Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic β-oxidation trifunctional enzyme complexes

Abstract

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

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

“…However, both exhibit lower T m (Figure 2c) than the EcTFE complex (T m = 43 ˚C and 45 ˚C, respectively, instead of 51 ˚C), showing that the heterotetrameric complex is the most stable form. In addition, the ECH and HAD specific activities of EcTFE-α with 2E-butenoyl-CoA are 13.8 ± 2.5 and 1.8 ± 0.3 µmole mg -1 min -1 respectively, whereas the corresponding activities in the complex are 29 ± 2 and 2.7 ± 0.4 µmole mg -1 min -1 (Sah-Teli et al, 2019). That is, EcTFE-α is less active than when present as part of the EcTFE complex (Figure 2e).…”

“…EcTFE-α-His and EcTFE-β-His were overexpressed in E. coli BL21DE3pLys. The overexpressed individual subunits as well as the complexes were purified as reported previously (Sah-Teli et al, 2019). Briefly, the soluble fraction containing the overexpressed protein was incubated with Ni-NTA beads, washed and the protein was then eluted with a buffer containing 50 mM Tris, 0.5 M NaCl, 10% glycerol, and 250 mM imidazole at pH 8.0.…”

“…High resolution structures of Pseudomonas fragi TFE (PfTFE) (Imamura et al, 1990, Ishikawa et al, 2004, Mycobacterium tuberculosis TFE (MtTFE) (Venkatesan and Wierenga 2013) and human mitochondrial TFE (HsTFE) (Liang et al, 2018, Xia et al, 2019 show that these three TFEs indeed have unique quaternary structures. Interestingly, in P. fragi, M. tuberculosis and human, there is only one TFE whereas in E. coli there are two TFEs (Campbell et al, 2003, Sah-Teli et al, 2019. Recently, the low resolution SAXS and EM structures of these two TFEs have been reported (Sah-Teli et al, 2019), showing that the overall tetrameric assemblies of EcTFE and anEcTFE are similar to the PfTFE and HsTFE assemblies, respectively.…”

“…Interestingly, in P. fragi, M. tuberculosis and human, there is only one TFE whereas in E. coli there are two TFEs (Campbell et al, 2003, Sah-Teli et al, 2019. Recently, the low resolution SAXS and EM structures of these two TFEs have been reported (Sah-Teli et al, 2019), showing that the overall tetrameric assemblies of EcTFE and anEcTFE are similar to the PfTFE and HsTFE assemblies, respectively. The kinetic properties of anEcTFE and HsTFE are also similar in that they prefer longer chain fatty acyl-CoAs whereas EcTFE prefers shorter to medium chain length fatty acyl-CoAs.…”

“…The kinetic properties of anEcTFE and HsTFE are also similar in that they prefer longer chain fatty acyl-CoAs whereas EcTFE prefers shorter to medium chain length fatty acyl-CoAs. Such complementary substrate specificities of EcTFE and anEcTFE allow for a complete degradation of fatty acyl-CoAs in E. coli under aerobic conditions (Sah-Teli et al, 2019) while in human mitochondria the shorter chain fatty acyl-CoAs are metabolized by monofunctional βoxidation enzymes (Houten et al, 2016).…”