Phytanoyl-CoA hydroxylase from rat liver: protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid α-oxidation

Gerbert A Jansen,Rob Ofman,Simone Denis,Sacha Ferdinandusse,Eveline M Hogenhout,Cornelis Jakobs,Ronald J.A Wanders

doi:10.1016/s0022-2275(20)32099-x

Gerbert A Jansen, Rob Ofman + Show 5 more

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https://doi.org/10.1016/s0022-2275(20)32099-x

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Abstract

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid α-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid α-oxidation is localized in peroxisomes. In rat, however, the α-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38.6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. ▪ The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid α-oxidation in rat and human.—Jansen, G. A., R. Ofman, S. Denis, S. Ferdinandusse, E. M. Hogenhout, C. Jakobs, and R. J. A. Wanders. Phytanoyl-CoA hydroxylase from rat liver: protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid α-oxidation.

Highlights

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid ␣-oxidation pathway
Measurement of PhyH activity was based on the conversion of radiolabeled phytanoyl-CoA into 2-hydroxyphytanoyl-CoA, using a radiochemical HPLC method allowing the detection of these CoA-esters [25]
Phytanoyl-CoA hydroxylase catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoylCoA

Summary

Introduction

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid ␣-oxidation pathway. Several studies have shown that in humans, phytanic acid ␣-oxidation is localized in peroxisomes. Phytanoyl-CoA hydroxylase from rat liver: protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid ␣-oxidation. Rainville, and Watkins [2] showed that the first step in the ␣-oxidation process, the conversion of phytanoyl-CoA into 2-hydroxyphytanoyl-CoA is catalyzed by phytanoyl-CoA hydroxylase (PhyH). This enzyme is a 2-oxoglutarate-dependent dioxygenase which requires Fe2ϩ and ascorbate as cofactors (Scheme 1). The individual enzymatic steps of the ␣oxidation pathway have recently been resolved, there is still considerable debate about the subcellular localization

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