Insights into the stability and substrate specificity of the <em>E. coli</em> aerobic β-oxidation trifunctional enzyme complex

Abstract

<div lang="en" class="abs"><p class="abs_header">Abstract</p><p>Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In <em>E. coli</em>, the last three steps of the β-oxidation are catalyzed by two heterotetrameric α₂β₂ enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has <em>2E</em>-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of <em>Pseudomans fragi</em> and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain <em>2E</em>-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in <em>E. coli</em>.</p></div>