Identification of Pristanal Dehydrogenase Activity in Peroxisomes: Conclusive Evidence That the Complete Phytanic Acid -Oxidation Pathway Is Localized in Peroxisomes G. A. Jansen,* , † D. M. van den Brink,* , † R. Ofman,* , † O. Draghici,* , † G. Dacremont,‡ and R. J. A. Wanders* , † ,1 *Department of Clinical Chemistry and †Department of Pediatrics, University of Amsterdam, Academic Medical Centre, Emma Children’s Hospital, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; and ‡University of Ghent, Ghent, Belgium Received April 10, 2001 Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which, due to the methyl-group at the 3-position, can not undergo -oxidation unless the terminal carboxyl-group is re- moved by -oxidation. The structure of the phytanic acid -oxidation machinery in terms of the reactions involved, has been resolved in recent years and in- cludes a series of four reactions: (1) activation of phy- tanic acid to phytanoyl-CoA, (2) hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, (3) cleav- age of 2-hydroxyphytanoyl-CoA to pristanal and formyl-CoA, and (4) oxidation of pristanal to pristanic acid. The subcellular localization of the enzymes in- volved has remained enigmatic, with the exception of phytanoyl-CoA hydroxylase and 2-hydroxyphytanoyl- CoA lyase which are both localized in peroxisomes. The oxidation of pristanal to pristanic acid has been claimed to be catalysed by the microsomal aldehyde dehydrogenase FALDH encoded by the ALDH10-gene. Making use of mutant fibroblasts deficient in FALDH activity, we show that phytanic acid -oxidation is completely normal in these cells. Furthermore, we show that pristanal dehydrogenase activity is not fully deficient in FALDH-deficient cells, implying the exis- tence of one or more additional aldehyde dehydroge- nases reacting with pristanal. Using subcellular local- ization studies, we now show that peroxisomes contain pristanal dehydrogenase activity which leads us to conclude that the complete phytanic acid -oxidation pathway is localized in peroxisomes. © 2001 Academic Press Key Words: peroxisomes; Refsum disease; phytanic acid fatty acid; fatty acid oxidation. Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a naturally occurring fatty acid which is present in ruminant fats and meat. Like any 3-methyl- branched fatty acid, phytanic acid cannot undergo straightforward -oxidation but first undergoes -oxidation to produce the (n-1) homologue pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) plus CO 2 . Interest in phytanic acid -oxidation dates back to the early 1960s when Klenk and Kahlke (1) reported their remarkable finding that phytanic acid accumu- lates in tissues and plasma from patients suffering from Refsum disease. Refsum patients show a series of abnormalities including retinitis pigmentosa, cerebel- lar ataxia, and chronic polyneuropathy plus a number of less constant features (2). The structure of the phytanic acid -oxidation path- way has long remained a mystery. Until a few years ago, it was believed that phytanic acid, and not phytanoyl-CoA, is the true substrate undergoing -oxidation. Furthermore, mitochondria were thought to be the site of -oxidation. Both postulates turned out to be wrong. A major breakthrough was the discovery of the enzyme phytanoyl-CoA hydroxylase (PhyH) by Mihalik et al. (3). This enzyme catalyses the 2-oxoglutarate, ascorbate and Fe 2+ -dependent forma- tion of 2-hydroxyphytanoyl-CoA from phytanoyl-CoA. Furthermore, the enzyme turned out to be localized in peroxisomes (3–5) and is deficient in Refsum disease Abbreviations used: ER, endoplasmic reticulum; FALDH, fatty aldehyde dehydrogenase; LACS, long-chain acyl-CoA synthetase; PTS, peroxisome targeting signal; SLS, Sjo ¨gren-Larsson syndrome; VLACS, very-long-chain acyl-CoA synthetase. 1 To whom correspondence and reprint requests should be ad- dressed at Department of Pediatrics, F0-224, Academic Medical Cen- tre, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Fax: + 31 20 6962596. E-mail: wanders@ amc.uva.nl. Biochemical and Biophysical Research Communications 283, 674 – 679 (2001) doi:10.1006/bbrc.2001.4835, available online at http://www.idealibrary.com on 674 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.