[47] Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle

Fong, Jim C.; Schulz, Horst

doi:10.1016/0076-6879(81)71049-8

Cited by 92 publications

(48 citation statements)

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“…sized from oleic acid, 5-cis-tetradecenoic acid, 2-trans-tetradecenoic acid, and 2-trans-dodecenoic acid, respectively, by the mixed anhydride method as described by Fong and Schulz (15). 2-trans-TetradecenoylCoA was partially converted to L-3-hydroxytetradecanoyl-CoA by hydration in the presence of crotonase in 0.1 M KP i (pH 8.0).…”

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confidence: 99%

An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

Ren

Aguirre²,

Ntamack

et al. 2004

Journal of Biological Chemistry

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The degradation of 2-trans,5-cis-tetradecadienoylCoA, a metabolite of oleic acid, by the purified complex of fatty acid oxidation from Escherichia coli was studied to determine how much of the metabolite is converted to 3,5-cis-tetradecadienoyl-CoA and thereby diverted from the classical, isomerase-dependent pathway of oleate ␤-oxidation. Approximately 10% of the 2,5-intermediate was converted to the 3,5-isomer. When the latter compound was allowed to accumulate, it strongly inhibited the flux through the main pathway. Since ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase was not detected in E. coli cells grown on oleate, the 3,5-intermediate cannot be metabolized via the reductase-dependent pathway. However, it was hydrolyzed by a thioesterase, which was most active with 3,5-cis-tetradecadienoyl-CoA as substrate and which was induced by growth of E. coli on oleate. An analysis of fatty acids present in the medium after growth of E. coli on oleate revealed the presence of 3,5-tetradecadienoate, which was not detected after cells were grown on palmitate or glucose. Altogether, these data prompt the conclusion that oleate is mostly degraded via the classical, isomerase-dependent pathway in E. coli but that a small amount of 2-trans,5-cistetradecadienoyl-CoA is diverted from the pathway via conversion to 3,5-cis-tetradecadienoyl-CoA by ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase. The 3,5-intermediate, which would strongly inhibit ␤-oxidation if allowed to accumulate, is hydrolyzed, and the resultant 3,5-tetradecadienoate is excreted into the growth medium. This study provides evidence for the novel function of a thioesterase in ␤-oxidation.

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An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

Ren

Aguirre²,

Ntamack

et al. 2004

Journal of Biological Chemistry

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“…Pawar and Schulz (25) (31). Also, both enzyme activities have been attributed to separate proteins in the mitochondria of higher organisms (7,9,30). Therefore, further purification of these two activities from C. crescentus cell extracts was carried out in an attempt to determine whether they are separate proteins.…”

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confidence: 99%

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus

1990

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Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from ,-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the ,8-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.Caulobacter crescentus is a gram-negative bacterium that exhibits a sequence of morphological changes at the cell surface during each cell cycle (27). These events involve the temporally and spatially defined biogenesis of a single flagellum, a stalk, pili, and DNA bacteriophage receptors. Several of the protein components of these surface structures are membrane associated and are targeted to specific cell surface locations. Because portions of the cell membrane are retained from one cell cycle to another, the possibility exists that the structure and biosynthesis of the membrane are involved in the positioning of these proteins. To determine whether the cell membrane has a role in this process, we are investigating membrane lipid metabolism.The phospholipid composition of the C. crescentus membrane is unusual in that it is predominantly composed of phosphotidylglycerol and cardiolipin (4). Neither phosphotidylethanolamine nor phosphotidylserine is synthesized by C. crescentus (4). The fatty acid composition is a mixture of saturated and monounsaturated 16-and 18-carbon fatty acids (15). These fatty acids are synthesized by an anaerobic pathway in a manner similar to that in Escherichia coli (15). The biogenesis of the membrane is being studied in wild-type cells and in mutants altered in phospholipid (3) and fatty acid (10, 11) synthesis. Some of these mutants are auxotrophic for glycerol-3-phosphate and lack glycerol-3-phosphate dehydrogenase activity (3); others require oleic acid for growth (10, 11). A common feature of these mutants is that they all block membrane lipid synthesis in the absence of supplement, coincident with the disruption of surface differentiation events.To determine how membrane lipid turnover might contribute to the expression of cell surface differentiation events in C. crescentus, we have begun an investigation of both longand short-chain fatty acid catabolism in this organism (22 3-hydroxyacyl-CoA dehydrogenase, and P-ketoacyl-CoA thiolase. Long-chain fatty acids are degraded by these five p-oxidation enzymes in E. coli (Fig. 1). Short-chain fatty acid catabolism in E. coli is known to require three Poxidation enzymes (crotonase, 3-h...

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“…Crotonyl-CoA was purchased from Sigma-Aldrich, MO. As described previously, octenoyl-CoA was synthesized from trans-2-octenoic acid (Tokyo Kasei, Tokyo, Japan) and CoA (Oriental Yeast, Osaka, Japan) (20) and purified using Sep-Pak C 18 columns (Waters, MA) (21). To initiate the hydration reaction, a 5-l aliquot of protein solution was added to quartz cells (1.0-cm path length) containing the enoyl-CoA solution.…”

Section: Methodsmentioning

confidence: 99%

Contribution of the Distal Pocket Residue to the Acyl-Chain-Length Specificity of ( R )-Specific Enoyl-Coenzyme A Hydratases from Pseudomonas spp

Tsuge

Sato

Hiroe

et al. 2015

Appl Environ Microbiol

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d(R)-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid ␤-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1 Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1 Pa from Pseudomonas aeruginosa, despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1 Pp , the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1 Pa , the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1 Pp and PhaJ1 Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1 Pp than in PhaJ1 Pa . Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1 Pp and PhaJ1 Pa , all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1 Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1 Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity. P olyhydroxyalkanoates (PHAs), which are synthesized by some bacterial taxa, act as energy and carbon stores for use during starvation and are desirable for use as bio-based polymer materials (1-3). PHA biosynthesis requires a number of specific enzymes, such as PHA synthases and monomer-supplying enzymes. PHA synthase polymerizes the (R)-3-hydroxyalkanoate moiety of (R)-3-hydroxyacyl coenzyme A (3HA-CoA) into PHA, whereas monomer-supplying enzymes synthesize (R)-3HA-CoA from various metabolic intermediates. To control the material properties of PHAs, it is necessary to regulate the PHA monomer composition, which depends strongly on the enzymatic properties of PHA synthases and monomer-supplying enzymes.(R)-Specific enoyl-CoA hydratase [PhaJ, (R)-hydratase] is a monomer-supplying enzyme that was first found to be involved in the biosynthesis of PHA from fatty acids in Aeromonas caviae (4, 5). Trans-2-enoyl coenzyme A (enoyl-CoA), an intermediate in fatty acid ␤-oxidation, undergoes stereospecific hydration by the function of PhaJ, resulting in the formation of (R)-3HA-CoAs. Currently, four types of phaJ genes (phaJ1 Pa to phaJ4 Pa ) have been identified in the genome of Pseudomonas aeruginosa DSM1707; their translational products were characterized...

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[47] Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle

Cited by 92 publications

References 9 publications

An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus

Contribution of the Distal Pocket Residue to the Acyl-Chain-Length Specificity of ( R )-Specific Enoyl-Coenzyme A Hydratases from Pseudomonas spp

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