Incomplete Beta Oxidation of Unsaturated Fatty Acids Facilitated by Thioesterases in Escherichia Coli
An investigation of thioesterases involved in a novel pathway of beta-oxidation, referred to as thioesterase-dependent pathway, led to the identification of a new thioesterase (thioesterase III) that is induced by growth of E. coli on oleic acid. This enzyme was partially purified and identified as the ybaW gene product by mass spectrometric analysis of tryptic peptides. The ybaW gene, which has a putative consensus sequence for binding the fatty acid degradation repressor, was cloned and expressed in E. coli. Thioesterase III was shown to be a long-chain acyl-CoA thioesterase that is most active with 3,5-tetradecadienoyl-CoA, a minor metabolite of oleate beta-oxidation. Its substrate specificity and induction of expression by fatty acids agree with its proposed function in this pathway by hydrolyzing metabolites of beta-oxidation that are resistant to further degradation and that would inhibit the flux through the pathway if allowed to accumulate. This pathway was further studied in E. coli with 9-cis,11-trans-octadecadienoic acid (conjugated linoleic acid) as substrate, which was shown to support growth of E. coli in the absence of any other carbon source. The identification of 3,5-dodecadienoic acid in the growth medium revealed the partial beta-oxidation of conjugated linoleic acid to 3,5-dodecadienoyl-CoA, which was hydrolyzed to 3,5-dodecadienoic acid and released from the cell. The involvement of acyl-CoA thioesterases in this process was evaluated by determining the substrate specificity of thioesterase II and comparing it with that of thioesterase III and by assessing mutant strains devoid of one or both of these thioesterases for growth on conjugated linoleic acid. Both thioesterases were highly active with 3,5-dodecadienoyl-CoA as substrate. A deficiency of either thioesterase decreased the growth rate of cells on conjugated linoleic acid, but not on palmitic acid. The absence of both thioesterases reduced growth even further, but did not abolish it completely. It is concluded that thioesterases II and III function in the partial degradation of conjugated linoleic acid via the thioesterase-dependent pathway of beta-oxidation, which provides all energy and carbon precursors required for the growth of E. coli.
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3,5-cis-tetradecadienoyl-CoA 3,5-dodecadienoic acid 3,5-dodecadienoyl-CoA 3,5-tetradecadienoyl-CoA 6xHis-tagged absence of thioesterases acetyl-CoA acyl acyl-CoA dehydrogenase assayed for thioesterase bands beta oxidation buffer centrifuged chloramphenicol coli cells coli strain YR1 coli thioesterase column chromatography completely degraded conjugated linoleic acid containing DEAE-cellulose degraded fatty acids dependent pathway dienoyl-CoA dodecadienoyl-CoA double bonds eluted encoding thioesterase enoyl-CoA isomerase enzyme extracted fadR fatty acid degradation fatty acyl-CoA glucose glycerol gradient grown on oleate growth medium hydrolysis hydrolyzed immunoblotting isomerase-dependent pathway KPi pH long-chain acyl-CoA thioesterase mass spectrometry material corresponding methyl esters mitochondria molecular mutant myristoyl-CoA as substrate myristoyl-CoA thioesterase activity NaCl oleate oleic acid oleoyl-CoA oxidation palmitic acid partially degraded fatty partially purified novel peptides plasmid purified novel thioesterase reductase-dependent pathway Schulz SCoA SDS-PAGE sequence shown in Fig sole carbon source substrate specificity tetradecadienoyl-CoA thioesterase II thioesterase mutants thioesterase-dependent pathway Tris-HCl pH 7.8 unsaturated fatty acids wild-type ybaW protein YR1 by column