje

Keyword(s):

Escherichia Coli ◽

Coenzyme A ◽

Acyl Carrier Protein ◽

Carrier Protein ◽

Gene Encoding ◽

Specific Expression ◽

Tobacco Seeds ◽

Transgenic Rape ◽

Organelle Targeting

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Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.3807-3813.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 3807-3813 ◽

Cited By ~ 35

Author(s):

Zhong Zheng ◽

Qiang Gong ◽

Tao Liu ◽

Ying Deng ◽

Jin-Chun Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Coenzyme A ◽

Acyl Carrier Protein ◽

Physiological Role ◽

Carrier Protein ◽

E Coli ◽

Thioesterase Ii ◽

Abnormal Accumulation ◽

Acyl Coenzyme A

ABSTRACT 3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene). By employing an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A.

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