Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2128–2133, March 1998 Biochemistry Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: Resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency ELISABETH G. VAN GRUNSVEN*, EMANUEL VAN BERKEL*, LODEWIJK IJLST*, PETER VREKEN*, JOHANNIS B. C. DE KLERK†,JERZY ADAMSKI‡,HUGH LEMONDE§,PETER T. CLAYTON§,DEAN A. CUEBAS¶, \ AND RONALD J. A. WANDERS* ** *University of Amsterdam, Academic Medical Centre, Department of Clinical Chemistry, Laboratory of Genetic Metabolic Diseases, Amsterdam, The Netherlands; †Erasmus University, Sophia Children’s Hospital, Department of Paediatrics, Rotterdam, The Netherlands; ‡GSF-Forzungszentrum, Institut fu¨r Sa¨ugetiergenetik,Neuherberg, D-85764 Oberschleissheim, Germany; §Institute of Child Health, University of London, London WC1N 1EH, United Kingdom; \ ¶Southwest Missouri State University, Department of Chemistry, Springfield, MO 65804; and University of Amsterdam, Academic Medical Centre, Department of Paediatrics, Emma Children’s Hospital, Amsterdam, The Netherlands Edited by Christian de Duve, International Institute of Cellular and Molecular, Brussels, Belgium, and approved December 2, 1997 (received for review September 3, 1997) ABSTRACT Peroxisomes play an essential role in a num- the b-oxidation of fatty acids, the a-oxidation of phytanic acid, ber of different metabolic pathways, including the b-oxidation the synthesis of cholesterol and other isoprenoids, the detox- of a distinct set of fatty acids and fatty acid derivatives. The ification of glyoxylate, and the synthesis of docosahexaenoic importance of the peroxisomal b-oxidation system in humans acid (3, 4). is made apparent by the existence of a group of inherited One of the most important functions of peroxisomes is the diseases in which peroxisomal b-oxidation is impaired. This b-oxidation of fatty acids and fatty acid derivatives (4, 5). Fatty includes X-linked adrenoleukodystrophy and other disorders acid oxidation in peroxisomes differs from that in mitochon- with a defined defect. On the other hand, many patients have dria in many respects, although the overall b-oxidative mech- been described with a defect in peroxisomal b-oxidation of anisms are the same. In both organelles, fatty acid oxidation unknown etiology. Resolution of the defects in these patients proceeds via a sequence of four steps involving a,b- requires the elucidation of the enzymatic organization of the dehydrogenation, hydration, 3-hydroxyacyl-CoA dehydroge- peroxisomal b-oxidation system. Importantly, a new peroxi- nation, and, finally, thiolytic cleavage. An important difference somal b-oxidation enzyme was recently described called D- is that the two b-oxidation systems display different substrate bifunctional protein with enoyl-CoA hydratase and 3-hy- specificities. Indeed, the major dietary fatty acids including droxyacyl-CoA dehydrogenase activity primarily reacting with palmitate, oleate, and linoleate are oxidized in mitochondria, a-methyl fatty acids like pristanic acid and di- and trihy- whereas peroxisomes are involved in the b-oxidation of a range droxycholestanoic acid. In this patient we describe the first of minor fatty acids including very-long-chain fatty acids case of D-bifunctional protein deficiency as resolved by enzyme (notably C26:0), and 2-methyl branched-chain fatty acids like activity measurements and mutation analysis. The mutation pristanic acid. Furthermore, peroxisomes are the sole site of found (Gly16Ser) is in the dehydrogenase coding part of the di- and trihydroxycholestanoic acid (DHC and THC) b-oxi- gene in an important loop of the Rossman fold forming the dation. b-Oxidation of the latter two cholestanoic acids results 1 NAD -binding site. The results show that the newly identified in the formation of the primary bile acids chenodeoxycholic D-bifunctional protein plays an essential role in the peroxi- acid and cholic acid, respectively (4, 5). somal b-oxidation pathway that cannot be compensated for by Several peroxisomal disorders have been described in which the L-specific bifunctional protein. peroxisomal fatty acid b-oxidation is defective. These include X-linked adrenoleukodystrophy (6), the most common perox- Although peroxisomes were initially believed to play only a isomal disorder, as well as pseudo-neonatal adrenoleukodys- minor role in mammalian metabolism, it is now clear that they trophy due to straight-chain acyl-CoA oxidase deficiency (7), catalyze essential reactions in a number of different metabolic L-bifunctional protein deficiency (8), and pseudo-Zellweger pathways and thus play an indispensable role in intermediary syndrome due to a deficiency of 41-kDa peroxisomal thiolase metabolism. (9, 10). Apart from these disorders with a defined defect in The importance of peroxisomes in humans is made apparent peroxisomal b-oxidation, many patients have been described by the existence of a group of inherited diseases, the peroxi- with a defect in peroxisomal b-oxidation of unknown etiology somal disorders, caused by an impairment in one or more (see references 52–59 in ref. 1). peroxisomal functions. The cerebro-hepato-renal (Zellweger) One of the major reasons for the difficulty in resolving the syndrome is generally considered to be the prototype of this underlying defect in these patients has been our insufficient group of diseases. Patients with this disease lack morpholog- knowledge about the functional organization of the peroxiso- ically distinguishable peroxisomes leading to the loss of virtu- mal b-oxidation system. Recent studies, however, have shed ally all peroxisomal functions. Clinically, patients with Zell- new light on the enzymology of the peroxisomal system. weger syndrome show a large variety of severe abnormalities Indeed, it is now clear that multiple enzymes are present for often leading to early death (1, 2). each of the b-oxidation steps. The existence of multiple The metabolic pathways in which peroxisomes are involved acyl-CoA oxidases had already been established (see ref. 4 for include the biosynthesis of ether phospholipids and bile acids, This paper was submitted directly (Track II) to the Proceedings office. The publication costs of this article were defrayed in part by page charge Abbreviations: DHC, dihydroxycholestanoic acid; THC, trihydroxy- cholestanoic acid. payment. This article must therefore be hereby marked ‘‘advertisement’’ in **To whom reprint requests and correspondence should be addressed accordance with 18 U.S.C. §1734 solely to indicate this fact. at: University of Amsterdam, Academic Medical Centre, Depart- © 1998 by The National Academy of Sciences 0027-8424y98y952128-6$2.00y0 ment of Clinical Chemistry (Room F0-226), Meibergdreef 9, 1105 PNAS is available online at http:yywww.pnas.org. AZ, Amsterdam, The Netherlands. 2128 Downloaded by guest on September 30, 2021 Biochemistry: van Grunsven et al. Proc. Natl. Acad. Sci. USA 95 (1998) 2129 review). Recently, however, a new peroxisomal b-oxidation acid b-oxidation, DHAPAT-activity, catalase immunofluores- enzyme was discovered containing both enoyl-CoA hydratase cence, and very-long-chain fatty acids were measured as and 3-hydroxyacyl-CoA dehydrogenase activity (11–15), which described before (19). was first identified as a 17b-estradiol dehydrogenase (16, 17). Measurement of the Enoyl-CoA Hydratase and 3-Hydroxy- Structural analysis of this protein has revealed three functional acyl-CoA Dehydrogenase Components of D-Bifunctional Pro- domains. The N-terminal part (amino acids 1–323) contains tein. The combined activity of the enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activity, whereas the 3-hydroxyacyl-CoA dehydrogenase components of the D- central part (amino acids 324–596) harbors the 2-enoyl-CoA bifunctional protein were measured in a medium of the hydratase activity. The C-terminal part (amino acids 597–737) following composition: 50 mM TriszHCl (pH 8.5), 1 mM shows strong homology with sterol carrier protein and cata- NAD1, 150 mM KCl, 0.1 mM (24E)-3a,7a,12a-trihydroxy-5b- lyzes the in vitro transfer of 7-dehydrocholesterol and phos- cholest-24-enoyl-CoA (24-ene-THC-CoA; prepared as de- phatidylcholine between membranes (11). scribed in ref. 21), 5 mM pyruvate, and 18 unitsyml lactate In contrast to the well known L-bifunctional protein (18) that dehydrogenase. Reactions were allowed to proceed for 60 min converts trans-enoyl-CoA thioesters to their 3-keto forms via at 37°C by using a protein concentration of 150 mgyml. the L-hydroxy-stereoisomer, this new bifunctional enzyme Reactions were terminated by addition of 2 M HCl to a final catalyzes these same transformations via D-hydroxyacyl-CoAs, concentration of 0.18 M followed by neutralization to a pH of which has prompted Hashimoto and coworkers (14, 15) to about 5.0 by using 0.6 M Mes plus 2 M KOH. Resolution of the name it D-bifunctional enzyme. different CoA-esters was achieved essentially as described by An important finding is that the two bifunctional proteins the method of Xu and Cuebas (21). have different substrate specificities. The D-bifunctional pro- RNA Isolation and cDNA Synthesis. Total RNA was isolated tein catalyzes the formation of 3-ketoacyl-CoA intermediates either from cultured skin fibroblasts (stored at 280°C) or from from both straight-chain and 2-methyl-branched-chain fatty freshly prepared lymphocytes by using the acid guanidinium acids, whereas the L-specific bifunctional protein is incapable thiocyanate-phenol-chloroform extraction