Structural and functional characterization of the recombinant human mitochondrial trifunctional protein.

Abstract

The α and β subunits of the human mitochondrial trifunctional protein (TFP), the multienzyme complex involved in fatty acid β-oxidation, were coexpressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography. The resulting α/His-β construct was analyzed by gel filtration, sedimentation velocity, and electron microscopy, indicating a predominance of α(2)β(2) and α(4)β(4) complexes, with higher order oligomers. Electron microscopy indicated that the elementary species α(2)β(2) had overall structural similarity with its bacterial homologue. As shown by cosedimentation and surface plasmon resonance analyses, recombinant TFP interacted strongly with cardiolipin and phosphatidylcholine, suggesting that the natural complex associates with the inner mitochondrial membrane through direct interactions with phospholipids. Recombinant TFP displayed 2-enoyl-CoA hydratase (ECH), l-3-hydroxyacyl-CoA dehydrogenase (HACD), and 3-ketoacyl-CoA thiolase (KACT) activities, and ECH and HACD each reached equilibrium when the downstream enzymes (HACD and KACT, respectively) were made inactive, indicating feed-back inhibition. The KACT activity was optimal at pH 9.5, sensitive to ionic strength, and inhibited at concentrations of its substrate 3-ketohexadecanoyl-CoA >5 μM. Its kinetic constants (k(cat) = 169 s(-1), K(m) = 4 μM) were consistent with those determined previously on a purified porcine TFP preparation. Using different assays, trimetazidine, an efficient antiaginal agent, had no significant inhibitory effect on any of the three enzymatic activities of the recombinant TFP preparation, in contrast with other reports. This study provides the first detailed structural and functional characterization of a recombinant human TFP preparation and opens the way to in-depth analyses through site-directed mutagenesis.

DOI: 10.1021/bi100742w

Cite this paper

@article{Fould2010StructuralAF, title={Structural and functional characterization of the recombinant human mitochondrial trifunctional protein.}, author={Benjamin Fould and Virginie Garlatti and Emmanuelle Neumann and Daphna Fenel and Christine Gaboriaud and G{\'e}rard J. Arlaud}, journal={Biochemistry}, year={2010}, volume={49 39}, pages={8608-17} }