Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Abstract

Peroxisomes play an essential role in a number of important metabolic pathways including ␤-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various ␤-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 ␤-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.Peroxisomes are organelles bound by a single membrane, which are present in almost all eukaryotic cells. Peroxisomes are involved in a variety of important metabolic processes including the ␤-oxidation of fatty acids, synthesis of plasmalogen, and bile acids (1). A specialized set of enzymes responsible for the peroxisomal functions are compartmentalized in the organelle, and the deficiency of peroxisomal enzymes causes severe metabolic disease such as acyl-CoA oxidase deficiency and D-bifunctional protein deficiency (2-4). Fibroblasts from patients with these deficiencies exhibit the disturbed peroxisomal ␤-oxidation of very long-chain fatty acids (VLCFAs), 4 and VLCFAs are accumulated in the plasma and tissues of these patients (5). Analyses of these peroxisomal diseases reflect the importance of the peroxisomal ␤-oxidation in organisms along with disclosing the enzymatic organization of the peroxisomal ␤-oxidation system. Peroxisomes are involved in the ␤-oxidation of VLCFAs, branched chain fatty acids such as pristanic acid, and bile acid intermediates such as dihydroxycholestanoic acid and trihydroxycholestanoic acid, which are incompatible with mitochondrial ␤-oxidation (6). Peroxisomal ␤-oxidation of fatty acids proceeds via a four-step pathway as in mitochondria, and multiple enzymes are involved in each step of the...

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

“…Kashiwayama et al [36] found that the area Trp249-Arg251 is essential for substrate binding and W249G variation leaves the capacity of the enzyme to bind substrates to a level of 30% of the native protein, a feature seen also in our results by increasing K m . In the yeast complementation experiment, both R248C and W249G enzymes could still utilize LCFAs (Figure 2).…”

“…This could loosen the structure locally, which is supported by the increased temperature sensitivity (Table 1), and disturb the binding of NAD + , seen in the activity measurements as a need for a large surplus of NAD + to achieve measurable data. In MD simulations the fluctuation extends also to the signature Ser-Tyr-Lys catalytic triad of the SDR superfamily [35], as well as to the cavity area (Figure 4) important for the substrate binding [36]. Most important seems to be, however, that the hydrogen bond not involving any side chain atoms (T15 N to V97 O ) seems to be more stable.…”

“…Variation sites are color coded with the fluctuation analysis as follows: N158D (green), E232K (violet), R248C (yellow) and W249G (light brown). Residues found essential for discussion on the D-BP deficiency mechanism are colored as follows: amino acids belonging to catalytic triad (S151, T164 and K168) in magenta, amino acids locating at the tip of the cavity leading to the active site (I180, Y156 [36] and I288) in purple blue and amino acids important for the substrate binding (E250 and R251) in orange [36]. NAD + is colored in yellow and presented in stick presentation.…”

“…These two latter areas have been shown to be essential for substrate binding also by Kashiwayama et al . [36] with N158D variation causing decreased capability to bind VLCFA substrates. Also the NAD + binding site seems to have increased fluctuation.…”

Multiple Cellular Transport and Binding Processes of Unesterified Docosahexaenoic Acid in Outer Blood–Retinal Barrier Retinal Pigment Epithelial Cells

“…The uptake of BSA-or RBPassociated [ 14 C] DHA (14 nM) by the capped cRNA-or waterinjected Xenopus laevis oocytes was examined as reported previously. 25) The uptake amount of [ 14 C] DHA in the oocytes was evaluated as the oocyte-to-medium (oocyte/medium) ratio (µL/oocyte) as reported previously. 25) Chemical Synthesis of Photoreactive DHA Analogue Bearing a Diazirine Moiety as a Photophore (Fig.…”