Structural and enzymological studies on human mitochondrial and Escherichia coli fatty acid β-oxidation trifunctional enzyme (TFE) complexes

Fatty acids are one of the major sources of energy in living organisms. They are oxidized mainly by the enzymes of β-oxidation pathway for energy production. The trifunctional enzyme (TFE) catalyzes the last three of the four reactions of the fatty acid β-oxidation cycle. TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities in the α subunit and 3-ketoacyl-CoA thiolase (KAT) activity in the β subunit. Mutations in TFE cause several fatty acid β-oxidation defects in human and may lead to severe disorders including sudden infant death syndrome. Human has only the mitochondrial trifunctional enzyme (HsTFE) whereas E. coli has two TFEs: EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions where oxygen molecule is the ultimate electron acceptor. However, anEcTFE is expressed under aerobic as well as under anaerobic conditions using nitrate or fumarate molecule as the ultimate electron acceptors instead of oxygen molecule. The functional unit of TFE is usually a α2β2-heterotetramer. In this study, biochemical, biophysical and structural properties of EcTFE, anEcTFE and HsTFE have been studied and compared with each other as well as with other TFEs. It was discovered in the current studies that the anEcTFE is membrane bound, like the HsTFE, whereas EcTFE is located in the cytosol. The systematic biophysical and kinetic characterization of these TFEs suggested that anEcTFE has more similarity to HsTFE than its aerobic homologue (EcTFE) in terms of their structure and substrate specificity. anEcTFE and HsTFE have a preference for medium- and long-chain enoyl-CoAs, whereas EcTFE prefers short chain enoyl-CoA substrates. These studies further demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for a complete degradation of long-chain enoyl-CoAs in E. coli under aerobic conditions. The crystal structures of α-subunit of EcTFE as well as anEcTFE were determined in this study and these structures were also compared with the recently published structures of HsTFE. The structural analysis shows that the EcTFE ECH active site has a relatively small fatty acyl tail binding pocket when compared to anEcTFE and HsTFE structures, which is in line to their substrate specificity properties. These analyses suggest that anEcTFE and HsTFE are evolutionarily closely related, whereas EcTFE belongs to a separate TFE subfamily. Further, the substrate channeling property of these TFEs has also been studied and the analysis suggests that the negatively charged CoA conjugated intermediates are captured in a positively charged reaction chamber, thereby facilitating the channeling of the intermediates between the active sites at least in EcTFE. Shiv Kumar Sah-Teli, 2020 Keywords: fatty acid β-oxidation, trifunctional enzyme, enoyl-CoA hydratase, 3S-hydroxyacyl-CoA dehydrogenase, thiolase, substrate specificity, substrate channeling