title: Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative.
abstract: Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.
authors.affiliation: Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kashiwayama
authors.forename: Takenori
authors.initials: T
authors.lastname: Tomohiro
authors.forename: Kotomi
authors.initials: K
authors.lastname: Narita
authors.forename: Miyuki
authors.initials: M
authors.lastname: Suzumura
authors.forename: Tuomo
authors.initials: T
authors.lastname: Glumoff
authors.forename: J Kalervo
authors.initials: JK
authors.lastname: Hiltunen
authors.forename: Paul P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.forename: Yasumaru
authors.initials: Y
authors.lastname: Hatanaka
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D003978:Diazomethane / Q000737:chemistry; D004746:Enoyl-CoA Hydratase / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver; D015336:Molecular Probe Techniques; D009097:Multienzyme Complexes / Q000032:analysis* / Q000235:genetics*; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000737:chemistry*; D064029:Peroxisomal Multifunctional Protein-2; D020675:Peroxisomes / Q000201:enzymology*; D019920:Photoaffinity Labels; D051381:Rats
chemical_list: D009097:Multienzyme Complexes; D019920:Photoaffinity Labels; D019308:Palmitic Acid; D003978:Diazomethane; D015067:17-Hydroxysteroid Dehydrogenases; C491286:Hsd17b4 protein, rat; D064029:Peroxisomal Multifunctional Protein-2; D004746:Enoyl-CoA Hydratase