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

Yoshinori Kashiwayama,Takenori Tomohiro,Kotomi Narita,Miyuki Suzumura,Tuomo Glumoff,J Kalervo Hiltunen,Paul P Van Veldhoven,Yasumaru Hatanaka,Tsuneo Imanaka

doi:10.1074/jbc.m110.104547

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.

Highlights

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme [11,12,13,14,15]
multifunctional enzyme type 2 (MFE2) has a broad substrate spectrum, and is able to hydrate the enoyl-CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids, and bile acid intermediates, and it can dehydrogenate the straight chain and branched chain 3-hydroxyacyl-CoAs (14, 16 –19)
In terms of the transport of fatty acid derivatives destined for peroxisomal ␤-oxidation and their metabolites across the peroxisomal membrane, peroxisomal ATP-binding cassette proteins are suggested to be involved in this process

Summary

Introduction

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme [11,12,13,14,15]. MFE2 has a broad substrate spectrum, and is able to hydrate the enoyl-CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids, and bile acid intermediates, and it can dehydrogenate the straight chain and branched chain 3-hydroxyacyl-CoAs (14, 16 –19). The final step in peroxisomal ␤-oxidation is the thiolytic cleavage of 3-ketoacyl-CoAs into chain-shortened acyl-CoAs and acetyl-CoA or propionyl-CoA It can be catalyzed by 3-ketoacyl-CoA thiolase or sterol carrier protein x, which cleaves straight chain ketoacyl-CoAs or both straight and 2-methyl-3-ketoacyl-CoAs, respectively [20]. In addition to these enzymes, peroxisomes contain other important proteins that are directly or indirectly involved in the ␤-oxidation. In terms of the transport of fatty acid derivatives destined for peroxisomal ␤-oxidation and their metabolites across the peroxisomal membrane, peroxisomal ATP-binding cassette proteins are suggested to be involved in this process. We synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed a photochemical approach with the novel photoreactive fatty acid analogue to identify fatty acid-binding proteins and analyze the substrate binding of the peroxisomal proteins

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Results

Conclusion