The fabJ-encoded beta-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates.

Siggaard-Andersen, Marie-Louise; Wissenbach, Margit; Chuck, Jo-Anne; Svendsen, Ib; Olsen, Johan G.; Wettstein-Knowles, Penny von

doi:10.1073/pnas.91.23.11027

Cited by 41 publications

(39 citation statements)

References 21 publications

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“…We show that the product of the recombinant fabB gene has the properties assigned to KAS I. Analysis of the fabFlfabJ gene product shows that it does not have the substrate specificity characteristics ascribed by Siggaard-Andersen et al [11] to the putative KAS IV, but does have the properties previously associated with KAS II [13]. We provide additional Chromatographie and substrate-range characterizations of these enzymes and demonstrate that thermal regulation of fatty acid synthesis is due solely to intrinsic properties of the KAS II enzyme.…”

Section: Introductionmentioning

confidence: 90%

“…Proteins in a partially purified fraction containing the putative KAS IV activity were resolved by SDS-PAGE and N-terminal amino acid sequences of several of the polypeptides were determined. One of the sequences showed homology to known condensing enzymes and was subsequently used to clone the corresponding gene which they designated fabJ [11]. Later, Magnuson et al [12] reported cloning oí fabF, a gene with DNA sequence identical to that of fab J.…”

Section: Introductionmentioning

confidence: 99%

“…In 1994, Siggaard-Andersen et al [11] reported identification of a fourth acyl-ACP condensing enzyme in E. coli with a substrate specificity for C4:0-and C6:0-ACP. They referred to this enzyme as KAS IV, and reported a 50% inhibition of its activity in the presence of 3 u.M cerulenin.…”

Section: Introductionmentioning

confidence: 99%

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Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Edwards¹,

Nelsen²,

Metz³

et al. 1997

FEBS Letters

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Analysis of the ß-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene. Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene. This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF. We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.

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Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

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Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Edwards¹,

Nelsen²,

Metz³

et al. 1997

FEBS Letters

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show abstract

“…The E. coli fabF gene (31,32) encoding KAS II was amplified by PCR using the primer set 5Ј-CATGCCATGGTGTCTAAGCGTCGTGTAGTT-G-3Ј and 5Ј-AACTGCAGAGGGTGGCAAATGACAACTTAG-3Ј. Genomic DNA extracted from E. coli JM109 strain functioned as the template.…”

Section: Construction Of a Cdna Library And Cloning Of The Mtkas Cdnamentioning

confidence: 99%

“…After centrifugation at 27,500 ϫ g for 20 min, the supernatant was recovered as the soluble protein extract. To visualize the presence of KAS proteins, the soluble protein extracts were subjected to SDS-PAGE followed by either Coomassie Brilliant Blue staining or transfer to a PVDF membrane for Western blot analysis with antibodies against mtKAS (see below), plus KAS I and KAS II of E. coli (31).…”

Section: Construction Of a Cdna Library And Cloning Of The Mtkas Cdnamentioning

confidence: 99%

Identification and Molecular Characterization of the β-Ketoacyl-[Acyl Carrier Protein] Synthase Component of the Arabidopsis Mitochondrial Fatty Acid Synthase

Yasuno

Wettstein-Knowles

Wada

2004

Journal of Biological Chemistry

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Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, ␤-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C 4 -C 18 fatty acids exhibiting a bimodal distribution with peaks at C 8 and C 14 -C 16 . Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.Fatty acids are synthesized by the enzymatic reactions of acetyl-CoA carboxylase (ACCase) 1 and fatty acid synthase (FAS). FAS enzyme complexes are classified into two groups based on their structural forms and organization. Complexes consisting of multifunctional polypeptides encoded by one or two genes (type I) are present in the cytoplasm of animals and fungi (1, 2), whereas those composed of monofunctional enzymes (type II) are present in most bacteria and plant plastids (3, 4) as well as in mitochondria, as will be detailed below. The incipient reaction of fatty acid synthesis is catalyzed by ACCase to form malonyl-CoA from acetyl-CoA, the initial carbon source. The first FAS activity, malonyl-CoA:ACP transacylase (MCAT), transfers the malonyl group from CoA to acyl carrier protein (ACP) to form the donor substrate malonyl-ACP that provides the C 2 -units for elongation. Repetitive elongation cycles accomplished by FAS start with condensation of the acyl primer substrate with the C 2 -unit to give a ␤-ketoacyl-ACP. The introduced ␤-keto group is then removed by three reactions, a ␤-keto reduction, a ␤-dehydration, and an enoyl reduction. The resulting saturated acyl-ACP serves as a substrate for the next extension. Most frequently seven or eight cycles yield palmitoyl (C 16 )-ACP and stearoyl (C 18 )-ACP, respectively. Additional FAS ac...

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ThegndGene Encoding a Novel 6-Phosphogluconate Dehydrogenase and Its Adjacent Region ofActinobacillus actinomycetemcomitansChromosomal DNA

Yamaguchi

Nakano

Yamashita

et al. 1997

Biochemical and Biophysical Research Communications

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The fabJ-encoded beta-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates.

Cited by 41 publications

References 21 publications

Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Identification and Molecular Characterization of the β-Ketoacyl-[Acyl Carrier Protein] Synthase Component of the Arabidopsis Mitochondrial Fatty Acid Synthase

ThegndGene Encoding a Novel 6-Phosphogluconate Dehydrogenase and Its Adjacent Region ofActinobacillus actinomycetemcomitansChromosomal DNA

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