Structure-function studies of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2)

Abstract

<div lang="en" class="abs"><p class="abs_header">Abstract</p><p>Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2<em>E</em>)-enoyl-CoA hydratase-2 and (3<em>R</em>)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2). </p><p>The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved.</p><p>The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer.</p><p>The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes.</p><p>The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid <em>in vivo</em> was tested with <em>Saccharomyces cerevisiae fox-2</em> cells (devoid of endogenous MFE-2). Subsequently, <em>in vitro</em> characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.</p></div>